The Qγ component of intra-membrane charge movement is present in mammalian muscle fibres, but suppressed in the absence of S100A1

S100A1 is a Ca²⁺ binding protein that modulates excitation–contraction (EC) coupling in skeletal and cardiac muscle. S100A1 competes with calmodulin for binding to the skeletal muscle SR Ca²⁺ release channel (the ryanodine receptor type 1, RyR1) at a site that also interacts with the C-terminal tail of the voltage sensor of EC coupling, the dihydropyridine receptor. Ablation of S100A1 leads to delayed and decreased action potential evoked Ca²⁺ transients, possibly linked to altered voltage sensor activation. Here we investigate the effects of S100A1 on voltage sensor activation in skeletal muscle utilizing whole-cell patch clamp electrophysiology to record intra-membrane charge movement currents in isolated flexor digitorum brevis (FDB) muscle fibres from wild-type and S100A1 knock-out (KO) mice. In contrast to recent reports, we found that FDB fibres exhibit two distinct components of intra-membrane charge movement, an initial rapid component ( Q _β), and a delayed, steeply voltage dependent 'hump' component ( Q _γ) previously recorded primarily in amphibian but not mammalian fibres. Surprisingly, we found that Q _γ was selectively suppressed in S100A1 KO fibres, while the Q _β component of charge movement was unaffected. This result was specific to S100A1 and not a compensatory result of genetic manipulation, as transient intracellular application of S100A1 restored Q _γ. Furthermore, we found that exposure to the RyR1 inhibitor dantrolene suppressed a similar component of charge movement in FDB fibres. These results shed light on voltage sensor activation in mammalian muscle, and support S100A1 as a positive regulator of the voltage sensor and Ca²⁺ release channel in skeletal muscle EC coupling.     

Voltage-controlled Ca2+ release and entry flux in isolated adult muscle fibres of the mouse

The voltage-activated fluxes of Ca²⁺ from the sarcoplasmic reticulum (SR) and from the extracellular space were studied in skeletal muscle fibres of adult mice. Single fibres of the interosseus muscle were enzymatically isolated and voltage clamped using a two-electrode technique. The fibres were perfused from the current-passing micropipette with a solution containing 15 m m EGTA and 0.2 m m of either fura-2 or the faster, lower affinity indicator fura-FF. Electrical recordings in parallel with the fluorescence measurements allowed the estimation of intramembrane gating charge movements and transmembrane Ca²⁺ inward current exhibiting half-maximal activation at −7.60 ± 1.29 and 3.0 ± 1.44 mV, respectively. The rate of Ca²⁺ release from the SR was calculated after fitting the relaxation phases of fluorescence ratio signals with a kinetic model to quantify overall Ca²⁺ removal. Results obtained with the two indicators were similar. Ca²⁺ release was 2–3 orders of magnitude larger than the flux carried by the L-type Ca²⁺ current. At maximal depolarization (+50 mV), release flux peaked at about 3 ms after the onset of the voltage pulse and then decayed in two distinct phases. The slower phase, most likely resulting from SR depletion, indicated a decrease in lumenal Ca²⁺ content by about 80% within 100 ms. Unlike in frog fibres, the kinetics of the rapid phase of decay showed no dependence on the filling state of the SR and the results provide little evidence for a substantial increase of SR permeability on depletion. The approach described here promises insight into excitation–contraction coupling in future studies of genetically altered mice.     

Effects of Mg2+ and SR luminal Ca2+ on caffeine-induced Ca2+ release in skeletal muscle from humans susceptible to malignant hyperthermia

Regulation of the ryanodine receptor (RYR) by Mg²⁺ and SR luminal Ca²⁺ was studied in mechanically skinned malignant hyperthermia susceptible (MHS) and non-susceptible (MHN) fibres from human vastus medialis. Preparations were perfused with solutions mimicking the intracellular milieu and changes in [Ca²⁺] were detected using fura-2 fluorescence. At 1 m m cytosolic Mg²⁺, MHS fibres had a higher sensitivity to caffeine (2-40 m m ) than MHN fibres. The inhibitory effect of Mg²⁺ on caffeine-induced Ca²⁺ release was studied by increasing [Mg²⁺] of the solution containing 40 m m caffeine. Increasing [Mg²⁺] from 1 to 3 m m reduced the amplitude of the caffeine-induced Ca²⁺ transient by 77 ± 7.4 % ( n = 8) in MHN fibres. However, the caffeine-induced Ca²⁺ transient decreased by only 24 ± 8.1 % ( n = 9) in MHS fibres. In MHN fibres, reducing the Ca²⁺ loading period from 4 to 1 min (at 1 m m Mg²⁺) decreased the fraction of the total sarcoplasmic reticulum (SR) Ca²⁺ content released in response to 40 m m caffeine by 90.4 ± 6.2 % ( n = 6). However, in MHS fibres the response was reduced by only 31.2 ± 17.4 % ( n = 6) under similar conditions. These results suggest that human malignant hyperthermia (MH) is associated with reduced inhibition of the RYR by (i) cytosolic Mg²⁺ and (ii) SR Ca²⁺ depletion. Both of these effects may contribute to increased sensitivity of the RYR to caffeine and volatile anaesthetics.     

Calmodulin kinase modulates Ca2+ release in mouse skeletal muscle

Activation of the contractile machinery in skeletal muscle is initiated by the action-potential-induced release of Ca²⁺ from the sarcoplasmic reticulum (SR). Several proteins involved in SR Ca²⁺ release are affected by calmodulin kinase II (CaMKII)-induced phosphorylation in vitro , but the effect in the intact cell remains uncertain and is the focus of the present study. CaMKII inhibitory peptide or inactive control peptide was injected into single isolated fast-twitch fibres of mouse flexor digitorum brevis muscles, and the effect on free myoplasmic [Ca²⁺] ([Ca²⁺]_i) and force during different patterns of stimulation was measured. Injection of the inactive control peptide had no effect on any of the parameters measured. Conversely, injection of CaMKII inhibitory peptide decreased tetanic [Ca²⁺]_i by ≈25 %, but had no significant effect on the rate of SR Ca²⁺ uptake or the force-[Ca²⁺]_i relationship. Repeated tetanic stimulation resulted in increased tetanic [Ca²⁺]_i, and this increase was smaller after CaMKII inhibition. In conclusion, CaMKII-induced phosphorylation facilitates SR Ca²⁺ release in the basal state and during repeated contractions, providing a positive feedback between [Ca²⁺]_i and SR Ca²⁺ release.     

Interplay between SERCA and sarcolemmal Ca2+ efflux pathways controls spontaneous release of Ca2+ from the sarcoplasmic reticulum in rat ventricular myocytes

Waves of calcium-induced calcium release occur in a variety of cell types and have been implicated in the origin of cardiac arrhythmias. We have investigated the effects of inhibiting the SR Ca²⁺-ATPase (SERCA) with the reversible inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (TBQ) on the properties of these waves. Cardiac myocytes were voltage clamped at a constant potential between −65 and −40 mV and spontaneous waves evoked by increasing external Ca²⁺ concentration to 4 m m . Application of 100 μ m TBQ decreased the frequency of waves. This was associated with increases of resting [Ca²⁺]_i, the time constant of decay of [Ca²⁺]_i and the integral of the accompanying Na⁺–Ca²⁺ exchange current. There was also a decrease in propagation velocity of the waves. There was an increase of the calculated Ca²⁺ efflux per wave. The SR Ca²⁺ content when a wave was about to propagate decreased to 91.7 ± 3.2%. The period between waves increased in direct proportion to the Ca²⁺ efflux per wave meaning that TBQ had no effect on the Ca²⁺ efflux per unit time. We conclude that (i) decreased wave frequency is not a direct consequence of decreased Ca²⁺ pumping by SERCA between waves but, rather, to more Ca²⁺ loss on each wave; (ii) inhibiting SERCA increases the chance of spontaneous Ca²⁺ release propagating at a given SR content.     

The effects of membrane potential, SR Ca2+ content and RyR responsiveness on systolic Ca2+ alternans in rat ventricular myocytes

Previous work has shown that small depolarizing pulses produce a beat to beat alternation in the amplitude of the systolic Ca²⁺ transient in ventricular myocytes. The aim of the present work was to investigate the role of changes of SR Ca²⁺ content and L-type Ca²⁺ current in this alternans. As the amplitude of the depolarizing pulse was increased from 10 to 30 mV the magnitude of alternans decreased. Confocal linescan studies showed that this was accompanied by an increase in the number of sites from which Ca²⁺ waves propagated. A sudden decrease in the depolarisation amplitude resulted in three classes of behaviour: (1) a gradual decrease in Ca²⁺ transient amplitude before alternans developed accompanied by a loss of SR Ca²⁺, (2) a gradual increase in Ca²⁺ transient amplitude before alternans accompanied by a gain of SR Ca²⁺, and (3) immediate development of alternans with no change of SR content. We conclude that alternans develops if the combination of decreased opening of L-type channels and change of SR Ca²⁺ content results in spatially fragmented release from the SR as long as there is sufficient Ca²⁺ in the SR to sustain wave propagation. Potentiation of the opening of the ryanodine receptor (RyR) by low concentrations of caffeine (100 μ m ) abolished alternans for a few pulses but the alternans then redeveloped once SR Ca²⁺ content fell to the new threshold for wave propagation. Finally we show evidence that inhibiting L-type Ca²⁺ current with 200 μ m Cd²⁺ produces alternans by means of a similar fragmentation of the Ca²⁺ release profile and propagation of mini-waves of Ca²⁺ release.     

Role of phosphate and calcium stores in muscle fatigue

Intensive activity of muscles causes a decline in performance, known as fatigue, that is thought to be caused by the effects of metabolic changes on either the contractile machinery or the activation processes. The concentration of inorganic phosphate (P_i) in the myoplasm ([P_i]_myo) increases substantially during fatigue and affects both the myofibrillar proteins and the activation processes. It is known that a failure of sarcoplasmic reticulum (SR) Ca²⁺ release contributes to fatigue and in this review we consider how raised [P_i]_myo contributes to this process. Initial evidence came from the observation that increasing [P_i]_myo causes reduced SR Ca²⁺ release in both skinned and intact fibres. In fatigued muscles the store of releasable Ca²⁺ in the SR declines mirroring the decline in SR Ca²⁺ release. In muscle fibres with inoperative creatine kinase the rise of [P_i]_myo is absent during fatigue and the failure of SR Ca²⁺ release is delayed. These results can all be explained if inorganic phosphate can move from the myoplasm into the SR during fatigue and cause precipitation of CaP_i within the SR. The relevance of this mechanism in different types of fatigue in humans is considered.     

Metabolic Regulation of Ca2+ Release in Permeabilized Mammalian Skeletal Muscle Fibres

In the present study, the link between cellular metabolism and Ca²⁺ signalling was investigated in permeabilized mammalian skeletal muscle. Spontaneous events of Ca²⁺ release from the sarcoplasmic reticulum were detected with fluo-3 and confocal scanning microscopy. Mitochondrial functions were monitored by measuring local changes in mitochondrial membrane potential (with the potential-sensitive dye tetramethylrhodamine ethyl ester) and in mitochondrial [Ca²⁺] (with the Ca²⁺ indicator mag-rhod-2). Digital fluorescence imaging microscopy was used to quantify changes in the mitochondrial autofluorescence of NAD(P)H. When fibres were immersed in a solution without mitochondrial substrates, Ca²⁺ release events were readily observed. The addition of l -glutamate or pyruvate reversibly decreased the frequency of Ca²⁺ release events and increased mitochondrial membrane potential and NAD(P)H production. Application of various mitochondrial inhibitors led to the loss of mitochondrial [Ca²⁺] and promoted spontaneous Ca²⁺ release from the sarcoplasmic reticulum. In many cases, the increase in the frequency of Ca²⁺ release events was not accompanied by a rise in global [Ca²⁺]_i. Our results suggest that mitochondria exert a negative control over Ca²⁺ signalling in skeletal muscle by buffering Ca²⁺ near Ca²⁺ release channels.     

Extra activation component of calcium release in frog muscle fibres

In addition to activating more Ca²⁺ release sites via voltage sensors in the t-tubular membranes, it has been proposed that more depolarised voltages enhance activation of Ca²⁺ release channels via a voltage-dependent increase in Ca-induced Ca²⁺ release (CICR). To test this, release permeability signals in response to voltage-clamp pulses to two voltages, –60 and –45 mV, were compared when Δ[Ca²⁺] was decreased in two kinds of experiments. (1) Addition of 8 m m of the fast Ca²⁺ buffer BAPTA to the internal solution decreased release permeability at –45 mV by > 2-fold and did not significantly affect Ca²⁺ release at –60 mV. Although some of this decrease may have been due to a decrease in voltage activation at –45 mV – as assessed from measurements of intramembranous charge movement – the results do tend to support a Ca-dependent enhancement with greater depolarisations. (2) Decreasing SR (sarcoplasmic reticulum) Ca content ([Ca_SR]) should decrease the Ca²⁺ flux through an open channel and thereby Δ[Ca²⁺]. Decreasing [Ca_SR] from > 1000 μ m (the physiological range) to < 200 μ m decreased release permeability at –45 mV relative to that at –60 mV by > 6-fold, an effect shown to be reversible and not attributable to a decrease in voltage activation at –45 mV. These results indicate a Ca-dependent triggering of Ca²⁺ release at more depolarised voltages in addition to that expected by voltage control alone. The enhanced release probably involves CICR and appears to involve another positive feedback mechanism in which Ca²⁺ release speeds up the activation of voltage sensors.     

Sarcoplasmic reticulum Ca2+ release and depletion fail to affect sarcolemmal ion channel activity in mouse skeletal muscle

In skeletal muscle, sarcoplasmic reticulum (SR) Ca²⁺ depletion is suspected to trigger a calcium entry across the plasma membrane and recent studies also suggest that the opening of channels spontaneously active at rest and possibly involved in Duchenne dystrophy may be regulated by SR Ca²⁺ depletion. Here we simultaneously used the cell-attached and whole-cell voltage-clamp techniques as well as intracellular Ca²⁺ measurements on single isolated mouse skeletal muscle fibres to unravel any possible change in membrane conductance that would depend upon SR Ca²⁺ release and/or SR Ca²⁺ depletion. Delayed rectifier K⁺ single channel activity was routinely detected during whole-cell depolarizing pulses. In addition the activity of channels carrying unitary inward currents of ∼1.5 pA at −80 mV was detected in 17 out of 127 and in 21 out of 59 patches in control and mdx dystrophic fibres, respectively. In both populations of fibres, large whole-cell depolarizing pulses did not reproducibly increase this channel activity. This was also true when, repeated application of the whole-cell pulses led to exhaustion of the Ca²⁺ transient. SR Ca²⁺ depletion produced by the SR Ca²⁺ pump inhibitor cyclopiazonic acid (CPA) also failed to induce any increase in the resting whole-cell conductance and in the inward single channel activity. Overall results indicate that voltage-activated SR Ca²⁺ release and/or SR Ca²⁺ depletion are not sufficient to activate the opening of channels carrying inward currents at negative voltages and challenge the physiological relevance of a store-operated membrane conductance in adult skeletal muscle.     

Effect of ADP on slow-twitch muscle fibres of the rat: implications for muscle fatigue

Slow-twitch mechanically skinned fibres from rat soleus muscle were bathed in solutions mimicking the myoplasmic environment but containing different [ADP] (0.1 μ m to 1.0 m m ). The effect of ADP on sarcoplasmic reticulum (SR) Ca²⁺-content was determined from the magnitude of caffeine-induced force responses, while temporal changes in SR Ca²⁺-content allowed determination of the effective rates of the SR Ca²⁺-pump and of the SR Ca²⁺-leak. The SR Ca²⁺-pump rate, estimated at pCa (−log₁₀[Ca²⁺]) 7.8, was reduced by 20% as the [ADP] was increased from 0.1 to 40 μ m , with no further alteration when the [ADP] was increased to 1.0 m m . The SR Ca²⁺-leak rate constant was not altered by increasing [ADP] from 0.1 to 40 μ m , but was increased by 26% when the [ADP] was elevated to 1.0 m m . This ADP-induced SR Ca²⁺-leak was insensitive to ruthenium red but was abolished by 2,5-di(tert-butyl)-1,4-hydroquinone (TBQ), indicating that the leak pathway is via the SR Ca²⁺-pump and not the SR Ca²⁺-release channel. The decrease in SR Ca²⁺-pump rate and SR Ca²⁺-leak rate when [ADP] was increased led to a 40% decrease in SR Ca²⁺-loading capacity. Elevation of [ADP] had only minor direct effects on the contractile apparatus of slow-twitch fibres. These results suggest that ADP has only limited depressing effects on the contractility of slow-twitch muscle fibres. This is in contrast to the marked effects of ADP on force responses in fast-twitch muscle fibres and may contribute to the fatigue-resistant nature of slow-twitch muscle fibres.     

Calcium-induced calcium release regulates action potential generation in guinea-pig sympathetic neurones

Experiments were done using guinea-pig sympathetic neurones dissociated from the stellate ganglia to establish whether calcium-induced calcium release (CICR) modulated action potential (AP) generation in mammalian neurones. Using measurements of intracellular calcium ([Ca²⁺]_i) with the Ca²⁺-sensitive dye fluo-3, we demonstrated that 10 m m caffeine activated ryanodine receptors and caused a rise in [Ca²⁺]_i in both Ca²⁺-containing and Ca²⁺-deficient solutions. We also demonstrated that combined treatment with caffeine and 1 μ m thapsigargin or caffeine and 20 μ m ryanodine blocked subsequent caffeine-induced elevations of [Ca²⁺]_i. Treatment with thapsigargin, ryanodine or 200 μ m Cd²⁺ to disrupt CICR decreased the latency to AP generation during 400 ms depolarizing current ramps using the perforated patch whole cell patch clamp in current clamp mode. Treatment with 500 μ m tetraethylammonium also decreased the latency to AP generation during depolarizing current ramps in control cells, but not in cells pretreated with thapsigargin to deplete internal Ca²⁺ stores. In summary, we propose that an outward current, carried at least in part through BK channels, is activated by CICR at membrane voltages approaching the threshold for AP initiation and that this current opposed depolarizing current ramps applied to guinea-pig sympathetic stellate neurones.     

Reorganized stores and impaired calcium handling in skeletal muscle of mice lacking calsequestrin-1

Calsequestrin (CS), the major Ca²⁺-binding protein in the sarcoplasmic reticulum (SR), is thought to play a dual role in excitation–contraction coupling: buffering free Ca²⁺ increasing SR capacity, and modulating the activity of the Ca²⁺ release channels (RyRs). In this study, we generated and characterized the first murine model lacking the skeletal CS isoform (CS1). CS1- null mice are viable and fertile, even though skeletal muscles appear slightly atrophic compared to the control mice. No compensatory increase of the cardiac isoform CS2 is detectable in any type of skeletal muscle. CS1- null muscle fibres are characterized by structural and functional changes, which are much more evident in fast-twitch muscles (EDL) in which most fibres express only CS1, than in slow-twitch muscles (soleus), where CS2 is expressed in about 50% of the fibres. In isolated EDL muscle, force development is preserved, but characterized by prolonged time-to-peak and half-relaxation time, probably related to impaired calcium release from and re-uptake by the SR. Ca²⁺-imaging studies show that the amount of Ca²⁺ released from the SR and the amplitude of the Ca²⁺ transient are significantly reduced. The lack of CS1 also causes significant ultrastructural changes, which include: (i) striking proliferation of SR junctional domains; (ii) increased density of Ca²⁺-release channels (confirmed also by ³H-ryanodine binding); (iii) decreased SR terminal cisternae volume; (iv) higher density of mitochondria. Taken together these results demonstrate that CS1 is essential for the normal development of the SR and its calcium release units and for the storage and release of appropriate amounts of SR Ca²⁺.     

Cross-linking of Both Types of IgG Fc Receptors, FcγyRI and FcγyRII, Enhances Intracellular Free Ca2+ in the Monocytic Cell Line U937

We have studied the effects of Fc receptor triggering on the free cytosolic Ca²⁺ concentration, [Ca²⁺] _i , in U937. These cells express two types of IgG Fc receptors, FcγRI and FcγRII. Binding of several anti-FcγRI and anti-FcγRII mouse monoclonal antibodies (MoAb) to Quin2- or Indo-I-loaded U937 cells had no direct effect on [Ca²⁺] _i . After addition of a bridging anti-mouse Ig antibody however, transient increases in [Ca²⁺] _i were observed for both types of FcγR. One of the anti-FcγRII MoAb, CIKM5, was exceptional in that it could induce Ca²⁺ increases in U937 cells by itself. Studies with F(ab')₂ fragments of CIK M5 revealed that this MoAb simultaneously binds to FcγRII, via both its Fab and Fc fragments, which might induce cross-linking of two FcγRII molecules. One anti-FcγRI MoAb, 197, a mouse (m)IgG2a antibody directed to an epitope outside the IgG-binding region, remarkably also caused an immediate increase in [Ca²⁺] _i , but only when added to U937 precultured with gamma interferon (IFN-γ). FcγRI can bind monomeric human IgG as well as mIgG2a, and cross-linking of cytophilic Ig induced an increase in [Ca²⁺] _i . Our results show that [Ca²⁺] _i increases can be induced only after cross-linking of FcγR, either via anti-FcγR MoAb or via Fc-FcR interactions. Furthermore, we show that FcγR cross-linking results in activation of the Ca²⁺/phosphatidylinositol (PI) signal transduction pathway.     

T-tubule disorganization and reduced synchrony of Ca2+ release in murine cardiomyocytes following myocardial infarction

In cardiac myocytes, initiation of excitation–contraction coupling is highly localized near the T-tubule network. Myocytes with a dense T-tubule network exhibit rapid and homogeneous sarcoplasmic reticulum (SR) Ca²⁺ release throughout the cell. We examined whether progressive changes in T-tubule organization and Ca²⁺ release synchrony occur in a murine model of congestive heart failure (CHF). Myocardial infarction (MI) was induced by ligation of the left coronary artery, and CHF was diagnosed by echocardiography (left atrial diameter >2.0 mm). CHF mice were killed at 1 or 3 weeks following MI (1-week CHF, 3-week CHF) and cardiomyocytes were isolated from viable regions of the septum, excluding the MI border zone. Septal myocytes from SHAM-operated mice served as controls. T-tubules were visualized by confocal microscopy in cells stained with di-8-ANEPPS. SHAM cells exhibited a regular striated T-tubule pattern. However, 1-week CHF cells showed slightly disorganized T-tubule structure, and more profound disorganization occurred in 3-week CHF with irregular gaps between adjacent T-tubules. Line-scan images of Ca²⁺ transients (fluo-4 AM, 1 Hz) showed that regions of delayed Ca²⁺ release occurred at these gaps. Three-week CHF cells exhibited an increased number of delayed release regions, and increased overall dyssynchrony of Ca²⁺ release. A common pattern of Ca²⁺ release in 3-week CHF was maintained between consecutive transients, and was not altered by forskolin application. Thus, progressive T-tubule disorganization during CHF promotes dyssynchrony of SR Ca²⁺ release which may contribute to the slowing of SR Ca²⁺ release in this condition.     

Calcium oscillations in interstitial cells of the rabbit urethra

Measurements were made (using fast confocal microscopy) of intracellular Ca²⁺ levels in fluo-4 loaded interstitial cells isolated from the rabbit urethra. These cells exhibited regular Ca²⁺ oscillations which were associated with spontaneous transient inward currents recorded under voltage clamp. Interference with d - myo -inositol 1,4,5-trisphosphate (IP₃) induced Ca²⁺ release using 100 μ m 2-aminoethoxydiphenyl borate, and the phospholipase C (PLC) inhibitors 2-nitro-4-carboxyphenyl N , N -diphenylcarbamate and U73122 decreased the amplitude of spontaneous oscillations but did not abolish them. However, oscillations were abolished when ryanodine receptors were blocked with tetracaine or ryanodine. Oscillations ceased in the absence of external Ca²⁺, and frequency was directly proportional to the external Ca²⁺ concentration. Frequency of Ca²⁺ oscillation was reduced by SKF-96365, but not by nifedipine. Lanthanum and cadmium completely blocked oscillations. These results suggest that Ca²⁺ oscillations in isolated rabbit urethral interstitial cells are initiated by Ca²⁺ release from ryanodine-sensitive intracellular stores, that oscillation frequency is very sensitive to the external Ca²⁺ concentration and that conversion of the primary oscillation to a propagated Ca²⁺ wave depends upon IP₃-induced Ca²⁺ release.     

Simultaneous imaging of Ca2+ signals in interstitial cells of Cajal and longitudinal smooth muscle cells during rhythmic activity in mouse ileum

Electrical rhythmicity in smooth muscle cells is essential for the movement of the gastrointestinal tract. Interstitial cells of Cajal (ICC) lie adjacent to smooth muscle layers and are implicated as the pacemaker cells. However, the pace making mechanism remains unclear. To study the intercellular interaction during electrical rhythm generation, we visualized changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in smooth muscle cells and myenteric ICC within segments of mouse ileum loaded with a fluorescent Ca²⁺ indicator, fluo-3. We observed rhythmic [Ca²⁺]_i changes in longitudinal smooth muscle cells travelling rapidly through the smooth muscle cell layer. Between the rhythmic Ca²⁺ transients, we found brief Ca²⁺ transients localized to small areas within smooth muscle cells. The amplitude but not the periodicity of rhythmic [Ca²⁺]_i transients in both cell types was partially inhibited by nicardipine, an L-type Ca²⁺ channel antagonist, suggesting that the rhythmic [Ca²⁺]_i transients reflect membrane potential depolarizations corresponding to both slow waves and triggered Ca²⁺ spikes. Longitudinal smooth muscle cells and myenteric ICC showed synchronous spontaneous [Ca²⁺]_i transients in eight out of 21 ileac preparations analysed. In the remaining preparations, the synchrony between ICC and smooth muscle cells was absent, although the rhythmicity of the smooth muscle cells was not disturbed. These results suggest that myenteric ICC may play multiple roles including pace making for physiological bowel movement.     

pH-dependent and -independent effects inhibit Ca2+-induced Ca2+ release during metabolic blockade in rat ventricular myocytes

We have investigated the role of changes of intracellular pH (pH_i) in the effects of metabolic blockade (cyanide plus 2-deoxyglucose) on Ca²⁺ release from the sarcoplasmic reticulum (SR) in rat ventricular myocytes. pH_i and cell length were measured simultaneously. Metabolic blockade decreased the frequency of Ca²⁺ waves, an effect previously shown to be due to inhibition of Ca²⁺ release from the SR. This was accompanied by an intracellular acidification. Intracellular acidification was produced in the absence of metabolic inhibition by application of sodium butyrate. A maintained intracellular acidosis produced a decrease of wave frequency. A hysteresis between pH_i and wave frequency was observed such that during the onset of the acidification the wave frequency decreased more than in the steady state. Comparison of the steady state relationship between pH_i and wave frequency showed that the decrease of wave frequency produced by metabolic blockade was greater than could be accounted for simply by the accompanying decrease of pH_i. In other experiments the buffering power of the solution was increased. Under these conditions, metabolic blockade produced no change of pH_i but the decrease of wave frequency persisted. We conclude that, although intracellular acidification occurs during metabolic blockade, it is not responsible for most of the inhibition of Ca²⁺ release from the SR.     

Properties of spontaneous Ca2+ transients recorded from interstitial cells of Cajal-like cells of the rabbit urethra in situ

Interstitial cells of Cajal-like cells (ICC-LCs) in the urethra may act as electrical pacemakers of spontaneous contractions. However, their properties in situ and their interaction with neighbouring urethral smooth muscle cells (USMCs) remain to be elucidated. To further explore the physiological role of ICC-LCs, spontaneous changes in [Ca²⁺]_i (Ca²⁺ transients) were visualized in fluo-4 loaded preparations of rabbit urethral smooth muscle. ICC-LCs were sparsely distributed, rather than forming an extensive network. Ca²⁺ transients in ICC-LCs had a lower frequency and a longer half-width than those of USMCs. ICC-LCs often exhibited Ca²⁺ transients synchronously with each other, but did not often show a close temporal relationship with Ca²⁺ transients in USMCs. Nicardipine (1 μ m ) suppressed Ca²⁺ transients in USMCs but not in ICC-LCs. Ca²⁺ transients in ICC-LCs were abolished by cyclopiazonic acid (10 μ m ), ryanodine (50 μ m ) and caffeine (10 m m ) or by removing extracellular Ca²⁺, and inhibited by 2-aminoethoxydiphenyl borate (50 μ m ) and 3-morpholino-sydnonimine (SIN-1; 10 μ m ), but facilitated by increasing extracellular Ca²⁺ or phenylephrine (1–10 μ m ). These results indicated that Ca²⁺ transients in urethral ICC-LCs in situ rely on both Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ influx through non-L-type Ca²⁺ channel pathways. ICC-LCs may not act as a coordinated pacemaker electrical network as do ICC in the gastrointestinal (GI) tract. Rather they may randomly increase excitability of USMCs to maintain the tone of urethral smooth muscles.     

External anions and cations distinguish between AMPA and kainate receptor gating mechanisms

Possible interactions between different intracellular Ca²⁺ release channels were studied in isolated rat gastric myocytes using agonist-evoked Ca²⁺ signals. Spontaneous, local Ca²⁺ transients were observed in fluo-4-loaded cells with linescan confocal imaging. These were blocked by ryanodine (100 μ m ) but not by the inositol 1,4,5-trisphosphate receptor (IP₃R) blocker, 2-aminoethoxydiphenyl borate (100 μ m ), identifying them as Ca²⁺ sparks. Caffeine (10 m m ) and carbachol (10 μ m ) initiated Ca²⁺ release at sites which co-localized with each other and with any Ca²⁺ spark sites. In fura-2-loaded cells extracellular 2-aminoethoxydiphenyl borate and intracellular heparin (5 mg ml⁻¹) both inhibited the global cytoplasmic [Ca²⁺] transient evoked by carbachol, confirming that it was IP₃R-dependent. 2-Aminoethoxydiphenyl borate and heparin also increased the response to caffeine. This probably reflected an increased Ca²⁺ store content since 2-aminoethoxydiphenyl borate more than doubled the amplitude of transients evoked by ionomycin. Ryanodine completely abolished carbachol and caffeine responses but only reduced ionomycin transients by 30 %, suggesting that blockade of carbachol transients by ryanodine was not simply due to store depletion. Double labelling of IP₃Rs and RyRs demonstrated extensive overlap in their distribution. These results suggest that carbachol stimulates Ca²⁺ release through co-operation between IP₃Rs and RyRs, and implicate IP₃Rs in the regulation of Ca²⁺ store content.