Spontaneous Calcium Release Research Articles

Autonomic nerve activity and atrial fibrillation

This review focuses on the importance of autonomic nervous system (ANS) activity in the induction of paroxysmal atrial fibrillation (PAF). Clinical studies suggest that both sympathetic and parasympathetic nervous systems are important in mediating PAF. Consistent with that hypothesis, heart rate variability analyses showed that sympathovagal imbalance is present before the onset of PAF episodes. The importance of the ANS in PAF is further supported by animal experiments and recent clinical studies showing that vagal denervation enhances the efficacy of circumferential pulmonary vein ablation in preventing AF recurrence. In vitro studies show that ANS activation facilitates early afterdepolarization and triggered activity by simultaneously prolonging the intracellular calcium (Ca(i)) transient (sympathetic effect) and shortening the action potential duration (parasympathetic effect). By simultaneously mapping the membrane potential and Ca(i) transient in canine pulmonary vein during sympathetic stimulation, we demonstrated that spontaneous (voltage-independent) sarcoplasmic reticulum calcium release underlies the mechanisms of focal discharges. We developed and studied canine models of PAF induced by electrical, structural, and neural remodeling. We also have developed methods for long-term continuous recording of sympathetic and vagal nerve activity in ambulatory dogs. Preliminary results show that simultaneous sympathovagal discharges precede the onset of PAF in these dogs. ANS activity and Ca(i) transient dynamics are important in the development of PAF. These studies suggest that new methods or drugs aimed at modification of cardiac ANS activity may lead to new opportunities for AF control.

The proarrhythmic antihistaminic drug terfenadine increases spontaneous calcium release in human atrial myocytes

Spontaneous calcium release from the sarcoplasmic reticulum in cardiac myocytes plays a central role in cardiac arrhythmogenesis. Compounds intended for therapeutical use that interfere with intracellular calcium handling may therefore have an undesired proarrhythmic potential. Here we have used isolated human atrial myocytes to compare the effect of the proarrhythmic antihistaminic drug terfenadine with the non-proarrhythmic antihistaminic drugs fexofenadine and rupatadine on intracellular calcium homeostasis. Perforated patch-clamp technique was used to measure ionic currents and to detect spontaneous calcium release from the sarcoplasmic reticulum. Our results show that the compound terfenadine, with known arrhythmogenic effects, inhibits L-type calcium current ( I Ca) with an IC 50 of 185 nM when cells are stimulated at 1.0 Hz. The inhibitory effect of 0.3 μM terfenadine increased from 19 ± 4% at stimulation frequency of 0.2 Hz to 63 ± 6% at 2.0 Hz. Moreover, terfenadine also increased spontaneous calcium release from the sarcoplasmic reticulum. At a concentration of 1 μM, terfenadine significantly increased the spontaneous Na–Ca exchange current ( I NCX) frequency from 0.48 ± 0.25 to 1.93 ± 0.67 s − 1 . In contrast, fexofenadine and rupatadine did not change I Ca or the frequency of spontaneous I NCX. We conclude that the proarrhythmic antihistaminic drug terfenadine alters intracellular calcium handling in isolated human atrial myocytes. This experimental model may be suitable to screen for potential arrhythmogenic side-effects of compounds intended for therapeutical use.

Adenosine A2A receptors are expressed in human atrial myocytes and modulate spontaneous sarcoplasmic reticulum calcium release

Alterations in the cyclic AMP-dependent regulation of the cardiac ryanodine receptor (RyR2) have been proposed to account for increased spontaneous calcium release from the sarcoplasmic reticulum (SR) in patients with heart failure, ventricular tachyarrhythmias and atrial fibrillation. While the adenosine A(2A) receptor (A(2A)R) is known to regulate cyclic AMP levels, expression and function of this receptor in human cardiac myocytes has not been investigated. PCR, western blotting and immunofluorescence were used to identify the A(2A)R, and functional effects of A(2A)R stimulation were measured with confocal calcium imaging and patch-clamp technique. The A(2A)R is expressed in the human right atrium and distributed in a banded pattern along the Z-lines, overlapping with the ryanodine receptor. A(2A)R stimulation caused a protein kinase A dependent increase in spontaneous SR calcium release in isolated human atrial myocytes. The A(2A)R agonist CGS21680 increased the frequency of calcium sparks from 0.12+/-0.03 to 0.31+/-0.08 sparks.mum min(-1) (p<0.05) and calcium waves from 0.65+/-0.31 to 5.11+/-1.84 waves.min(-1) (p<0.03). Moreover, spontaneous Na-Ca exchange currents (I(NCX)) increased from 1.19+/-0.17 to 2.50+/-0.42 min(-1) (p<0.001). In contrast, CGS21680 did not alter caffeine inducible calcium release (6.98+/-0.52 vs. 6.82+/-0.57 amol pF(-1), p=0.6) or the spontaneous I(NCX) amplitude (0.32+/-0.05 vs. 0.29+/-0.04 pA pF(-1), p=0.2). Current-voltage relationship and amplitude of the L-type calcium current (1.62+/-0.18 vs. 1.80+/-0.18 pA pF(-1)) were not altered, but calcium release dependent inactivation was faster with CGS21680 (13.4+/-0.7 vs. 15.8+/-1.0 ms, p<0.001). Adenosine A(2A) receptors are expressed in the human atrial myocardium and modulate the frequency of spontaneous calcium release from the SR.

P4-5: In vivo evidence of localized spontaneous calcium release in NCX1 knockdown zebrafish myocardium

Abnormal intracellular Ca2+ handling has been implicated in human cardiac diseases. Many animal models with perturbed Ca2+ regulation are embryonic lethal, preventing detailed physiologic analysis. The zebrafish cardiac mutant tremblor displays a characteristic loss of coordinated contraction resulting from a defect in the Na+/Ca2+ exchanger NCX1. We used this in vivo model to investigate whether impaired myocyte Ca2+ extrusion resulted in a propensity for spontaneous Ca2+ release as a mechanism for the observed phenotype.

Late‐Phase 3 EAD. A Unique Mechanism Contributing to Initiation of Atrial Fibrillation

Early (EAD) and delayed (DAD) afterdepolarizations-induced triggered activity is capable of initiating and maintaining cardiac arrhythmias. EAD-induced triggered responses are traditionally thought to be involved in the generation of ventricular arrhythmias under long QT conditions and are precipitated by bradycardia or long pauses. In contrast, DAD-induced triggered activity commonly underlies arrhythmias precipitated by tachycardia. Spontaneous release of calcium from the sarcoplasmic reticulum (SR) secondary to cellular calcium overload induces DADs and some forms of EADs. Recent studies from our laboratory have uncovered a novel mechanism giving rise to triggered activity, termed "late-phase 3 EAD," which combines properties of both EAD and DAD, but has its own unique character. Late-phase 3 EAD-induced triggered extrasystoles represent a new concept of arrhythmogenesis in which abbreviated repolarization permits "normal SR calcium release" to induce an EAD-mediated closely coupled triggered response, particularly under conditions permitting intracellular calcium loading. This review briefly describes the mechanisms and properties of late-phase 3 EADs, how they differ from conventional EADs and DADs, as well as their role in the initiation of cardiac arrhythmias, such as atrial fibrillation.

Calcium overload, spontaneous calcium release, and ventricular arrhythmias

Calcium overload, spontaneous calcium release, and ventricular arrhythmias

Intracellular Calcium Dynamics and Anisotropic Reentry in Isolated Canine Pulmonary Veins and Left Atrium

Rapid activations due to either focal discharge or reentry are often present during atrial fibrillation (AF) in the pulmonary veins (PVs). The mechanisms of these rapid activations are unclear. We studied 7 isolated, Langendorff-perfused canine left atrial (LA) and PV preparations and used 2 cameras to map membrane potential alone (Vm, n=3) or Vm and intracellular calcium simultaneously (Ca(i), n=4). Rapid atrial pacing induced 26 episodes of focal discharge from the proximal PVs in 5 dogs. The cycle lengths were 223+/-52 ms during ryanodine infusion (n=13) and 133+/-59 ms during ryanodine plus isoproterenol infusion (n=13). The rise of Ca(i) preceded Vm activation at the sites of focal discharge in 6 episodes of 2 preparations, compatible with voltage-independent spontaneous Ca(i) release. Phase singularities during pacing-induced reentry clustered specifically at the PV-LA junction. Periodic acid-Schiff (PAS) stain identified large cells with pale cytoplasm along the endocardium of PV muscle sleeves. There were abrupt changes in myocardial fiber orientation and increased interstitial fibrosis in the PV and at the PV-LA junction. PV muscle sleeves may develop voltage-independent Ca(i) release, resulting in focal discharge. Focal discharge may also be facilitated by the presence of PAS-positive cells that are compatible with node-like cells. During reentry, phase singularities clustered preferentially at sites of increased anisotropy such as the PV-LA junction. These findings suggest that focal discharge caused by spontaneous calcium release and anisotropic reentry both contribute to rapid activations in the PVs during AF.

Can Novel Therapies for Arrhythmias Caused by Spontaneous Sarcoplasmic Reticulum Ca 2+ Release be Developed Using Mouse Models?

See related article, pages e77–e82 Ventricular arrhythmias are a significant cause of premature death in Western society and occur both in the absence and presence of heart disease. For example, in heart failure close to 50% of patients die of lethal forms of ventricular arrhythmias. In spite of a large amount of basic and clinical investigations, current pharmacotherapy for ventricular arrhythmias is inadequate, demonstrating the need for novel therapeutic approaches. Abnormal automaticity is responsible for induction and maintenance of different types of ventricular tachycardias. The form of abnormal automaticity that results from abnormal release of Ca2+ from a Ca2+ overloaded sarcoplasmic reticulum (SR), referred to as a “triggered arrhythmia”, is the topic of this editorial. In vitro studies have shown that increasing the SR Ca2+ load beyond a critical level causes spontaneous opening of the SR Ca2+ release channel (ryanodine receptor; RYR) and results in spontaneous SR Ca2+ release.1 The subsequent increase in cytosolic [Ca] activates an inward current through the sarcolemmal Na+/ Ca2+ exchanger which causes membrane depolarization.1,2 Spontaneous SR Ca2+ release during the diastolic interval causes depolarization (delayed after depolarizations; DADs) which has been shown to result in action potentials in single cells and in the intact heart when the aberrant release is adequately synchronized within and among cardiac myocytes.3,4 Factors that lead to DADs include increased heart rate and catecholamine stress, which both induce triggered arrhythmias by increasing SR Ca2+ loading.3 Whereas many molecules are involved in SR Ca2+ overload (DAD) related arrhythmias, abnormal opening …

Cellular Mechanism of Calcium-Mediated Triggered Activity in the Heart

Calcium overload due to enhanced calcium entry is a mechanism for spontaneous calcium release (SCR) from the sarcoplasmic reticulum, delayed-afterdepolarizations (DAD), and triggered activity. However, the exact mechanistic relationship between elevated intracellular calcium levels and triggered activity originating from a specific location remains unclear. We hypothesize that under conditions of enhanced calcium entry, elevation of intracellular calcium will result in multiple calcium release events of which only one is more likely to initiate a triggered beat. We used optical mapping of action potentials and ratiometric calcium transients in an electromechanically-uncoupled canine wedge model of enhanced calcium entry, using I(Ks) blockade with beta-adrenergic stimulation. Under conditions of enhanced calcium entry, the rate of calcium uptake was faster compared with control conditions; however, during rapid pacing, cytoplasmic calcium elevation at the endocardium was significantly increased (15+/-4%) compared with control (10+/-3, P<0.04). Rapid pacing induced multiple simultaneous SCR events with largest amplitude and earliest onset near the endocardium compared with the epicardium. Furthermore, SCR events with largest amplitude and earliest onset served as a focus for DAD-mediated triggered activity. Interestingly, polymorphic VT occurred in some experiments when multiple SCR events occurred. In conclusion, multiple, simultaneous SCR events occur over a broad region of relatively slower calcium uptake and elevated diastolic calcium levels. However, SCR events closer to the endocardium have the largest amplitude and earliest onset and are, thereby, more likely to initiate DAD-mediated triggered activity. Finally, multiple SCR events may be a mechanism of polymorphic VT under calcium overload conditions.

Delayed Afterdepolarization‐Mediated Triggered Activity Associated with Slow Calcium Sequestration Near the Endocardium

Previously, we have shown that cells near the endocardium are more prone to elevated diastolic intracellular calcium levels than cells near the epicardium. The arrhythmogenic consequence of such regional differences in calcium handling is not clear. Using optical mapping techniques, calcium transients and action potentials were recorded simultaneously from ventricular sites across the transmural wall of the arterially perfused canine left ventricular wedge preparation during control conditions, and under conditions of increased calcium entry (I(K) blockade and beta-adrenergic stimulation). Under conditions of enhanced calcium entry, the decay of the calcium transient and diastolic calcium levels during rapid pacing were slower (38%, P < 0.01) and higher (215%, P < 0.02), respectively, near (within approximately 3 mm) the endocardium compared to the epicardium (n = 9). Immediately after termination of rapid pacing under conditions of increased calcium entry, ectopic activity and simultaneous delayed after depolarizations and spontaneous calcium release events were observed. Over all experiments, ectopic activity occurred more frequently closer to the endocardium compared to the epicardium. Under conditions of enhanced calcium entry, myocytes closer to the endocardium exhibit a higher level of diastolic calcium and greater ectopic activity compared to the epicardium. We show for the first time simultaneous delayed after depolarization and spontaneous calcium release events from myocytes in a normally coupled multicellular preparation. These data combined suggest that myocytes near the endocardium are more susceptible to calcium-mediated triggered activity.

Biophysical adaptation of the theory of photo-induced phase transition: model of cooperative gating of cardiac ryanodine receptors

Theory of photo-induced phase transitions has been adapted to describe the cooperative dynamics of the lattice of ryanodine receptors/channels (RyR) in cardiac muscle which regulate the release of the intracellular activator calcium from calcium stores in the sarcoplasmic reticulum (SR) by a process of Ca2+-induced Ca2+ release (CICR). We introduce two main degrees of freedom for RyR channel, fast electronic and slow conformational ones. The RyR lattice response to the L-type channel triggering evolves due to a nucleation process with a step-by-step domino-like opening of RyR channels. Typical mode of RyR lattice functioning in a CICR process implies the fractional release with a robust termination due to the depletion of SR with a respective change in effective conformational strain. The SR overload leads to an unconventional auto-oscillation regime with a spontaneous calcium release. The model is believed to consistently describe the main features of CICR, that is its gradedness, coupled gating, irreversibility, inactivation/adaptation, and spark termination.

Atrial fibrillation is associated with increased spontaneous calcium release from the sarcoplasmic reticulum in human atrial myocytes.

Spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) can generate afterdepolarizations, and these have the potential to initiate arrhythmias. Therefore, an association may exist between spontaneous SR Ca2+ release and initiation of atrial fibrillation (AF), but this has not yet been reported. Spontaneous Ca2+ release from the SR, manifested as Ca2+ sparks and Ca2+ waves, was recorded with confocal microscopy in atrial myocytes isolated from patients with and those without AF. In addition, the spontaneous inward current associated with Ca2+ waves was measured with the use of the perforated patch-clamp technique. The Ca2+ spark frequency was higher in 8 patients with AF than in 16 patients without (6.0+/-1.2 versus 2.8+/-0.8 sparks/mm per second, P<0.05). Similarly, the spontaneous Ca2+ wave frequency was greater in patients with AF (2.8+/-0.5 versus 1.1+/-0.3 waves/mm per second, P<0.01). The spontaneous inward current frequency was also higher in 10 patients with AF than in 13 patients without this arrhythmia (0.101+/-0.028 versus 0.031+/-0.007 per second, P<0.05, at a clamped potential of -80 mV). In contrast, both the Ca2+ released from the SR and the Na+-Ca2+ exchange rate induced by a rapid caffeine application were comparable in patients with and without AF. The observed increase in spontaneous Ca2+ release in patients with AF probably is due to an upregulation of the SR Ca2+ release channel activity, which may contribute to the development of AF.

Effects of Ryanoids on Spontaneous and Depolarization-Evoked Calcium Release Events in Frog Muscle

The effects of ryanoids on calcium sparks and transients were studied in voltage-clamped cut frog muscle fibers with a laser scanning confocal microscope. For each ryanoid employed, several sequential effects were observed, including: a), transient increases in spontaneous spark frequency; b), conversions of sparks to long-lasting steady glows; and c), occasional interruptions of the glows. The ratio of the amplitude of the glow induced by a ryanoid to that of the precursory spark followed the order: ryanodol > ryanodine > C 10-O eq-glycyl-ryanodine > C 10-O eq- β-alanyl-ryanodol. This sequence of glow amplitudes parallels that of the subconductances induced by these ryanoids in single-channel studies, suggesting that the glows reflect Ca 2+ fluxes through semiopen calcium release channels. Ryanoids also abolished depolarization-evoked sparks elicited with small pulses, and transformed the calcium release during depolarization to a uniform nonsparking fluorescence signal. The ratio of this signal, averaged spatially, to that of the control followed the order: ryanodol < ryanodine < C 10-O eq-glycyl-ryanodine < C 10-O eq- β-alanyl-ryanodol, implying an inverse relationship with the amplitudes of ryanoid-induced glows. The observation that depolarization-evoked calcium release can occur after ryanoid suppression of calcium sparks suggests the possibility of a new strategic approach for treating skeletal muscle diseases resulting from leaky calcium release channels.

Electrophysiological changes in heart failure and their relationship to arrhythmogenesis.

This review focuses mainly on studies in non-ischemic animal models of heart failure. These animals develop ventricular arrhythmias, mostly non-sustained ventricular tachycardia, and often die suddenly. Clinical studies suggest that sudden death is due to ventricular tachycardia or fibrillation in about 50% of cases, the other half to bradyarrhythmias or electromechanical dissociation. Electrophysiologic changes in heart failure are not confined to the ventricles: the intrinsic sinus rate is reduced due to a downregulation of If and sensitivity to acetylcholine is enhanced by upregulation of the muscarinic receptor. Reduction of heart rate may be a protective mechanism since at rapid rates contractility is reduced and the likelihood for triggered activity due to delayed afterdepolarizations is enhanced. The beneficial effect of beta-adrenergic blockade in patients may be partly due to the reduction in sinus rate. Although the results of different studies often vary, the most consistent electrophysiological changes in the ventricles are prolongation of the action potential, especially at slow rates, a reduction in the transient outward current Ito, the rapid and slow components of the delayed rectifier Ikr and Iks, and the inward rectifier Ik1. Abnormalities in intracellular calcium handling play a major role in the genesis of delayed afterdepolarizations. Triggered activity based on delayed afterdepolarizations has been demonstrated in failing myocardium and are caused by spontaneous release of calcium from the sarcoplasmic reticulum (SR), especially in the presence of noradrenaline. Three factors combine to the enhanced propensity for the occurrence of delayed afterdepolarizations: (1) increased activity of the Na/Ca exchanger, (2) a reduced inward rectifier, (3) residual beta-adrenergic responsiveness required to raise the reduced sarcoplasmic calcium content to a level where spontaneous calcium release occurs. Early afterdepolarizations have also been demonstrated, especially in human myocytes from failing hearts in the presence of noradrenaline. Mapping experiments have shown that the ventricular arrhythmias are mainly due to non-reentrant mechanisms, most likely triggered activity based on delayed afterdepolarizations.

Localised calcium release events in cells from the muscle of guinea-pig gastric fundus.

After enzymatic dispersion of the muscle of the guinea-pig gastric fundus, single elongated cells were observed which differed from archetypal smooth muscle cells due to their knurled, tuberose or otherwise irregular surface morphology. These, but not archetypal smooth muscle cells, consistently displayed spontaneous localized (i.e. non-propagating) intracellular calcium ([Ca(2+)](i)) release events. Such calcium events were novel in their magnitude and kinetic profiles. They included short transient events, plateau events and events which coalesced spatially or temporally (compound events). Quantitative analysis of the events with an automatic detection programme showed that their spatio-temporal characteristics (full width and full duration at half-maximum amplitude) were approximately exponentially distributed. Their amplitude distribution suggested the presence of two release modes. Carbachol application caused an initial cell-wide calcium transient followed by an increase in localized calcium release events. Pharmacological analysis suggested that localized calcium release was largely dependent on external calcium entry acting on both inositol trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) to release stored calcium. Nominally calcium-free external solution immediately and reversibly abolished all localized calcium release without blocking the initial transient calcium release response to carbachol. This was inhibited by 2-APB (100 microm), ryanodine (10 or 50 microm) or U-73122 (1 microm). 2-APB (100 microm), xestospongin C (XeC, 10 microm) or U-73122 (1 microm) blocked both spontaneous localized calcium release and localized release stimulated by 10 microm carbachol. Ryanodine (50 microm) also inhibited spontaneous release, but enhanced localized release in response to carbachol. This study represents the first characterization of localized calcium release events in cells from the gastric fundus.

SR calcium handling and calcium after-transients in a rabbit model of heart failure.

After-depolarization associated arrhythmias are frequently observed in heart failure and associated with spontaneous calcium release from sarcoplasmic reticulum (SR), calcium after-transients. We hypothesize that disturbed SR calcium handling underlies calcium after-transients in heart failure (HF). We measured the stimulation rate dependence (0.2-3 Hz) of diastolic calcium, calcium transient amplitude and SR calcium content in left ventricular myocytes isolated from hearts of rabbits with pressure and volume overload-induced HF and age-matched control animals. Cytosolic calcium was measured with indo-1. In some experiments, delayed after-depolarizations (DADs) were monitored with the voltage sensitive dye di-4-Annepps. SR calcium content was estimated from the response to rapid cooling (RC). After-transients were elicited in the presence of norepinephrine (100 nmol/l) after cessation of burst pacing. With increasing stimulation rate (0.2-3.0 Hz): (1) steady state diastolic [Ca](i) increased from 102 to 174 nmol/l in HF and from 44 to 103 nmol/l in control, (2) calcium transient amplitudes decreased from 310 to 254 nmol/l in HF and increased from 186 to 429 nmol/l in control, (3) SR calcium content decreased from 1.25 to 1.09 mmol/l in HF and increased from 1.51 to 2.48 mmol/l in control, (4) in HF and control, the end diastolic SR membrane calcium gradient decreased by about 30%; at any stimulation rate, the magnitude of gradient in HF was one-third of control, (5) systolic depletion of SR was 85% in HF and 60% in control. In HF, noradrenaline (100 nmol/l) increased SR calcium content and SR membrane gradient by 40% versus about 7% in control. Calcium after-transients were observed in 14 out of 18 HF rabbits, and none in eight control animals and were associated with DADs. Calcium after-transients were associated with a 35% decrease in SR calcium content. The frequency of occurrence of calcium after-transients was related to diastolic calcium. in HF, diastolic calcium is increased and both SR calcium content and SR membrane calcium gradient are decreased in a stimulation rate-dependent manner. In HF, beta-adrenergic stimulation can partly restore the SR calcium content and SR membrane gradient at higher stimulation rates in a meta-stable condition; upon transition to low stimulation rates, the SR membrane can no longer maintain this high unbalanced SR calcium load at increased diastolic calcium, the magnitude of which is causally related to the occurrence of calcium after-transients.

5-Hydroxytryptamino-induced calcium sparks in cultured rat stomach fundus smooth muscle cells

With a new fluorescence probe of Ca2+, STDIn-AM, 5-hydroxytryptamino (5-HT)-induced spontaneous calcium release events (calcium sparks) in cultured rat stomach fundus smooth muscle cells (SFSMC) are investigated by laser scanning confocal microscope. The mechanisms of initiation of Ca2+ sparks, propagating Ca2+ waves and their relation to E-C coupling are discussed. After the extracellular [Ca2+] is increased to 10 mmol/L, addition of 5-HT causes hot spots throughout the cytoplasm, which is brighter near the plasmalemma. The amplitude of the event is at least two times greater than the standard deviation of fluorescence intensity fluctuations measured in the neighboring region and the duration of the Ca2+ signal is over 100 ms. The results suggest that 5-HT acts by the way of 5-HT2 receptors on SFSMC, then through 5-HT2 receptors couples IP3/Ca2+ and DG/PKC double signal transduction pathways to cause Ca2+ release from intracellular Ca2+ stores and followed Ca2+ influx possibly through calcium release-activated calcium influx. The acceptor of activated 5-HT2 can also cause membrane depolarization, which then stimulates the L-type Ca2+ channels leading to Ca2+ influx. Then the local Ca2+ entry mentioned above activates ryanodine-sensitive Ca2+ release channels (RyR) on sarcoplasmic reticulum (SR) to cause local Ca2+ release events (Ca2+ sparks) through calcium-induced calcium release (CICR).

The effects of HCl and CaCl(2) injections on intracellular calcium and pH in voltage-clamped snail (Helix aspersa) neurons.

To investigate the mechanisms by which low intracellular pH influences calcium signaling, I have injected HCl, and in some experiments CaCl2, into snail neurons while recording intracellular pH (pHi) and calcium concentration ([Ca2+]i) with ion-sensitive microelectrodes. Unlike fluorescent indicators, these do not increase buffering. Slow injections of HCl (changing pHi by 0.1–0.2 pH units min−1) first decreased [Ca2+]i while pHi was still close to normal, but then increased [Ca2+]i when pHi fell below 6.8–7. As pHi recovered after such an injection, [Ca2+]i started to fall but then increased transiently before returning to its preinjection level. Both the acid-induced decrease and the recovery-induced increase in [Ca2+]i were abolished by cyclopiazonic acid, which empties calcium stores. Caffeine with or without ryanodine lowered [Ca2+]i and converted the acid-induced fall in [Ca2+]i to an increase. Injection of ortho-vanadate increased steady-state [Ca2+]i and its response to acidification, which was again blocked by CPA. The normal initial response to 10 mM caffeine, a transient increase in [Ca2+]i, did not occur with pHi below 7.1. When HCl was injected during a series of short CaCl2 injections, the [Ca2+]i transients (recorded as changes in the potential (VCa) of the Ca2+-sensitive microelectrode), were reduced by only 20% for a 1 pH unit acidification, as was the rate of recovery after each injection. Calcium transients induced by brief depolarizations, however, were reduced by 60% by a similar acidification. These results suggest that low pHi has little effect on the plasma membrane calcium pump (PMCA) but important effects on the calcium stores, including blocking their response to caffeine. Acidosis inhibits spontaneous calcium release via the RYR, and leads to increased store content which is unloaded when pHi returns to normal. Spontaneous release is enhanced by the rise in [Ca2+]i caused by inhibiting the PMCA.

Membrane cross-talk in the early distal tubule segment of frog kidney: role of calcium stores and chloride.

The activities of transport mechanisms in epithelial cells are generally coordinated in order to minimise disturbances in cellular ion content and volume. Furosemide, a potent inhibitor of transport in the renal diluting segment, up-regulates apical K+ channel activity following the release of calcium from intracellular stores. The signal pathway between furosemide application and this calcium release is not known. Single early distal tubule segments from frog kidney were permeabilised with saponin in order to monitor calcium levels within cytoplasmic stores using the calcium-sensitive dye, mag-fura. The uptake (or release) of calcium to (or from) stores was initiated by adding agents to the bath solution, which is in direct contact with the intracellular organelles. ATP promoted calcium uptake into stores, whereas ATP removal led to a slower, spontaneous calcium release. Following loading, calcium stores could be rapidly depleted by inositol 1,4,5-trisphosphate (IP3), but not ryanodine. Calcium release was evident upon lowering the "intracellular" chloride concentration from 12 to 4 mM, equivalent to the fall in chloride induced by furosemide in intact cells. These results suggest that intracellular chloride may function as a second messenger, mediating cross-talk between the apical membrane and intracellular calcium stores.

Relative importance of SR load and cytoplasmic calcium concentration in the genesis of aftercontractions in cardiac myocytes.

To determine whether calcium overload of the sarcoplasmic reticulum underlies drive train-induced aftercontractions in cardiac myocytes. Sarcoplasmic reticulum calcium contents were measured immediately prior to drive train-induced aftercontractions in isolated guinea pig cardiac myocytes, using caffeine application under voltage clamp conditions. Cell shortening during caffeine exposure and cell shortening during the final stimulated beat of the drive train and the delay between caffeine exposure and the onset of inward current were also used as indirect measures of sarcoplasmic reticulum load. At the threshold for aftercontractions, all four measures of sarcoplasmic reticulum load showed interruption of the positive relationship between stimulation frequency and sarcoplasmic reticulum content, the sarcoplasmic reticulum being no more loaded prior to an aftercontraction than following subthreshold drive trains. Intracellular calcium concentration, estimated with the calcium-sensitive dye indo-1, was higher in cells showing aftercontractions than those not. We conclude that calcium overload of the sarcoplasmic reticulum does not underlie spontaneous calcium release in this situation and the primary trigger for spontaneous release may instead be raised cytoplasmic calcium concentration.