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I have in my notes that a decrease in free Ca2+ increases membrane permeability to Na+ so that it is brought closer to threshold, but no further details. So how does this work?
The thing is that the Voltage Gated Channels,they are closed in resting state because Ca2+ is bound to it.So,in the resting stage,Ca2+ somewhat blocks the Na+ - K+ channel.But since there is low Ca2+ so there won't be much Ca2+ for guarding of channels and so it will lead to an increase in permeability.See the photo and imagine the gate being guarded by a Ca2+.I hope this helps.
Jack bean urease modulates neurotransmitter release at insect neuromuscular junctions
Background: Plants have developed a vast range of mechanisms to compete with phytophagous insects, including entomotoxic proteins such as ureases. The legume Canavalia ensiformis produces several urease isoforms, of which the more abundant is called Jack Bean Urease (JBU). Previews work has demonstrated the potential insecticidal effects of JBU, by mechanisms so far not entirely elucidated. In this work, we investigated the mechanisms involved in the JBU-induced activity upon neurotransmitter release on insect neuromuscular junctions.
Methods: Electrophysiological recordings of nerve and muscle action potentials, and calcium imaging bioassays were employed.
Results and conclusion: JBU (0.28 mg/animal/day) in Locusta migratoria 2nd instar through feeding and injection did not induce lethality, although it did result in a reduction of 20% in the weight gain at the end of 168 h (n = 9, p ≤ 0.05). JBU (0.014 and 0.14 mg) injected direct into the locust hind leg induced a dose and time-dependent decrease in the amplitude of muscle action potentials, with a maximum decrease of 70% in the amplitude at the highest dose (n = 5, p ≤ 0.05). At the same doses JBU did not alter the amplitude of action potentials evoked from motor neurons. Using Drosophila 3rd instar larvae neuromuscular preparations, JBU (10 -7 M) increased the occurrence of miniature Excitatory Junctional Potentials (mEJPs) in the presence of 1 mM CaCl2 (n = 5, p ≤ 0.05). In low calcium (0.4 mM) assays, JBU (10 -7 M) was not able to modulate the occurrence of the events. In Ca 2+ -free conditions, with EGTA or CoCl2, JBU induced a significant decrease in the occurrence of mEPJs (n = 5, p ≤ 0.05). Injected into the 3rd abdominal ganglion of Nauphoeta cinerea cockroaches, JBU (1 μM) induced a significant increase in Ca 2+ influx (n = 7, p ≤ 0.01), similar to that seen for high KCl (35 mM) condition. Taken together the results confirm a direct action of JBU upon insect neuromuscular junctions and possibly central synapses, probably by disrupting the calcium machinery in the pre-synaptic region of the neurons.
Keywords: Calcium influx Insects Neuromuscular junction Neurotoxicity Plant ureases.
Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality (Crawford and Pardo, 1996), is an autosomal recessive degenerative disease of lower motor neurons. Symptoms include muscular weakness and atrophy of limb and trunk muscles. SMA is caused by mutations or loss of the SMN1 gene and retention of the SMN2 gene (Lefebvre et al., 1995) both genes encode for the survival motor neuron (SMN) protein. The best-characterized SMN function is its participation in the assembly of small nuclear ribonucleoproteins (Fischer et al., 1997 Liu et al., 1997 Meister et al., 2001 Pellizzoni et al., 2002). In the absence of a functional SMN1 gene, the severity of the disease depends on the amount of full-length SMN (SMN-FL) produced by SMN2. The majority of transcripts from the SMN2 gene lack exon 7 (SMNΔ7), but a small amount of the transcript is SMN-FL (Gennarelli et al., 1995 Lorson et al., 1999 Monani et al., 1999). Although SMN is a ubiquitous protein, deficient levels of SMN predominantly damage lower motor neurons (Monani et al., 2000). It has been proposed that SMA is a motor neuron “dying back” axonopathy, supported by the finding that in SMA there is substantial accumulation of neurofilaments in terminal axons followed by a major loss of motor neurons (Cifuentes-Diaz et al., 2002 Kariya et al., 2008). It has also been postulated that the reduction of SMN produces a motor neuron synaptopathy manifested by the arrest of the postnatal development of the neuromuscular junction (NMJ) and a functional deficit (Kariya et al., 2008 Kong et al., 2009). Electrophysiological analysis in the tibialis anterior (TA) muscle of a relatively severe SMA mouse model (Le et al., 2005 Butchbach et al., 2007 Murray et al., 2008 Kong et al., 2009) shows that the number of synaptic vesicles that release neurotransmitter during an action potential is decreased (Kong et al., 2009).
We sought to address the cellular mechanism of the pathogenesis of SMA by studying further the electrophysiological properties of neuromuscular synaptic transmission in the SMNΔ7 SMA mouse mutant. We have studied two proximal muscles, a pure fast-twitch muscle [Levator auris longus (LAL), located at the dorsal surface of the head and innervated by the facial nerve] and a predominantly slow twitch muscle [Transversus abdominis (TVA), a postural muscle from the anterior abdominal wall innervated by lower intercostal nerves]. We examined neuromuscular transmission at two stages, at an early age [postnatal days 7–8 (P7–8)], where the motor dysfunction is already evident (Le et al., 2005), and at a later age (P14–15), coincident with the end of the mouse life period. We hypothesized that in SMA there is an early synaptic dysfunction in motor nerve terminals. We first studied the ability of mutant muscular fibers to trigger action potentials upon stimulation from the presynaptic terminal. Next, we investigated the functionality of multiple nerve terminals innervating the same myofiber.
Finally, we investigated the capacity of presynaptic terminals to release neurotransmitters in three modes: (1) at rest (spontaneous release), (2) upon a single action potential (synchronous evoked release), and (3) during intraterminal Ca 2+ accumulation resulting from prolonged electrical stimulation (asynchronous release). In SMA we found that in addition to severe evoked neurotransmission defects, there was an abnormal increase in the amount of Ca 2+ -dependent asynchronous release during prolonged stimulations, suggesting an altered intraterminal bulk Ca 2+ concentration in SMA synapses.
Efficacy, reliability and paired-pulse modulation of synaptic transmission in connections between L2/3 pyramidal cells and interneurones
Experiments were performed on pairs or triplets of neurones (Fig. 1A) that form small local circuits in layer 2/3 of rat neocortex ( Reyes et al. 1998 ). Presynaptic neurones were always pyramidal cells, identified by the shape of the soma and the pattern of frequency accommodation of APs developing upon depolarizing somatic current injection. Target neurones were non-pyramidal, bitufted neurones and/or multipolar neurones. They too were identified by their soma shape and the characteristic AP pattern developing upon current injection ( Reyes et al. 1998 ). The peak amplitude of the unitary EPSP, evoked by a single presynaptic AP (the first AP in the train Fig. 1B) was taken as an index of efficacy, the percentage of failures as a measure of the reliability of a particular connection. As with previous experiments ( Reyes et al. 1998 ) made at 35°C, our experiments performed at 22-24°C, found large differences between the two types of connection (Fig. 1B). Unitary EPSPs recorded in bitufted cells were smaller and increased upon repetitive stimulation (Fig. 1B, left), whereas unitary EPSPs in multipolar cells were larger and decreased in amplitude (Fig. 1B, right).
Simultaneous, triple whole-cell voltage recording from pyramidal neurones and two classes of interneurone
A, schematic drawing of the recording configuration. A presynaptic L2/3 pyramidal neurone (P) was stimulated by brief pulses of intracellular current injection. Simultaneous whole-cell voltage recordings were made of unitary EPSPs from a bitufted (B) and a multipolar (M) neurone. B, simultaneous recordings of presynaptic APs evoked in a pyramidal neurone (top) and the unitary EPSPs in a bitufted and a multipolar neurone. Five consecutive traces recorded in the bitufted (left) and multipolar (right) neurone are shown. Lines above the records indicate the time of presynaptic APs in the train. The lowermost traces represent averages of 100 sweeps. The ratio of peak amplitudes of the second and first mean EPSP (EPSP2/EPSP1) was 1.43 in the bitufted neurone and 0.67 in the multipolar neurone.
The mean amplitude of unitary EPSPs was considerably smaller in bitufted cells compared to that recorded in multipolar cells. Figure 2A shows that the amplitude distribution in bitufted cells was narrow, with the mean being 0.92 ± 0.49 mV (n = 66). In contrast, in multipolar cells, the amplitude of EPSPs was larger and their distribution was wider, with a mean amplitude of 3.3 ± 1.9 mV (n = 41). The distributions of amplitudes recorded in the two classes of target cells show a small overlap in the region 0.5-2 mV (Fig. 2A).
Efficacy, reliability and paired-pulse ratio of synapses made by pyramidal neurones with bitufted or multipolar cells
A, efficacy of synaptic transmission. Distribution of average unitary EPSP amplitudes recorded in bitufted (left, n= 66) and multipolar (right, n= 41) neurones following stimulation of the presynaptic pyramidal cell. B, reliability of connections as measured by the percentage of failures. Failure distributions for recordings from bitufted (left, n= 72) and multipolar (right, n= 56) neurones. C, PPR as measured by the ratio EPSP2/EPSP1 recorded in bitufted (left, n= 76) and multipolar (right, n= 56) neurones.
A further clear difference between the two types of connection is their reliability as measured by the percentage of failures of the presynaptic AP to evoke an EPSP. Figure 2B shows the distribution of the percentage of failures in pyramid-to-bitufted and pyramid-to-multipolar connections. Whereas the presynaptic AP frequently failed to elicit an EPSP in bitufted neurones, indicating an unreliable synaptic connection, APs rarely failed to elicit an EPSP in multipolar neurones. The mean failure rates were 42 ± 18 %(n = 72) for the pyramid-to-bitufted connection and 1.63 ± 3.5 %(n = 56) for the pyramid-to-multipolar connection (Fig. 2B). Again the two distributions showed only a very small overlap.
A further property of synapses that is thought to be determined mostly by presynaptic mechanisms, the frequency-dependent change in efficacy, also showed a clear difference between the two types of connection. Figure 2C illustrates the distribution of the paired-pulse ratios (PPRs) of EPSPs in connections between pyramidal cells and bitufted or multipolar cells. EPSP amplitudes were normalized to the amplitude of the first EPSP (EPSP1) in a train. Whereas in the pyramid-to-bitufted connections the size of the second (EPSP2) and third EPSP (EPSP3) evoked by a train of presynaptic APs increased, it decreased in the pyramid-to-multipolar connections. The mean PPRs were 195 ± 59 %(n = 76) in bitufted cells and 53 ± 12 %(n = 56) in multipolar cells.
The differences in efficacy of pyramid-to-bitufted and pyramid-to-multipolar connections can reflect both pre- and postsynaptic factors whereas the differences in reliability and paired-pulse modulation are likely to be of presynaptic origin. As Ca 2+ influx into boutons drives evoked release, we investigated the effects of manipulating Ca 2+ dynamics in the two classes of nerve terminal.
Calcium channel subtypes
Differences in the Ca 2+ channel subtypes that are present in the two classes of nerve terminal could account for the different effectiveness of the coupling between Ca 2+ influx and transmitter release ( Wu et al. 1998 , 1999 ). We determined the contribution of different Ca 2+ channel subtypes to release by application of Ca 2+ channel blockers that are subtype specific.
In control conditions, when no exogenous buffer was loaded into pyramidal cells, EPSPs in both types of target cell were blocked completely by addition of Cd 2+ (100 μ m ) to the bath solution. Application of the P/Q-type calcium channel blocker ω-agatoxin IVA (Aga, 100 n m ) also reduced the amplitude of the EPSP, but only to about 40 % of the control value (Fig. 3A). This toxin blocks P-type channels with a KD of about 1 n m and Q-type channels with a KD of 100 n m .
Effect of calcium channel-blocking toxins on synaptic efficacy
A, time course of the effect of the calcium channel blocker ω-agatoxin IVA on unitary EPSP peak amplitude measured in a multipolar neurone during extracellular toxin application as indicated by the bar. Each point represents the mean of 10 responses. B, effects of Cd 2+ , Q/P-type, N-type and P/Q/N-type calcium channel-specific toxins on unitary EPSPs in bitufted (□) and multipolar ( ) neurones. The ordinate represents (in %) the ratio of mean EPSP amplitude in the steady state during and before toxin application (dashed lines in A).
Application of ω-conotoxin MVIIC (MVIIC), a blocker of P/Q- and N-type channels ( Wu et al. 1999 ), also very effectively reduced EPSP amplitude. At 1 μ m of MVIIC, unitary EPSP amplitudes were reduced to < 3 % of the control value in both facilitating and depressing terminals (Fig. 3B). This toxin blocks P-type channels with a KD of about 500 n m and Q-type channels with a KD of about 5 n m ( Zhang et al. 1993 ), suggesting that Q-type channels significantly contribute to release. The EPSP that remained after adding saturating toxin concentrations was blocked by 50 μ m Cd 2+ in both facilitating and depressing terminals. This residual release is probably due to Ca 2+ influx via R-type calcium channels ( Wu et al. 1998 ) as it was blocked by Ni 2+ (100 μ m , not shown).
Application of ω-conotoxin GVIA (Ctx), which is specific for N-type channels, blocked release to about 20 % of the control value in both types of terminal, indicating that N-type channels strongly contribute to evoked release from pyramidal cell terminals (Fig. 3B). Thus mostly P/N/Q-type calcium channels mediate release in both classes of pyramidal cell terminal. Note that facilitating terminals on average were slightly but not significantly (P > 0.1, Student's t test) more sensitive to any toxin.
Calcium concentration dependence of efficacy
We next measured the dependence of EPSP amplitude on [Ca 2+ ]o to identify possible differences in the relationship between facilitating and depressing terminals. Such differences might reflect different degrees of saturation of Ca 2+ sensors or of other limiting factors. Figure 4 illustrates that release was very differently dependent on [Ca 2+ ]o in the range between 1 and 4 m m . In facilitating terminals the relationship between EPSP amplitude and [Ca 2+ ]o was very steep, showing only moderate saturation, whereas in depressing terminals the EPSP amplitude was close to saturation at the normal [Ca 2+ ]o of 2 m m . The data points for facilitating and depressing synapses could be fitted satisfactorily by a Hill equation with an exponent of 4 and half-effective concentrations (a1/2) of 2.79 and 1.09 m m , respectively. Smaller exponents or model equations for independent binding to several sites could not simultaneously fit (with the same exponent) the steep relation of the curve for facilitating terminals and the saturating character of the curve for depressing terminals, in agreement with Reid et al. (1998). Below, we will discuss the differences between the two types of terminal in terms of differences in the saturation of Ca 2+ sensors, although it should be kept in mind that mechanisms other than saturation of sensors might be responsible for limiting the release.
Effect of [Ca 2+ ]o on synaptic efficacy
Relative change of unitary EPSP amplitude following changes of [Ca 2+ ]o in connections of pyramidal cells with bitufted cells (▪) and multipolar cells (•) plotted on a double logarithmic scale. In each experiment [Ca 2+ ]o was either increased or decreased from control (2 m m ). [Mg 2+ ]o was kept constant at 1 m m . EPSPs measured at different [Ca 2+ ]o were normalized to the values obtained at 2 m m [Ca 2+ ]o in the same experiment. Data are least-square fits to Hill isotherms with nH= 4, according to:with ymax and K1/2 as free parameters. K1/2 values were 2.79 and 1.09 m m for data on the connections of pyramidal cells with bitufted cells (showing facilitation) and multipolar cells (showing depression), respectively.
Latency of EPSCs and asynchronous release
A lower degree of saturation of the Ca 2+ sensor in terminals on bitufted cells could be explained by either a lower affinity of the sensor or a longer diffusional distance between release sites and Ca 2+ channels. If the first is true, one might expect release from the facilitating synapse to be more synchronized than that from the depressing one. The rationale for this prediction is that [Ca 2+ ]RS, the Ca 2+ concentration at the release site, drops below the high threshold level within a shorter time. We, therefore, measured the latency of unitary EPSCs evoked by pyramidal cell stimulation in the two types of interneurone. The latency of EPSCs recorded in bitufted and multipolar cells was 2.97 ± 0.42 (n = 4) and 1.17 ± 0.19 ms (n = 4), respectively. In this analysis we took the first EPSC evoked within a 5 ms time window after the peak of the AP to be a phasic response. The distribution of latencies was also much wider for the responses measured from bitufted cells than that for multipolar cells (Fig. 5A), consistent with a longer diffusional distance in boutons contacting bitufted cells. In addition to this we observed prominent asynchronous or delayed release from both facilitating and depressing terminals (Fig. 5B, events measured in the interval from 5 to 20 ms after an AP) lasting for tens of milliseconds after an AP. In bitufted interneurones evoked delayed EPSCs can be investigated with minimal interference from spontaneous EPSCs, which occur at a very low frequency. In multipolar cells, which have a relatively high rate of spontaneous activity, we compared the number of spontaneous EPSCs before an AP with the number of asynchronous events after an AP, counted within the same time window (15 ms). In all cases the asynchronous events were more frequent than spontaneous EPSCs. For instance, in the experiment shown in Fig. 5B, analysis of 100 sweeps revealed 16 spontaneous EPSCs, which occurred before stimulation of the presynaptic pyramidal cell, and 183 asynchronous responses following the AP initiation. This ratio between the rates of phasic and asynchronous release is much smaller than that in many other types of synapse ( Borst & Sakmann, 1996 Ravin et al. 1999 ). This suggests that the increase in [Ca 2+ ]RS driving phasic release may not be as large as in other synapses.
Latency of EPSCs and asynchronous release from pyramidal cell terminals contacting bitufted or multipolar cells
A, representative distributions of latencies of phasic EPSCs measured within the first 5 ms after AP initiation in a bitufted cell (left) and a multipolar cell (right). Continuous lines represent Gaussian fits. B, time distribution of evoked (phasic and delayed) release from the facilitating (upper panel) and the depressing (lower panel) terminals measured in the same cell pairs as in A. Each bar represents the number of EPSCs recorded at a given time point after AP initiation.
Exogenous ca 2+ buffers affect efficacy
Another way to interfere with presynaptic Ca 2+ dynamics is to load terminals with Ca 2+ buffers having different rates of Ca 2+ binding such as EGTA and BAPTA, which compete with the endogenous Ca 2+ buffer.
Differential effects of EGTA and BAPTA on release.
We first determined the effect of loading pyramidal neurones with EGTA or BAPTA on synaptic efficacy, measured by the mean amplitude of the first unitary EPSP evoked during a 10 Hz train of three presynaptic APs. The individual trains of APs were separated by long intervals (5-7 s). The protocol used to measure the concentration dependence of the reduction in EPSP amplitude during loading of a pyramidal neurone with Ca 2+ buffer is shown in Fig. 6. First, 100 responses with buffer-free intracellular control solution were collected to establish a baseline. Subsequently, the presynaptic pipette was retracted and the same pyramidal cell was accessed once more with a new pipette, filled with a buffer-containing intracellular solution (Ohana & Sakmann, 1998 ). After 7-10 min, required for loading the buffer into the terminals, another 100 unitary EPSPs were recorded. The relative reduction in the size of EPSP1 in a train was estimated as a ratio of the mean amplitudes (EPSP1B/EPSP1C in %) measured after buffer loading (B) and in control conditions (C).
Effects of BAPTA loading on release from pyramidal cell terminals contacting bitufted or multipolar cells
A, effect of presynaptic BAPTA loading of a pyramidal cell on unitary EPSPs recorded from a bitufted cell. •, control EPSPs •, EPSPs after loading of pyramidal neurone with 0.1 m m BAPTA. Arrow indicates the time when a new whole-cell recording configuration with a buffer-containing pipette (P2) was established. Records below the graph represent averaged presynaptic APs (pre Vm) and unitary EPSPs (post Vm). B, effect of presynaptic BAPTA (0.5 m m ) loading of a pyramidal neurone on unitary EPSPs recorded from a multipolar neurone.
Figure 6 A illustrates the effect of 0.1 m m BAPTA when loaded into facilitating terminals of a pyramidal cell. The mean amplitude of the EPSP recorded in the bitufted neurone following BAPTA loading was approximately 50 % of control (lower traces). In contrast, the EPSPs recorded in multipolar neurones, which were innervated by depressing terminals, were barely reduced by this concentration of BAPTA (not shown). To produce a 50 % reduction of the EPSP amplitude in multipolar cells, a much higher concentration of BAPTA (0.5 m m ) had to be loaded into the depressing terminals (Fig. 6B).
Buffer concentration dependence.
Figure 7 shows the buffer concentration-dependent reduction of mean EPSP amplitude normalized to the control EPSP amplitude (before buffer loading) in bitufted (Fig. 7A) and multipolar neurones (Fig. 7B) when pyramidal cells were loaded with either BAPTA or EGTA. Release from terminals on bitufted neurones was significantly more sensitive to both buffers. The half-effective concentrations (which reduced EPSPs in bitufted cells to one-half of the control value) were 0.1 m m for BAPTA and 1 m m for EGTA. The half-effective concentrations for the terminals on multipolar cells were much higher, being 0.5 m m for BAPTA and 7 m m for EGTA.
Concentration-dependent effect of internal BAPTA or EGTA on release from pyramidal cell terminals contacting bitufted or multipolar cells
A, graph of the presynaptic BAPTA and EGTA concentration-dependent effect on the mean amplitude of unitary EPSP evoked by AP stimulation of terminals contacting bitufted cells. Mean EPSP amplitude was normalized to control values determined in the same connection before loading with buffer. The half-effective concentrations (when the mean EPSP amplitude was reduced to 50 % of the control dashed lines) were 0.1 m m BAPTA and 1 m m EGTA, respectively. B, graph of the presynaptic BAPTA and EGTA concentration-dependent effect on the mean amplitude of EPSPs evoked by AP stimulation of terminals contacting multipolar cells. The half-effective concentrations (dashed lines) were 0.5 m m BAPTA and 7 m m EGTA. The lines connecting the data points were calculated by double exponential fits.
In synapses between pyramidal and bitufted neurones the concentration dependence of EPSP reduction by BAPTA loading was steepest in the range between 0.02 and 0.7 m m . At 1 m m BAPTA or higher the EPSP amplitude was at most 5-10 % of the control value. The remaining level of evoked release gradually dropped with further elevation of the BAPTA concentration to 3 m m (Fig. 7A). In terminals on multipolar neurones a comparable residual level of evoked release (< 10 % of control) was observed only at higher (≥ 3 m m ) BAPTA concentrations.
The result that BAPTA, at relatively low concentrations, reduced transmitter release in both types of terminal may suggest that their concentration of endogenous mobile buffer is low. The average diffusional distance for Ca 2+ between the Ca 2+ domain and the Ca 2+ sensor at the vesicle release site (RS) is long enough so that the exogenous buffers can intercept Ca 2+ . Nerve terminals forming synapses on bitufted neurones were more sensitive to both of the exogenous buffers. The higher effectiveness of either buffer loaded into terminals contacting bitufted cells could indicate that in these terminals the diffusional distance between Ca 2+ channels and the vesicular Ca 2+ sensor is longer or, alternatively, that the affinity of the sensor for Ca 2+ is lower (see below for a detailed discussion).
Exogenous ca 2+ buffers affect short-term modification of efficacy
Synaptic efficacy is modulated in the time range of tens of milliseconds when several presynaptic APs occur successively. The efficacy can either increase or decrease. Some of these forms of short-term modulation of release have been attributed to presynaptic Ca 2+ -dependent mechanisms ( Katz & Miledi, 1968 Zucker, 1996 Debanne et al. 1996 ).
In contrast to the effects of buffers on phasic release, where differences in the Ca 2+ -binding kinetics of BAPTA and EGTA caused large differences, one might expect similar effects of the two buffers on facilitation, which happens on a relatively long time scale. Synaptic depression, on the other hand, being only indirectly dependent on intracellular Ca 2+ ( Elmqvist & Quastel, 1965 ) may mirror the buffer effects on phasic release. We tested these hypotheses by quantifying the degree of facilitation and depression when EGTA or BAPTA was loaded into the two classes of terminal.
The effect of EGTA (0.5 m m ) loaded into a pyramidal neurone on facilitation of EPSPs in bitufted cells is shown in Fig. 8A. In this experiment the degree of facilitation, measured as the ratio EPSP2/EPSP1 was 2.15 in control conditions. In the presence of EGTA (0.5 m m ) EPSP1 was on average reduced to 60 % of the control value, i.e. EGTA caused a relatively moderate reduction in efficacy, whereas facilitation was completely blocked (EPSP2/EPSP1 = 1.02). The effect of EGTA on facilitation was concentration dependent. Complete block was observed at EGTA concentrations of 0.2 m m or higher (Fig. 8B) suggesting that accumulation of residual Ca 2+ contributes to facilitation.
Concentration-dependent effect of internal EGTA on facilitation and depression
A, unitary EPSPs were recorded in bitufted cells before (Control) and after loading of presynaptic pyramidal cells with EGTA during repetitive stimulation of pyramidal cells evoking trains of 3 APs (10 Hz). Lines above the records indicate the time of occurrence of presynaptic APs in the train. Upper trace, presynaptic neurone loaded with control solution. Lower trace, same neurone after loading with EGTA (0.5 m m ). B, concentration-dependent effect of internal EGTA in the presynaptic pyramidal cell on facilitation of EPSPs recorded from bitufted cells. The amplitude of the second EPSP relative to that of the first (EPSP2/EPSP1) is plotted for different concentrations of EGTA (⋄). The ratio for control pipette solution (no Ca 2+ buffer) is also shown (◆). C, concentration-dependent effect of internal EGTA in the presynaptic pyramidal cell on depression of EPSPs recorded from multipolar cells. The EPSP2/EPSP1 ratio is given for different EGTA concentrations (•). The ratio for control pipette solution (no Ca 2+ buffer) is also given (•).
Loading of a pyramidal cell with BAPTA (0.5 m m ) strongly reduced the amplitude of the first EPSP to 25 % of control. In contrast to EGTA loading, however, facilitation was almost unaffected as the EPSP2/EPSP1 ratio was 2.3 in control versus 2.2 after loading (Fig. 9A). The effect of BAPTA was concentration dependent and BAPTA blocked facilitation only at concentrations of 1 m m or larger (Fig. 9B). Thus, contrary to the expectation that the two buffers should be equivalent in reducing facilitation, the faster-acting buffer BAPTA was much less effective.
Concentration-dependent effect of internal BAPTA on facilitation and depression
A, unitary EPSPs recorded from bitufted cells before (Control) and after loading of presynaptic pyramidal cells with BAPTA. Lines above the records indicate the time of occurrence of presynaptic APs. Upper trace, control EPSPs before BAPTA loading. Lower trace, EPSPs after loading with 0.5 m m BAPTA. B, concentration-dependent effect of presynaptic BAPTA on facilitation of EPSPs recorded in bitufted cells. The EPSP2/EPSP1 ratio is plotted as a function of BAPTA concentration in the pipette recording from the presynaptic pyramidal cell (◆). The ratio for control pipette solution (no Ca 2+ buffer) is also shown (⋄). C, concentration-dependent effects of presynaptic BAPTA on depression of EPSPs recorded in multipolar cells (•). The ratio for control pipette solution (no Ca 2+ buffer) is also given (•).
Depression was not reduced strongly in the presence of EGTA (Fig. 8C), even at high concentrations (10 m m ). When BAPTA was loaded into the pyramidal cell, depression of EPSPs in multipolar cells was also not reduced at concentrations that strongly reduced efficacy (Fig. 9C). With higher BAPTA concentrations (> 0.5 m m ), depression became smaller concomitantly with the reduction of efficacy. Above 2 m m , when efficacy was reduced to < 10 % of control, in some experiments a small facilitation was observed. This clearly suggests that in depressing terminals the main effect of the Ca 2+ buffers on depression was a reduction in the fraction of vesicles that are released by the first AP, and consequently an increase in the pool of remaining ‘release-ready’ vesicles.
Mixtures of BAPTA and EGTA.
The blocking effect of EGTA on the facilitation of EPSPs recorded from bitufted cells is consistent with the view that accumulation of free Ca 2+ underlies this process ( Hochner et al. 1991 Kamiya & Zucker, 1994 ). On the other hand, BAPTA binds Ca 2+ as efficiently as EGTA, but at low BAPTA concentrations (0.02-0.7 m m ) facilitation was still present or even enhanced. A key to understanding the different effects of the two buffers may be the finding that BAPTA in this concentration range strongly reduced EPSP1 but EGTA did not. Presumably, BAPTA effectively chelated Ca 2+ near the Ca 2+ sensor, and a fraction of the BAPTA remained bound to Ca 2+ after the first AP had occurred (as discussed by Winslow et al. 1994 ). Therefore, less free BAPTA would have been available when the next AP occurred and the concentration of free Ca 2+ reaching the Ca 2+ sensor would have risen to higher values. As a result, the EPSPs in response to the second and third APs would have been increased. One might ask whether facilitation in unperturbed terminals could be due to the saturation of the endogenous buffer.
To test this hypothesis we compared facilitation in terminals loaded with 0.2 m m BAPTA alone with that in terminals loaded with a mixture of 0.2 m m BAPTA and 0.2 m m EGTA. Facilitation was similar in the two cases (229 ± 68 %(n = 5) and 228 ± 4 %(n = 3), respectively), indicating that in the presence of BAPTA, the addition of EGTA did not block facilitation (Fig. 10A). When terminals were loaded with 0.2 m m EGTA and lower concentrations of BAPTA (0.05 and 0.02 m m ), facilitation was only reduced by EGTA when BAPTA was 0.02 m m . In this case facilitation was about one-half of the control value (Fig. 10A). Therefore, with a mixture of a fast and a slowly acting buffer, the faster one determined whether facilitation occurred or not if its concentration was high enough to significantly reduce the first EPSP in a train of APs. Thus, one can conclude that the mechanism of EGTA-sensitive endogenous facilitation is different from that induced by partial saturation of a fast acting mobile buffer like BAPTA. Nevertheless, the ‘partial Ca 2+ buffer saturation’ hypothesis as a mechanism of facilitation might also apply to native terminals, if they contained a fast saturable endogenous buffer. However, the facilitating terminals of pyramidal cells, studied here, seem to have a low concentration of endogenous mobile buffer.
Endogenous facilitation and pseudofacilitation in terminals on bitufted cells are driven by different mechanisms
A, EPSP2/EPSP1 ratios measured in bitufted cells after loading of presynaptic pyramidal cells with BAPTA alone, a mixture of BAPTA and EGTA, and EGTA alone. Note that with the mixtutre of BAPTA and EGTA the latter has no or little effect on facilitation induced by the ‘partial buffer saturation’ mechanism. B, the effect of changes in [Ca 2+ ]o on the facilitation ratio when BAPTA (0.2 m m ) was loaded into the presynaptic pyramidal cell. A decrease in [Ca 2+ ]o abolishes facilitation whereas an increase in [Ca 2+ ]o increases facilitation. C, the effect of changes in [Ca 2+ ]o on the facilitation ratio of EPSPs recorded in bitufted cells. Presynaptic terminals were dialysed with control solution without added calcium buffer. An increase in [Ca 2+ ]o reduced facilitation, a decrease in [Ca 2+ ]o increased facilitation.
Effects of [Ca 2+ ]o on terminals loaded with exogenous Ca 2+ buffers.
The partial Ca 2+ buffer saturation hypothesis also predicts that reduction of the buffer saturation by lowering [Ca 2+ ]o would result in a smaller facilitation. At higher [Ca 2+ ]o the fraction of buffer bound to calcium will increase after the first AP, resulting in increased facilitation. This, however, may be counteracted by depression, which might develop when the fraction of vesicles released is increased by elevated Ca 2+ influx. To test these ideas we compared facilitation in control and in buffer-loaded terminals at different [Ca 2+ ]o.
Facilitation in terminals loaded with 0.2 m m BAPTA was higher than in control (at 2 m m [Ca 2+ ]o) and lowering [Ca 2+ ]o to 1.5 m m decreased facilitation whereas increasing [Ca 2+ ]o to 3 m m increased facilitation (Fig. 10B). This result is readily explained by the concept of partial buffer saturation. It predicts that BAPTA saturation caused by Ca 2+ inflow during the first AP is higher at high [Ca 2+ ]o. When no exogenous buffers were loaded into the terminals, facilitation was progressively reduced as [Ca 2+ ]o was increased (3 and 4 m m ). Lowering of [Ca 2+ ]o to 1.5 and 1 m m resulted in an increased facilitation relative to control (2 m m [Ca 2+ ]o, Fig. 10C). This result contradicts the partial Ca 2+ buffer saturation hypothesis and will be discussed below.
To show the specificity of the effects of changing [Ca 2+ ]o on facilitating terminals loaded with BAPTA, we made similar measurements with depressing terminals on multipolar cells. The reduction of release by loading the terminal with 0.2 m m BAPTA was followed by a decrease in [Ca 2+ ]o from 2 to 1.5 m m . This resulted in an amplitude reduction of the first EPSP and in less depression (not shown). These results are consistent with the view that depletion of vesicles from the release-ready pool or depletion of release sites is the major mechanism underlying depression ( Zucker, 1994 ).
Changes in cytosolic Ca 2+ concentration ([Ca 2+ ]c) provide signals to control crucial events such as muscle contraction, neurotransmitter release, alterations in gene transcription and cell death (Berridge et al., 2003 Bootman et al., 2001 Orrenius et al., 2003). Ca 2+ signals, physiological and pathological, are modulated by the activity of mitochondria (Berridge, 2002 Hajnoczky et al., 2000 Jacobson and Duchen, 2004). These organelles accumulate Ca 2+ via an electrogenic uniporter when [Ca 2+ ]c exceeds ∼100 nM, using the potential, ΔΨm, generated across the inner mitochondrial membrane by the activity of the electron-transport chain (Becker et al., 1980 Nicholls and Crompton, 1980). Mitochondria release Ca 2+ more slowly back to the cytosol via a Na + - or H + -antiporter (Crompton et al., 1978 Rottenberg and Marbach, 1990). In this way, mitochondria act as localized [Ca 2+ ]c buffering units and might modulate Ca 2+ feedback-inhibition or activation mechanisms (Collins et al., 2000 Hajnoczky et al., 1999 Mackenzie et al., 2004), For example, mitochondrial Ca 2+ accumulation might determine whether or not a localized Ca 2+ signaling event, such as a `puff' (Parker and Ivorra, 1990) or `spark' (Cheng et al., 1993), proceeds to a global [Ca 2+ ]c oscillation or wave.
Mitochondria, being the site of aerobic ATP production, also affect cellular signaling by modulating the cytosolic ATP:ADP ratio (Nicholls and Ferguson, 2002). ATP production is itself stimulated by the uptake of Ca 2+ . The activity of the mitochondrially located citric acid (Krebs)-cycle enzymes pyruvate dehydrogenase, oxoglutarate dehydrogenase and NAD + -isocitrate dehydrogenase (McCormack and Denton, 1980) as well as that of the F1F0-ATP synthase are stimulated by Ca 2+ (Territo et al., 2000). Conversely, a decrease in ΔΨm, which provides the driving force for the ATP synthase, inhibits ATP production (Leyssens et al., 1996). Uptake of positively charged Ca 2+ ions should depolarize ΔΨm to some extent and so might restrict ATP production. Therefore, Ca 2+ signals might limit cellular energy production.
In some cells, mitochondria are located close to sites of initiation of [Ca 2+ ]c signals, such as at voltage-gated Ca 2+ channels on the plasmalemma (Barstow et al., 2004) or at inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors [Ins(1,4,5)P3Rs] on the endoplasmic/sarcoplasmic reticulum (ER/SR) (Csordas and Hajnoczky, 2003 Hajnoczky et al., 1999 Rizzuto et al., 1998). Indeed, mitochondria might be tethered to be within 10 nm of Ins(1,4,5)P3Rs (Csordas et al., 2006). At these locations, mitochondria are exposed to a local [Ca 2+ ]c that exceeds bulk average values (Rizzuto et al., 1998), and hence might be expected to both have a greater influence over the [Ca 2+ ]c signal itself (because of their low affinity for Ca 2+ ) and, in turn, be more likely to exhibit a decrease in ΔΨm due to the larger mitochondrial Ca 2+ current generated during these local [Ca 2+ ]c transients.
Although transient decreases in ΔΨm have been observed in isolated mitochondria that have been exposed to sudden increases in [Ca 2+ ] (Brustovetsky and Dubinsky, 2000 Huser et al., 1998), it is less clear whether or not this occurs during [Ca 2+ ]c signaling in intact cells. Transient, or `flickering', ΔΨm changes have been observed in some (Buckman and Reynolds, 2001 Drummond et al., 2000 Duchen et al., 1998 Haak et al., 2002 Jacobson and Duchen, 2002 Kaftan et al., 2000 O'Reilly et al., 2004 Petronilli et al., 2001) but not all (Collins et al., 2001 Csordas and Hajnoczky, 2003) intact cell types, although their relationship to [Ca 2+ ]c changes is uncertain. Whether or not transient ΔΨm depolarizations are seen during cellular Ca 2+ signals might depend on the cell type, the origin and the characteristics of the Ca 2+ signal. For example, inhibition of Ca 2+ release from the SR inhibits transient ΔΨm depolarizations in some circumstances (Jacobson and Duchen, 2002) but not in others (O'Reilly et al., 2004).
Transient ΔΨm depolarizations have also been attributed to the increased levels of oxidative species generated from a combination of intense excitation light and high concentrations of certain mitochondrial dyes (Szado et al., 2003), rather than to changes in [Ca 2+ ]c. In yet other studies, neither the intensity of the excitation light nor the concentration of mitochondrial dyes contributed to the generation of transient ΔΨm depolarizations (Buckman and Reynolds, 2001 O'Reilly et al., 2004 Vergun et al., 2003). Evidently, the mechanism(s) underlying the generation of ΔΨm depolarizations and, in particular, whether or not Ca 2+ signals arising from either influx or release modulate ΔΨm is unclear.
Cellular Ca 2+ signaling imposes an energy burden on the cell (in order to rebalance resting ion concentrations) and paradoxically also impedes the production of ATP by mitochondria if Ca 2+ signals result in ΔΨm depolarization. The question of whether or not mitochondrial Ca 2+ uptake depolarizes mitochondria was addressed in the present study. To examine how mitochondria affect Ca 2+ signals and whether or not changes in ΔΨm occur, ΔΨm was measured during [Ca 2+ ]c transients evoked by Ca 2+ influx across the plasmalemma and following Ca 2+ release from the SR by localized photolysis of caged Ins(1,4,5)P3, in voltage-clamped single smooth muscle cells. Voltage clamp was used because changes in plasmalemmal potential might compromise the ability to resolve mitochondrial ΔΨm changes (Ward et al., 2000). The use of caged Ins(1,4,5)P3 should minimize the number of second-messenger systems that are operative, which otherwise might also alter ΔΨm independently of Ca 2+ changes. With attenuated light intensity and low concentrations of the ΔΨm indicator tetramethylrhodamine ethyl ester (TMRE), only minimal ΔΨm changes were observed under resting conditions. ΔΨm depolarizations did not occur when [Ca 2+ ]c was increased by Ca 2+ influx via voltage-gated Ca 2+ channels or by release from Ins(1,4,5)P3Rs, even though mitochondrial Ca 2+ uptake took place. However, ΔΨm depolarizations were observed on increasing TMRE concentration or light intensity even though no change in [Ca 2+ ]c took place. These depolarizations were blocked by antioxidants or the mitochondrial permeability transition pore (mPTP)-inhibitor cyclosporin A (CsA). Very occasionally, transient ΔΨm depolarization was seen in cells undergoing sustained, spontaneous [Ca 2+ ]c oscillations. In this case, ΔΨm depolarizations were neither synchronized throughout the mitochondrial complement nor with changes in [Ca 2+ ]c. Therefore, during physiological Ca 2+ signaling, mitochondrial Ca 2+ uptake modifies the amplitude and duration of Ca 2+ signals without significantly altering ΔΨm, unless [Ca 2+ ]c oscillations are repetitive and sustained.
Abstract The present study tested two hypotheses: (1) that a receptor for extracellular Ca 2+ (Ca 2+ receptor [CaR]) is located in the perivascular sensory nerve system and (2) that activation of this receptor by physiological concentrations of extracellular Ca 2+ results in the release of vasodilator substance that mediates Ca 2+ -induced relaxation. Reverse transcription-polymerase chain reaction using primers derived from rat kidney CaR cDNA sequence showed that mRNA encoding a CaR is present in dorsal root ganglia but not the mesenteric resistance artery. Western blot analysis using monoclonal anti-CaR showed that a 140-kD protein that comigrates with the parathyroid CaR is present in both the dorsal root ganglia and intact mesenteric resistance artery. Immunocytochemical analysis of whole mount preparations of mesenteric resistance arteries showed that the anti-CaR–stained perivascular nerves restricted to the adventitial layer. Biophysical analysis of mesenteric resistance arteries showed that cumulatively raising Ca 2+ from 1 to 1.25 mol/L and above relaxes precontracted arteries with an ED50 value of 2.47±0.17 mmol/L (n=12). The relaxation is endothelium independent and is unaffected by blockade of nitric oxide synthase but is completely antagonized by acute and subacute phenolic destruction of perivascular nerves. A bioassay showed further that superfusion of Ca 2+ across the adventitial surface of resistance arteries releases a diffusible vasodilator substance. Pharmacological analysis indicates that the relaxing substance is not a common sensory nerve peptide transmitter but is a phospholipase A2/cytochrome P450–derived hyperpolarizing factor that we have classified as nerve-derived hyperpolarizing factor. These data demonstrate that a CaR is expressed in the perivascular nerve network, show that raising Ca 2+ from 1 to 1.25 mol/L and above causes nerve-dependent relaxation of resistance arteries, and suggest that activation of the CaR induces the release of a diffusible hyperpolarizing vasodilator. We propose that this system could serve as a molecular link between whole-animal Ca 2+ balance and arterial tone.
The observation that Ca 2+ ion, acting at the cell exterior, modulates vascular tone and resistance to blood flow has long intrigued smooth muscle physiologists. In 1911, Cow 1 showed that elevation of Ca 2+ above normal levels depresses reactivity of isolated arteries. More than 40 years later, Holman, 2 using isolated guinea pig taenia coli, reported that raising extracellular Ca 2+ transiently decreases electrical activity of the guinea pig taenia coli, contrary to the predicted augmentation. Bohr 3 subsequently showed that elevated concentrations of extracellular Ca 2+ depress the early rapid response of rat aorta to epinephrine, whereas the same levels of Ca 2+ increase the steady state response. Nearly a decade later, Holloway and Bohr 4 reported a method for quantifying Ca 2+ -induced relaxation of isolated arteries and suggested that Ca 2+ induces relaxation via a membrane stabilizing action. Subsequent work by Bohr and colleagues 5 6 has further characterized the vessel response to extracellular Ca 2+ .
Although these experiments established the fact that extracellular Ca 2+ suppresses vascular force generation, acceptance of this phenomenon as a physiological regulatory mechanism has been limited for two reasons. One is the fact that it has generally been assumed that the concentrations of Ca 2+ required to alter vascular function cannot be achieved in vivo. New evidence suggests that this is not true and indicates that in tissues involved in transcellular Ca 2+ movement (eg, intestine, kidney, and bone), the concentration of Ca 2+ in the interstitial space can be significantly higher than is present in the mixed venous plasma. 7
A second reason for limited acceptance is that no viable molecular mechanism explaining Ca 2+ -induced relaxation has been demonstrated. The identification of a cellular mechanism for this action has been a focus of our laboratory, 8 9 and we recently demonstrated that Ca 2+ -induced relaxation of the rat mesenteric resistance artery is attenuated by blockade of Ca 2+ -activated K + channels and is associated with a decrease in myofilament Ca 2+ sensitivity. 10 Because extracellular Ca 2+ should not directly activate K + channels and regulation of myofilament Ca 2+ sensitivity is a G protein-coupled process, 11 we proposed that extracellular Ca 2+ induces relaxation by activating a membrane spanning receptor for extracellular Ca 2+ that is similar to that which has been reported in other tissues, including parathyroid, thyroid, and kidney. 12 13 14 15
Our initial attempts at identifying the CaR in arterial tissue using RT-PCR amplification of CaR mRNA failed to show the appropriate transcript 16 and confirmed earlier reports. 12 13 15 More recently, however, it was demonstrated that message for the CaR is expressed in neural tissue. 17 With the knowledge that small arteries of the rat contain a dense perivascular sensory nerve network that is functionally linked with vasodilation 18 19 and that the cell bodies of the sensory fibers that are the sites of mRNA processing and protein synthesis are located in the DRG, 20 we postulated that the perivascular sensory nerve network houses a vascular CaR that mediates Ca 2+ -induced relaxation of small arteries. We now demonstrate that a receptor for extracellular Ca 2+ is present in sensory nerves of the mesenteric resistance artery wall and present physiological and pharmacological evidence that is consistent with the hypothesis that Ca 2+ modulates vascular reactivity by activation of this receptor. Preliminary reports of this work have been published in abstract form. 21 22
Peptide antagonists, including CGRP8–37, Spantide II, galantide, 4 Cl- d -Phe, 6 Leu 17 -VIP, and α-helical CRF 9–41, were purchased from Phoenix Pharmaceuticals nor-binaltorphimine was purchased from Research Biochemicals and SR48968 and SR140333 were generously supplied by Sanofi Recerche. Unless otherwise noted, all other chemicals used in these studies were of analytical grade and were purchased from the Sigma Chemical.
All procedures involving animals were performed in accordance with approval of the Institutional Animal Care and Use Committee. Male Wistar rats (6 to 10 weeks of age) were obtained from Harlan-Sprague Dawley and on arrival in our animal care facility were maintained in colony rooms with fixed light/dark cycles and constant temperature and humidity and provided with Purina rodent chow and water ad libitum. Vascular or other tissue was isolated while the rats were anesthetized with a mixture of ketamine and xylazine (100/5 mg/kg). Subchronic phenolic denervation of mesenteric arteries was achieved by applying 10% ethanolic phenol onto branch I mesenteric arteries through a midline abdominal incision while the animals are under anesthesia. The wound was then closed, and arteries distal to the site of phenol application were isolated 72 hours later.
Total RNA was isolated as recently described, 23 and 2 μg was reverse-transcribed using AMV reverse transcriptase and deoxynucleotides provided in a kit from Boehringer-Mannheim Biochemicals that was used according to the manufacturer’s instructions. Resulting cDNA was amplified through 35 cycles using Taq DNA polymerase (Promega) with an annealing temperature of 55°C and a polymerization period of 7 minutes at 72°C. The products were size-separated on 1.0% agarose gels and stained with ethidium bromide. The sequence of the primers, which were designed to amplify CaR cDNA corresponding to the region 941 to 1993, were 5′-GAACCTGGACGAGTTCTG-3′ for the forward primer and 5′-CCATGTTGTTGGTGAAG-3′ for the reverse primer. Sequence analysis of the 1052-bp PCR product from both DRG and kidney was performed using cycle sequencing with a dye terminator (Perkin-Elmer Cetus) and a 3703A Applied Biosystems DNA sequence analyzer and showed complete sequence homology with the published rat kidney sequence. 15
Western Blot Analysis
Protein was extracted and size-separated using 8% SDS-PAGE and then electroblotted onto nitrocellulose membrane as recently described. 23 The membrane was incubated overnight with a monoclonal mouse anti-CaR (0.13 μg/mL) raised against the conserved parathyroid CaR sequence ADDDYGRPGIEKFREEAEERDI (provided by NPS Pharmaceuticals in conjunction with Drs Allen Spiegel and Paul Goldsmith, NIDDK). In some experiments, anti-CaR was preabsorbed with 50 mg/mL excess antigen. The membrane was then incubated with horseradish peroxidase-coupled anti-mouse IGG (Amersham), and the resulting protein/antibody complex was visualized on x-ray film using the enhanced chemiluminescence method (ECL Amersham).
Vessels were cleaned of fat and connective tissue, and luminal blood was removed by flushing with cold PSS. After brief fixation in ice-cold methanol, segments were rinsed in TBS, intrinsic peroxidase activity was quenched using a mixture of H2O2 and NaN3 (DAKO), and nonspecific binding was blocked using a serum-free blocker (DAKO). The tissues were then incubated overnight with TBS alone (negative control) or anti-CaR (20 μg/mL). After washing, intrinsic biotin was blocked using a biotin blocking kit (DAKO), and the segments were incubated with biotin-conjugated secondary antibody (1:250) in the presence of the 2% serum from the appropriate species. The segments were then stained using horseradish peroxidase-conjugated avidin (Vector Labs) using 3–3′-diaminobenzene (Sigma) as substrate, counterstained with hematoxylin, dehydrated through sequential ethanol and xylene, and permanently mounted. The preparations were then visually examined using a Nikon Microphot FXA research microscope, and photographs were taken with the focus on either the adventia, media, or intima as judged by the pattern of nuclear staining.
Isometric force generation was measured using previously described methods. 10 Branch II mesenteric resistance arteries were isolated, cleaned of fat and connective tissue, and placed in ice-cold PSS of the following composition (in mol/L) NaCl 140, KCl 4.7, MgSO47H2O 1.17, NaHCO3 5, KH2PO4 1.15, Na2HPO4 1.10, CaCl2 1.0, HEPES 20, and glucose 5, pH 7.4. The vessels were then mounted on a wire myograph warmed to 37°C and gassed with 95% air/5% CO2, stretched to a predetermined length that was equivalent to an internal diameter of 200 to 225 μm, and allowed to equilibrate for a 30-minute period. After the equilibration period, the vessels were induced to contract with 5 μmol/L norepinephrine until reproducible contractile responses were obtained (three or four times).
Some vessels were studied after removal of the endothelium using a human hair, and functional denudation was verified in subsequent protocols by the absence of a relaxation response to the addition of 1 μmol/L acetylcholine. 24 Other vessels underwent a procedure to chemically damage perivascular nerves. 25 PSS was drained away from the segment, and 50 μL of a 0.75% solution of ethanolic phenol (90:10) in PSS was directly applied for a 15-second period (time empirically derived in preliminary experiments) followed by extensive washing over a 30-minute period. This treatment results in a nearly complete loss of a contractile response to activation of perivascular nerves using electrical field stimulation at 30 Hz, 70 V, and 2 ms duration.
Ca 2+ -induced relaxation was assessed by cumulatively adding Ca 2+ to vessels that were precontracted with 5 μmol/L norepinephrine the magnitude of relaxation was expressed as the percentage of the initial tension. When phasic activity was present, measurements were made from the precontraction baseline to the trough of the phasic transition.
A bioassay was used to determine whether a diffusible vasodilator substance is elicited by Ca 2+ from the adventitial surface of mesenteric resistance arteries. The bioassay segment was a 1.5- to 2-mm-long ring of mesenteric resistance artery mounted on a wire myograph donor tissue consisted of a section of the mesenteric resistance artery arcade with a mass 20 to 30 times that of the bioassay segment that was affixed above the preparation by means of a cannula placed in a section of the mesenteric trunk. PSS that was gassed with 95% air/5% CO2 and kept at 35°C was allowed to drop onto the bioassay segment at a flow rate of 1.5 mL/min either directly from a cannula or after superfusion across the donor segment. To quantify the relaxation response, the experimental trace was digitized using UN-SCAN-IT software (Silk Scientific Corp), and the area under the relaxation curve was determined for each treatment.
All data are presented as mean±SEM, and statistical analysis was performed using the SYSTAT software package. Comparisons among groups were performed using ANOVA with a repeated measures design when appropriate. A value of P<.05 was taken to indicate a statistically significant difference.
CaR Gene Expression
To assess CaR gene expression, total RNA was isolated from rat kidney (positive control, dorsal root ganglia, and mesenteric resistance artery). Complementary DNA was synthesized by RT and amplified by PCR with primers designed to amplify a 1052 bp region of the rat kidney CaR. 15 mRNA encoding the CaR was present in the kidney and DRG but not the mesenteric resistance artery (Fig 1A ). Control experiments in which PCR was performed in the absence of the RT step revealed no product, indicating that there was no contamination of the sample with genomic DNA. Sequence analysis of the amplimer isolated from both the kidney and DRG indicated complete homology with the published rat kidney sequence. These data indicate that mRNA for the CaR is present in cell bodies of sensory nerves but is not detectable in the arterial wall. Possible explanations are that CaR protein is synthesized in the DRG cell body and transported peripherally to perivascular neurons or that the concentration of CaR mRNA is too low to be detected in nerves of the arterial wall using the RT-PCR method.
CaR Protein Expression
Western blot analysis was used to test the hypothesis that protein immunoreactive with the parathyroid CaR is present in DRG and the mesenteric resistance artery wall. Protein extracted from thyroparathyroid (positive control, dorsal root ganglia, and mesenteric resistance arteries) was used in Western blot analysis as described in “Methods.” Consistent with previous reports, 12 13 the parathyroid CaR migrated as a doublet with molecular masses of ≈140 and ≈160 kD (Fig 1B ). Immunoreactive protein from DRG and mesenteric resistance arteries appeared as a single band that comigrated with the lower band of the parathyroid doublet (Fig 1B ). Preabsorption of the anti-CaR with excess antigen significantly reduced CaR signal from all three preparations.
Immunocytochemical analysis was used to determine the site of expression of the CaR in the mesenteric resistance artery wall. Whole-mount preparations were immunostained with anti-CaR as described in “Methods” and counterstained with hematoxylin to visualize nuclei in the various layers of the artery. A fine nerve-like pattern of staining was present in the adventitial layer (Fig 2A ) but not in the medial (Fig 2B ) or endothelial (Fig 2C ) layers. Moreover, no staining was observed when secondary antibody was used alone (Fig 2D ).
Experiments were also performed to test the hypothesis that activation of the perivascular CaR is linked with Ca 2+ -induced modulation of arterial reactivity. Because there are no known antagonists of the CaR, these experiments were designed to establish the Ca 2+ sensitivity and neuronal dependence of the Ca 2+ relaxation event. As illustrated in Fig 3A , the addition of 5 μmol/L norepinephrine to the mesenteric resistance artery induces an increase in tone that persists at a stable level for ≥30 minutes and exhibits vasomotion. When extracellular Ca 2+ is cumulatively added, dose-dependent relaxation was observed (Fig 3B ) with an ED50 value of 2.47±0.17 mmol/L determined from vessels from 12 separate animals. Moreover, the addition of 3 or 5 mmol/L Ca 2+ often caused a transient suppression of rhythmic activity, which returned after a 3- to 5-minute delay.
Because it is possible that Ca 2+ -induced relaxation is mediated by an effect of Ca 2+ to induce the production of NO, from either the endothelium 6 26 or nitroxidergic nerves, 27 the effect of inhibition of NO synthase on Ca 2+ -induced relaxation was assessed by pretreatment with 0.3 mmol/L L-NAME, which is a dose 24 that effectively inhibits acetylcholine-induced relaxation of segments precontracted with 100 mmol/L K + (control, 32.5±8% relaxation L-NAME, 14.5±7.2% P<.05, n=6). Blockade of NO synthase had no effect on the sensitivity to Ca 2+ or the magnitude of relaxation induced by Ca 2+ , indicating that neither endothelial nor neuronal NO mediates the relaxation (Fig 4 ).
Endothelial cells release vasodilator substances other than NO(ie, cyclooxygenase products such as prostacyclin and the putative endothelium-derived hyperpolarizing factor). 28 A role for the endothelium was therefore assessed by studying vessel segments that were mechanically denuded of endothelium. Removal of the endothelium significantly reduced acetylcholine-induced relaxation of vessels precontracted with 5 μmol/L norepinephrine (relaxationintact=90.5±3.9% versus relaxationdenuded=8.3±2.5, P<.005, n=5) but had no effect on relaxation induced by Ca 2+ (Fig 4 ).
To test the hypothesis that Ca 2+ -induced relaxation is dependent on functionally intact perivascular nerves, the effect of phenolic nerve destruction was assessed. 25 Acute denervation was achieved by applying a 0.75% solution of phenol directly to the vessel for 15 seconds followed by extensive washing. After treatment with phenol, there was generally a reduction in the force response to norepinephrine and in contrast to the relaxation response observed before the addition of phenol, cumulative addition of Ca 2+ resulted in an incremental increase in active force (Fig 5A ). To test whether the intrinsic ability of the vessel to relax was affected by phenol, the relaxation responses were assessed to the NO donor sodium nitroprusside and the K + channel opener (pinacidil). In contrast with the response to Ca, 2+ both agonists relaxed precontracted arteries. The response to nitroprusside was intact compared with prior results, 24 whereas the response to pinacidil was shifted to the right (pD2 [mol/L] for pinacidilcont=−5.81±0.2 versus pinacidilphenol=−4.67±0.19, P<.05, n=5], indicating a slight inhibitory effect on the K + channel agonist.
As a test for possible nonspecific phenol-induced damage to the artery, the effect of applying phenol 3 days before harvesting the vascular tissue was assessed as described in “Methods.” As with the acute application of phenol, increasing Ca 2+ caused contraction of vessels that were taken from animals treated with phenol, whereas vessels from sham/vehicle–treated animals relaxed in response to Ca 2+ (Fig 5B ). It should be noted that the relaxation response of sham-treated animals was decreased relative to the responses shown in Figs 3 to 5 , and we suspect that this is the result of a neurotoxic effect of the vehicle (100% ethanol).
Because these data indicated that extracellular Ca 2+ induces relaxation through a nerve-dependent mechanism, a bioassay was used to test the hypothesis that extracellular Ca 2+ elicits the release of a diffusible vasodilator substance from the adventitial surface of mesenteric resistance arteries. Direct addition of norepinephrine to the bioassay vessel through a cannula caused an increase in force and increasing concentrations of extracellular Ca 2+ caused a graded decrease in tension (Fig 6 ). When the procedure was repeated, allowing the solution to first superfuse the donor arcade, two effects were observed. The first was that the contractile response of the bioassay vessel to norepinephrine was significantly attenuated by superfusion of the medium across the donor (peak response was 68±8.9% of control, P=.009), suggesting the presence of a basal vasodilator factor the second was that the relaxation response to elevated Ca 2+ was significantly enhanced, particularly at the lowest concentrations of Ca 2+ (Fig 6 ).
Experiments were also performed to test the hypothesis that the vasodilator substance that mediates Ca 2+ -induced relaxation is one of the primary sensory nerve transmitters (ie, either CGRP or a member of the tachykinin family). 29 Blockade of CGRP receptors by preincubation with CGRP8–37 (1 μmol/L), which is a selective CGRP antagonist 30 that significantly depresses CGRP-induced relaxation in this preparation (data not shown), was without effect on Ca 2+ -induced relaxation (Fig 7 ). Similarly, blockade of the substance P (neurokinin 1) receptor with either the peptide antagonist Spantide II (10 μmol/L) 31 or the nonpeptide antagonist SR140333 (0.3 μmol/L) 32 was without effect (Fig 7 ). Moreover, blockade of the neurokinin 2 (tachykinin A) receptor with the nonpeptide antagonist SR48968 (0.3 μmol/L) 32 also was without effect on Ca 2+ -induced relaxation (Fig 7 ). Because these data and preliminary experiments using peptide antagonists for other possible neurotransmitters, including 4 Cl- d -Phe, 6 Leu 17 -VIP, nor-binaltorphimine (dynorphin), α-helical CRF, and galantide (galanin), 33 indicated that the mediator might not be a peptide transmitter, we tested the hypothesis that the mediator is a hyperpolarizing factor.
Pretreatment with 10 mmol/L TEA, which is a broad range K + channel blocker, 34 completely inhibited Ca 2+ -induced relaxation (Fig 8 ). Similarly, when mesenteric resistance arteries were precontracted with 100 mmol/L K + in the presence of 1 μmol/L phenotolamine, the relaxation response to cumulative addition of Ca 2+ was completely blunted (Fig 8 ). Because these data supported the hypothesis that Ca 2+ may induce the production of a hyperpolarizing factor and there is considerable evidence that endothelium-derived relaxing factors are cytochrome P450–derived metabolites of arachidonic acid, 35 36 we examined the effect of miconazole, which is a cytochrome P450 blocker. 37 Pretreatment of the mesenteric resistance arteries with 3 μmol/L miconazole, which had no effect on relaxation induced by pinacidil (data not shown), significantly inhibited Ca 2+ -induced relaxation (Fig 8 ). Finally, because arachidonic acid is liberated in many cells by activation of phospholipase A2, we assessed the effect of 5 μmol/L quinacrine, which is a phospholipase A2 antagonist. 38 This concentration of quinacrine, which had no effect on pinacidil-induced relaxation (data not shown), antagonized Ca 2+ -induced relaxation of isolated mesenteric resistance arteries (Fig 8 ).
The present report describes several new and potentially important findings regarding the regulation of vascular reactivity by extracellular Ca. 2+ One finding is that DRG, which house cell bodies of sensory nerve fibers, express mRNA for the CaR, and this message is apparently processed such that CaR protein is transported from DRG to the perivascular nerve network of peripheral arteries. The second finding is that raising Ca 2+ from 1.0 to 1.25 mmol/L and above relaxes isolated resistance arteries, and the relaxation is completely eliminated by phenolic destruction of perivascular nerves. The third finding, based on the results of the bioassay, is that the adventitial surface of resistance arteries releases a diffusible vasodilator substance in response to increasing concentrations of Ca. 2+ The fourth is that Ca 2+ -induced relaxation appears to be mediated by production or release of a nerve-derived hyperpolarizing factor, which is a cytochrome P450–generated metabolite of arachidonic acid. These findings have been collectively synthesized to form a novel hypothesis that states that alterations in extracellular Ca 2+ modulate vascular reactivity via activation of a perivascular CaR that is localized to sensory nerves and responds to changes in extracellular Ca 2+ with the release of a hyperpolarizing vasodilator.
Our findings concerning perivascular localization of the CaR confirm the report of Ruat et al, 17 who showed that cerebral arteries stain positively for a perivascular CaR. Moreover, our data provide an important extension of this finding by demonstrating that the peripheral/perivascular sensory nerve system expresses a Ca 2+ -sensing receptor. A key implication of this observation is that in contrast with the current view that the distribution of the CaR is limited to a few tissues (parathyroid, thyroid, kidney, and brain), the receptor might be present in an extensive network throughout the organism. Our findings also mesh neatly with the concept that sensory nerve fibers have a sensory efferent motor function through which neurotransmitter is released in response to either reflex activation or local stimuli, including cytokines and hydrogen ions. 19 29 39 These observations should have important implications in terms of our understanding of how whole-animal Ca 2+ homeostasis modulates regional function.
Several aspects of our experiments merit further discussion, including the data that support the perivascular localization of the CaR. The demonstration is based on two independent pieces of evidence. One is the RT-PCR analysis that shows that message for the CaR is present in the DRG but not in tissue from the arterial wall. This is important because it explains why prior attempts at identifying the CaR using Northern blot analysis were not successful. The second set of supportive data are the results of the Western blot and immunocytochemical analyses. Interpretation of the RT-PCR data are strengthened significantly by the demonstration that antibody specific for the CaR revealed a perivascular nerve localization of the protein.
One point that needs to be addressed is the data that link activation of the CaR with modulation of vascular tone. The primary evidence that serves to establish the linkage are the fact that Ca 2+ -induced relaxation is not dependent on an intact endothelium, the finding that Ca 2+ -induced relaxation is abolished by acute and subchronic phenolic denervation, and the results of the bioassay that showed that raising extracellular Ca 2+ induces the release of a diffusible vasodilator substance. Although provocative, we recognize that these findings are all indirect, and absolute proof of a linkage between the CaR and relaxation will depend on specific pharmacological blockade or tissue-specific deletion of the CaR in a relevant animal model. To our knowledge, however, no selective antagonists of the CaR are available, and the murine deletion mutant that has been developed does not grow past the neonatal stage and thus cannot be used for this type of cardiovascular study. 40
A related question is the identity of the vasodilator substance that mediates Ca 2+ -induced relaxation the present study indicates that it is neither NO nor a common sensory nerve peptide transmitter such as CGRP or a member of the tachykinin peptide family but rather a nerve-derived hyperpolarizing factor. Among the data that support this conclusion are our findings that two maneuvers that antagonize the action of endothelium-derived hyperpolarizing factors (eg, precontraction of the artery with a depolarizing concentration of K + and pretreatment with TEA) completely block Ca 2+ -induced relaxation (Fig 8 ). In addition, the results of the pharmacological analysis suggest further that Ca 2+ -induced production of the hyperpolarizing factor is associated with the release of arachidonic acid via activity of phospholipase A2 and subsequent metabolism by a cytochrome P450 enzyme. Of note, the brain CaR has been shown to be coupled to the release of arachidonic acid when expressed in Chinese hamster ovary cells, 41 and epoxyeicosatrienoic acids, which are cytochrome P450–generated compounds, have been shown to be hyperpolarizing vasodilators. 42 43 To our knowledge, this is the first demonstration that an exogenous ligand (ie, Ca 2+ ) can elicit the production of a nerve-derived hyperpolarizing factor from sensory nerves.
Another point that warrants discussion is the relationship between the apparent Ca 2+ sensitivity of Ca 2+ relaxation and the Ca 2+ sensitivity of the CaR in other tissues. The CaR of the parathyroid gland is thought to regulate PTH secretion in response to changes in serum-ionized Ca 2+ across the range of 0.8 to 2 mmol/L. The Ca 2+ relaxation event is also sensitive over this range, but relaxation clearly occurs at concentrations as high as 5 mmol/L. There are two possible explanations. One is that the sensitivity of the perivascular CaR is similar to that of the parathyroid and a separate, non-CaR–linked mechanism is operative at the higher levels of Ca. 2+ The second possibility is that the Ca 2+ sensitivity of the perivascular CaR exhibits a broader range than that for PTH.
Aside from the fact that our results provide a mechanism by which increases in extracellular Ca 2+ relax isolated arteries—a phenomenon that has intrigued vascular physiologists for >85 years 1 2 3 —they may also provide insight into the physiological mechanisms by which alterations in whole-animal Ca 2+ balance may be linked with blood pressure regulation. Although there is abundant evidence supporting the hypothesis that Ca 2+ supplementation lowers blood pressure in hypertensive humans and in animal models of high blood pressure, 44 45 46 the mechanism remains unclarified. 16 The present data permit the formulation of a testable theoretical paradigm that describes a mechanism by which Ca 2+ supplementation might modulate vascular reactivity and blood pressure. Specifically, we propose that increased Ca 2+ intake causes elevations in interstitial Ca 2+ in tissues involved in transcellular Ca 2+ movement (eg, the intestine and kidney), and this induces the release of a local hyperpolarizing vasodilator, which in turn decreases vascular resistance and lowers blood pressure.
Other potential implications of this work are based on the observations that mutations in the CaR have already been linked with familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. 47 48 If gene polymorphisms exist in the perivascular CaR, they could contribute to the inherited component of human essential and experimental genetic hypertension. 49 Finally, because the CaR is a proven target for pharmacological manipulation, 50 our data also provide the rationale for exploring the application of CaR agonists and antagonists to a variety of human pathologies, including hypertension, and the management of pain and inflammation.
The population of collagenase-isolated fibres
It is well documented that skeletal muscles of adult mdx mice have a high percentage of centrally nucleated fibres (CNF) reflecting the continuous process of regeneration. This percentage varies from muscle to muscle ( Boland et al. 1995 Pastoret & Sebille, 1995 ). We observed that the percentage of CNF in the population of collagenase-isolated mdx fibres was around 25 % and was not significantly different from the values we obtained on standard histological sections. Thus, the collagenase digestion of the muscle did not produce a selection of fibres on the basis of their state of differentiation (assessed by the nuclei position). Moreover, the same percentage of CNF was maintained over the 6 days of culture.
Mn 2+ influx in collagenase-isolated fibres: changes from d0 to d5
Individual quenching rates of Fura-PE3 fluorescence in the presence of external Mn 2+ ions are shown in Fig. 1. At d0, the influx remained below 5 % min −1 and no significant difference was detected between normal and mdx fibres. At d1, most of the values remained within the 5 % limits, while a small fraction of the fibres (8/27 and 9/25 for normal and mdx respectively) showed larger and widely dispersed values: from 6.1 to 21.5 % min −1 for normal fibres, and from 5.3 to 95 % min −1 for those from mdx muscles (we checked that this was not due to particularly high levels of Fura-PE3 loading). At d2, most values were again concentrated within the 5 % min −1 limit with only 1/7 (normal) and 1/14 (mdx) showing values around 10–13 % min −1 . At d3, the mean values were 2.5 and 5 % min −1 for normal and mdx fibres respectively, a significant difference (P < 0.01). These latter rates are very similar to those reported previously for collagenase-isolated fibres after 3 days of culture ( Tutdibi et al. 1999 ) at this age of culture, these authors never observed fluxes over 10 % min −1 in a set of more than 150 individual observations of both normal and mdx fibres (H. Brinkmeier, personal communication). However, when we extended observations to d4 and d5, the difference of Ca 2+ influx between normal and mdx fibres was no longer significant.
The correlation between Mn 2+ influx and [Ca 2+ ]i
The cytosolic [Ca 2+ ] was measured for each fibre before the Mn 2+ quenching rate was determined (see Methods) in order to assess the relationship between Ca 2+ influx (estimated from the Mn 2+ influx) and the steady-state value of [Ca 2+ ]i. Among the 132 fibres studied (Fig. 1), 130 individual pairs of values were obtained over the 6 days of study (68 mdx, 62 normal). They are displayed in Fig. 2A, without reference to the day of observation. The largest fraction of the results (116/130) is clustered within 0–10 % min −1 quenching rate and 25–85 n m [Ca 2+ ]i, as extreme limits. Results from normal and mdx fibres overlap. This densely populated cluster at the far left of Fig. 2A is presented as an expanded view in Fig. 2B. The whole set of data (Fig. 2A) exhibits an extremely skewed distribution, rendering calculation of arithmetic means inappropriate. Instead, the medians and the 25–75 percentile ranges were determined for both the quenching rates and the cytosolic [Ca 2+ ] as reported in Table 1. Medians were compared by a non-parametric statistical analysis (Mann-Whitney Rank sum test). Although the median of the quenching rate was significantly larger in mdx fibres (P < 0.01), the median of cytosolic [Ca 2+ ] did not differ from normal (P/ 0.2). Outside the main cluster of results, a small fraction of the fibres (12/130) showed both higher [Ca 2+ ]i values in the 100–140 n m range and a wide dispersion of the influx rate from > 5 to 95 % min −1 . All but one of the results of this second fraction were obtained at d1. Here, results from normal and mdx no longer overlap, due to the much higher values for Mn 2+ influx in four mdx fibres (as already seen in Fig. 1). Notwithstanding the very high influx values in some mdx fibres, [Ca 2+ ]i values in this group were not different from those obtained with the normal fibres of this high Mn 2+ influx fraction and remained within the 100–140 n m range, without reaching the contraction threshold, for all fibres were relaxed with visible striations at the time of measurement. We checked that these results did not come from the same animal. Moreover, the four fibres showing high Mn 2+ influx rates came from different cultures each of which also contained fibres having quenching rates as low as 2 % min −1 . Thus, the dispersion of the results reflected the heterogeneity of the fibre population and not the differences among animals.
The relationship between the Ca 2+ influx rate and the cytosolic [Ca 2+ ] was further studied by artificially increasing the Ca 2+ influx with low concentrations of the Ca 2+ ionophore 4-bromo A23187. We restricted our study to fibres cultured for 2 and 3 days, when those fibres with elevated influx had been naturally eliminated (see below). As seen in Fig. 3, we obtained influx rates ranging from 2 to 44 % min −1 while [Ca 2+ ]i remained within the 30–78 n m limits, with no difference between normal and mdx fibres. The results of Fig. 3 show that, within the duration of the experiment (< 120 min), the ‘robustness’ of the cytosolic Ca 2+ homeostasis in response to a large increase of Ca 2+ influx is as good in mdx fibres as in normal ones, even when this influx was up to 10-fold above naturally occurring values.
Correlation between Mn 2+ quenching rate and cytosolic [Ca 2+ ] in the presence of 4-bromo-A23187
Open symbols, normal fibres filled symbols, mdx fibres. Key: day of measurement. Fibres were obtained from 11 normal and 3 mdx mice. A23187 concentrations: 25–75 n m .
Fibre survival and Mn 2+ influx
The appearance of some fairly high values of Mn 2+ influx at d1, indicated that abnormally high Ca 2+ influx was occurring for some mdx and normal fibres. This prompted us to examine the survival of the fibres. However, for technical reasons, it was impossible to follow the fate of individual fibres where Mn 2+ influx had been measured. We thus had to rely on a study of the survival of a population of fibres. Six cultures were run in parallel and the number of remaining fibres were counted from d0 to d3. Only well elongated fibres (thus maintaining the resting state), showing no structural alterations, were counted. As shown in Fig. 4A, mdx fibres show a 10 % decline in number from d0 to d1, followed by a sharper decline such that survival was reduced to 51 %, at d2. This rate of decline subsequently slowed. In contrast, normal fibres showed a steady decline of 10–12 % per day (Fig. 4A, dotted line) so that survival at d2 was still 78 %. Thus, taking the loss rate of normal fibres as reference, mdx fibres showed an excess loss of 27 % (i.e. the difference between 78 and 51 %) from d0 to d2. For normal fibres, survival declined smoothly in spite of the fact that a fraction of the fibres (8/27) displayed, at d1, values of Ca 2+ influx in the > 5 to 21 % min −1 range. In Fig. 4A and B, statistical significance (marked *) concerns the difference between mdx and normal fibres.
To see if the sharp decrease of survival of mdx fibres from d1 to d2 could be attenuated, fibres were isolated and maintained in a medium containing one-tenth of the normal [Ca 2+ ]o of the culture medium, i.e. 0.08 m m , assuming that the reduction of [Ca 2+ ]o would similarly reduce the influx of Ca 2+ . Under these conditions, survival of mdx fibres decreased regularly from d0 to d3, as seen in Fig. 4B, without any sharp decrease between d1 and d2. The excess of mdx fibre death from d0 to d2 was now reduced from 27 % (at 0.8 m m [Ca 2+ ]o) to 10 %. For normal fibres, the reduction of external [Ca 2+ ] did not affect the survival time course. To facilitate the comparison between the 0.8 and 0.08 m m Ca 2+ conditions, data from d1, d2 and d3 of Fig. 4A and B were compiled in Fig. 4C where the statistical analysis now concerns differences of survival in the two [Ca 2+ ]o media.
On return to normal [Ca 2+ ]o, neither type of fibre showed any sudden significant change of survival rate. Both survival profiles from d0 to d4 approximate to a smooth exponential decay, with time constants of 225 for normal and 147 h for mdx fibres. Thus, even at low [Ca 2+ ]o, elimination of mdx fibres proceeded about 1.5 times faster than for normal fibres. When comparing survival at d2 in Fig. 4A and B, it is clear that a fraction of cell death (≈one-third) observed in 0.8 m m [Ca 2+ ]o also occurred at 0.08 m m when the size of the Ca 2+ entry was reduced proportionately. From d4 to d5, however, no further significant decrease of survival could be detected for either type of fibre.
Voltage-independent Ca 2+ channels: characteristics and progression from d0 to d5
Channel activity was observed on collagenase-isolated FDB fibres, by the patch clamp technique (cell-attached configuration) at holding potential of −60 mV (Fig. 5A). Results displayed in Table 2 give the single-channel conductance and the reversal potential for different solutions within the patch pipette. The I-V relationship is strictly ohmic, showing no sign of voltage activation or inactivation and is identical in mdx and normal fibres (not shown). Cationic selectivity is poor and allows fluxes of Ca 2+ , Ba 2+ , Mn 2+ and Na + (and most likely K + from within the cell). The estimated PCa/PNa ratio ( Lee & Tsien, 1984 ) is around 0.7. Moreover, the open probability is greatly reduced by trivalent cations (e.g. 50 μM La 3+ ). Open times showed a Poisson-like distribution, approximating to an exponential decay, the time constant of which is also reported in Table 2. These characteristics bear strong resemblance to those of the mechanosensitive channels already described in similar preparations ( Franco-Obregón & Lansman, 1994 ). Indeed, when gentle suction was applied to the patches, we observed, in all fibres tested (10 mdx and 10 C57), a significant increase of the mean open probabilities which amounted to 69 ± 8 % and 53 ± 8 % ( s.e.m. ), for mdx and C57 fibres respectively (with no significant difference between mouse lines, P/ 0.7, t test). Interestingly, the mechanosensitivity of these channels was present in the absence of dystrophin. All these gating properties were identical in normal and mdx fibres. Moreover, we observed that they remained unchanged from d0 to d5.
In contrast with this stability, the occurrence of these channels and the open probabilities are different between normal and mdx fibres and showed typical patterns of change from d0 to d5. Channel occurrence was estimated as the percentage of the fibres that, when sampled by the patch pipette, show channel activity (each fibre was sampled only once). Soon after isolation, channel activity was recorded in 80 % of the fibres, both in normal and mdx fibres. As shown in Fig. 5B, the occurrence steadily decreased from d0 to d5, but the decrease was always less marked for mdx fibres. Eventually, by d5, occurrence had declined to 20 and 32 % for normal and mdx fibres, respectively.
Figure 5C gives the change in the average channel open probabilities from d0 to d5. At d0, the values for normal and mdx fibres are very similar to that reported previously, with a significant difference between the two types of fibres. Surprisingly, for both types of fibres, the average open probability nearly doubled at d1 and recovered the d0 values by d3, remaining stable thereafter. At d5, the average value of mdx fibre was about twice the value for normal fibres (P < 0.05). These transient changes are still better illustrated by the histograms of the open probabilities from d0 to d5 (Fig. 6). At d0, the values did not exceed 0.1, while at d1, values for mdx fibres spread up to 0.27. Gradually both distributions narrowed again and recovered their d0 patterns by d5.
Pattern of change from d0 to d5 of the distribution of the open probabilities of voltage-insensitive Ca 2+ channels
Open columns, normal fibres filled columns, mdx fibres.
By integration of the current records over the 120 s of observation, the quantity of charge (pA s) passing through the membrane patch was calculated, when using a patch pipette filled with 110 m m CaCl2. As seen in Fig. 5D, a large increase at d1 followed by a progressive return towards the d0 values was observed for both types of fibre. For each day, values for mdx fibres were about twice those for normal fibres, except at d1 when the mean quantity of charge was tripled in mdx.
In spite of the large difference in experimental conditions, estimates of Ca 2+ influx by either the Mn 2+ fluorescence quenching technique (Fig. 1) or by electrical measurements with a Ca 2+ filled patch pipette (Fig. 5 and Fig. 6) provide remarkably consistent information on three points: (1) a large increase of Ca 2+ influx had developed at d1 (2) this was accompanied by large fibre-to-fibre differences, over the same d1-d2 period (3) a progressive return towards the low d0 values took place within the next 2 days (d2-d4). However, in contrast with the measurements of charges passing through the patch pipettes which were systematically higher in mdx fibres (Fig. 5D), estimation of Ca 2+ influx by the Mn 2+ quenching rate detected no difference at d0, d4 and d5 (Fig. 1). We think that the latter measurements better reflect the physiological situation since they were obtained in near-physiological conditions (in the presence of 140 m m Na + ), while the patch technique artificially amplified the Ca 2+ influx, as pipettes were filled with 110 m m Ca 2+ .
Although calcium is the principal signaling pathway controlling cellular processes of sensory neurons, there has been only minimal examination of effects of peripheral nerve injury on [Ca 2+ ] c . In this study, we demonstrate that axonal injury depresses resting calcium concentrations. This decrease is greater in directly axotomized neurons than in adjacent neurons exposed to degenerating axonal segments. The influence of injury is not uniform across neuronal types but is dominant in units presumed to have a nonnociceptive modality, as indicated by large somatic diameter and insensitivity to capsaicin.
We are not aware of other reports of resting [Ca 2+ ] c in painful neuropathic conditions, except for experimental diabetes mellitus. Although some studies of streptozotocin-induced diabetes have found increased resting [Ca 2+ ] c ,23,24the relevance to traumatic or inflammatory neuropathy is uncertain. For example, diabetes is associated with increased voltage-activated calcium currents in the DRG neuronal membrane,31whereas we have determined that traumatic neuropathy decreases these currents.12,13Also, there are contrasting reports that show unchanged sensory neuronal resting [Ca 2+ ] c in streptozotocin-induced diabetic rats32–34and in a spontaneously diabetic rat strain.35
Response of [Ca 2+ ] c to Capsaicin
Our intent in examining capsaicin response was to categorize cells according to sensory modality.17We also confirmed an EC 50 for [Ca 2+ ] c response to capsaicin in the 55–72 nm range,36,37which is much less than the 350–728 nm necessary to produce a membrane current.38–41Sensory neurons express the capsaicin receptor TRPV1 on endoplasmic reticulum,42so release of calcium stored in endoplasmic reticulum43may explain the responses to low capsaicin concentrations we observed. Supporting our belief that the [Ca 2+ ] c response to capsaicin is a valid indicator of nociceptive modality, we noted a higher response rate in small cells, which tend to be nociceptors. Further, the response rate was substantially decreased in the axotomized neurons of the L5 DRG after SNL but increased in L4, consistent with immunohistochemical observations of TRPV1 expression.44,45A generally higher resting [Ca 2+ ] c in control cells tested with capsaicin compared with those categorized only by size may be due to an unexplained effect of capsaicin on ionophore calibration. Nonetheless, injury effects between groups persisted in cells treated this way.
Technique of Measuring [Ca 2+ ] c
Determination of resting [Ca 2+ ] c is highly sensitive to technical details. The use of a ratiometric indicator such as Fura-2 largely corrects for variations in dye loading, bleaching, or differences in fluorescent signal due to cell thickness. We have noted substantial variability between neurons in R min , R max , and β. This requires the determination of calibration parameters for each neuron if [Ca 2+ ] c is to be compared between cells, although averaged calibration parameters may be adequate if repeated measures for a given cell are compared (e.g. , response to an agonist).
Technical differences may in part explain the wide range of resting [Ca 2+ ] c reported for sensory neurons that spans from 60 to 207 nm.24,36,46–48Further factors may influence measured [Ca 2+ ] c , however, including choice of K d , age of the animal,47and variability in neuronal size and sensory modality of the sample population, as we have shown in the current study. Our finding of higher resting [Ca 2+ ] c in large-diameter sensory neurons than in small neurons contrasts with previous findings in mice32,49but is in accord with findings in rats.24
Potential Mechanisms of Depressed [Ca 2+ ] c after Injury
Cytosolic calcium is regulated by the balanced actions of various membrane processes that acquire, store, and expel calcium. Although calcium entry through voltage-gated calcium channels does not influence [Ca 2+ ] c of the inactive neuron,50influx of calcium through voltage-independent channels regulated by calcium stores (capacitative calcium entry) is active at rest and sensitive to resting membrane potential.50,51Uptake into endoplasmic reticulum via the sarcoplasmic or endoplasmic reticulum calcium adenosine triphosphatase pump and expulsion of calcium from the cell by the plasma membrane calcium adenosine triphosphatase pump both regulate resting [Ca 2+ ] c ,52and mitochondria also sequester calcium in the inactive neuron.53In most sensory neurons, the plasmalemmal Na + –Ca 2+ exchanger is constitutively active such that removal of extracellular Na + increases resting [Ca 2+ ] c .54The decrease in resting [Ca 2+ ] c after peripheral nerve injury may result from a disturbance in any of these regulatory processes, but these have not been examined.
Withdrawal of target-derived neurotrophins may be an upstream trigger for depression of [Ca 2+ ] c , because nerve growth factor,55brain-derived neurotrophic factor, and neurotrophin-356support resting [Ca 2+ ] c . However, nerve growth factor in the ligated L5 DRG recovers after 2 days,57and brain-derived neurotrophic factor expression is increased after nerve injury.58,59An alternate cause of depressed resting [Ca 2+ ] c may be decreased neuronal firing, because activation of neurons increases resting [Ca 2+ ] c .60,61Decreased afferent traffic due to axotomy or disuse of the limb combined with injury-induced depression of voltage-activated calcium currents12,13may decrease the cytoplasmic calcium load. Although spontaneous activity develops in injured DRG neurons,8,62this is typically at very low rates.63,64The normal or higher levels of resting [Ca 2+ ] c in neurons from SNL rats that lacked hyperalgesia might reflect particularly high levels of spontaneous activity in these subjects. Differences we noted between putative nociceptive and low-threshold neurons may likewise be due to differences in neuronal activity between these modality categories.
Possible Consequences of Decreased [Ca 2+ ] c
A role for decreased resting [Ca 2+ ] c in generation of neuropathic pain is supported by our observation of a normal or increased resting [Ca 2+ ] c in a small number of rats lacking hyperalgesia after SNL. Although the functional consequence of dynamic [Ca 2+ ] c transients is well established, the role of resting [Ca 2+ ] c has been less studied. In various neuronal tissues, decreased [Ca 2+ ] c precipitates cell loss, including programed cell death by apoptosis.60,61,65DRG neuronal loss has been noted as a feature of neuropathy after SNL,66,67and induced cell activity may prevent cell loss after axotomy in the central nervous system, presumably by increasing resting [Ca 2+ ] c .68Therefore, the decrease in resting [Ca 2+ ] c we have observed may directly lead to loss of sensory neurons after injury.
A second role of resting [Ca 2+ ] c is control of responsiveness to ligand stimulation. Increased resting [Ca 2+ ] c attenuates receptor-triggered calcium signaling in lymphocytes69and central nervous system microglia.70In DRG neurons, increased [Ca 2+ ] c inactivates responses to capsaicin.37,38,71In contrast, responsiveness to heat is potentiated when [Ca 2+ ] c is increased by capsaicin or a calcium ionophore,72but this is not found after [Ca 2+ ] c increase by membrane depolarization.73Depressed resting [Ca 2+ ] c , as we have measured in sensory neurons after injury, may thus modulate the transduction of membrane receptor activation into intracellular calcium signals for a broad range of ligands and stimuli, including catecholamines, purines, pH, and inflammatory mediators such as bradykinin, complement, and cytokines. Altered expression of receptors may have a competing influence, however, as in the injury-induced changes in capsaicin responsiveness measured in this study. Low resting [Ca 2+ ] c may itself produce complex genetic effects, increasing expression of inducible nitric oxide synthase in chondrocytes while depressing synthesis of cyclooxygenase II,74and increasing the expression of sodium channels in the surface membrane of adrenal chromaffin cells.75
Finally, enzymatic signaling cascades sensitive to calcium, such as calmodulin, phosphatases, and protein kinases A and C, modulate neuronal activity. Through regulation of calcium-activated K + channels76and hyperpolarization-activated cation channels,77resting [Ca 2+ ] c controls membrane excitability and neuronal activity. Calcium/calmodulin-dependent protein kinase II has particularly diverse phosphorylation targets and may be an important pathway generating functional and genetic changes from shifts in resting [Ca 2+ ] c .78
6. Targeting sarcoplasmic reticulum Ca 2+ transport genes to improve contractility
The prospects of gene therapy for heart failure in particular restoring Ca 2+ transport have received much attention. 46, 53, 110–112 Data from failing human hearts suggest that a decrease in SR Ca 2+ transport is responsible for the negative force frequency seen in the failing hearts. Therefore, recent studies have focused on restoring SERCA pump activity, either by adenoviral mediated gene transfer of SERCA2a or by inhibiting PLB. Adenoviral gene transfer studies showed that overexpression of SERCA2a in failing human ventricular myocytes increased Ca 2+ transport and restored contraction and relaxation velocity. 47 The negative frequency response was normalized in cardiomyocytes overexpressing SERCA2a. 47 Encouraged by these in vitro studies, Hajjar and co-workers developed a catheter-based technique to introduce genes into the myocardium. 46, 53, 54, 111, 112 SERCA2a gene transfer in a rat model of pressure-overload hypertrophy (where SERCA2a levels were decreased and severe contractile dysfunction was evident) restored both systolic and diastolic dysfunction to normal levels. SERCA2a overexpression decreased left ventricular size and restored the slope of the end-diastolic pressure–dimension relationship to control levels. Further, SERCA2a gene transfer was used to abrogate ventricular arrhythmias in a rat model of ischaemia reperfusion (I/R) injury. 113, 114 SERCA2a overexpression significantly decreased ventricular arrhythmias during I/R and 24 h later reduced infarct size and improved wall thickening in the anterior wall. These studies suggest that a decrease in diastolic Ca 2+ and better handling of intracellular ions are both associated with improved survival of the cardiomyocytes. Therefore, restoring Ca 2+ transport by increasing SERCA2a activity appears to be critical for maintaining cardiac inotrophy and for preventing the pathological effects of Ca 2+ overload.
Phospholamban ablation was shown to prevent structural and functional abnormalities in mouse models. 8, 115 Therefore, strategies to suppress PLB inhibition and enhance SERCA activity have been tested. An approach to increase SERCA pump activity is to decrease the levels of PLB. Del monte et al. 116 showed that adenoviral gene transfer of antisense PLB in failing human cardiomyocytes restored contractility, Ca 2+ handling, and the force frequency response. In addition, a pseudophosphorylated mutant PLB peptide (S16EPLN) was tested in BIO14.6 CM hamsters (showing a progressive stage of dilated cardiomyopathy and heart failure). Expression of mutant PLB enhanced myocardial SR Ca 2+ uptake and suppressed progressive impairment of left ventricular (LV) systolic function and contractility upto 28−30 weeks. 117 However, chronic inhibition of PLB activity may not be beneficial in human hearts, since loss of PLB function leads to dilated cardiomyopathy at a young age in humans. 87, 88 These experimental studies provide us promising initial results and move us a step forward towards manipulating SERCA pump activity as a therapeutic strategy to rescue contractile function in diseased human hearts. These studies also suggest that maintaining a physiological ratio of SERCA/PLB may be necessary for the hearts ability to meet various physiological demands.
The calcium ion has a very important role to play in homeostasis. It is particularly relevant in physiological processes involved during the conduct of anaesthesia. It is also becoming a very important consideration during the conduct of cardiopulmonary bypass and the management of the critically ill patient on an intensive care unit. Current research also shows that the calcium ion is an integral part of the processes that produce both analgesia and anaesthesia. The main recommendation regarding the anaesthetist's role in managing patients with abnormal calcium metabolism is to try to restore and keep the ionised serum calcium within normal limits.