Relationship between asynchronous Ca2+ waves and force development in intact smooth muscle bundles of the porcine trachea

Fluctuations in intracellular calcium concentration ([Ca2+]i) constitute the main link in excitation-contraction coupling (E-C coupling) in airway smooth muscle cells (ASMC). It has recently been reported that ACh induces asynchronous recurring Ca2+ waves in intact ASMC of murine bronchioles. With the use of a novel technique allowing us to simultaneously measure subcellular [Ca2+]i and force generation in ASMC located within an intact tracheal muscle bundle, we examined a similar pattern of Ca2+ signaling in the trachea. We found that application of ACh resulted in the generation of recurring intracellular Ca2+ waves progressing along the longitudinal axis of the ribbon-shaped intact ASMC. These Ca2+ waves were not synchronized between neighboring cells, and induction of wave-like [Ca2+]i oscillations was temporally associated with development of force by the tracheal muscle bundle. By comparing the concentration dependence of force generation and the parameters characterizing the [Ca2+]i oscillations, we found that the concentration-dependent increase in ACh-induced force development by the tracheal smooth muscle bundle is achieved by differential recruitment of intact ASMC to initiate Ca2+ waves and by enhancement in the frequency of [Ca2+]i oscillations and elevation of interspike [Ca2+]i once the cells are recruited. Our findings demonstrate that asynchronous recurring Ca2+ waves underlie E-C coupling in ACh-induced contraction of the intact tracheal smooth muscle bundle. Furthermore, in contrast to what was reported in enzymatically dissociated ASMC, Ca2+ influx through the L-type voltage-gated Ca2+ channel was not an obligatory requirement for the generation of [Ca2+]i oscillations and development of force in ACh-stimulated intact ASMC.     

Ca2+]i-independent contractile force generation by rat hepatic stellate cells in response to endothelin-1

The contractile force generated by hepatic stellate cells in response to endothelin-1 contributes to sinusoidal blood flow regulation and hepatic fibrosis. This study's aim was to directly test the widely held view that changes in cytosolic Ca2+ concentration ([Ca2+]i) mediate stellate cell force generation. Contractile force generation by primary cultures of rat hepatic stellate cells grown in three-dimensional collagen gels was directly and quantitatively measured using a force transducer. Stellate cell [Ca2+]i, myosin activation, and migration were quantified using standard techniques. [Ca2+]i was modulated using ionomycin, BAPTA, KCl, and removal of extracellular Ca2+. Removal of extracellular Ca2+ did not alter endothelin-1-stimulated force development or [Ca2+]i. Ionomycin, a Ca2+ ionophore, triggered an increase in [Ca2+]i that was three times greater than that stimulated by endothelin-1, but only induced 16% of the force and 38% of the myosin regulatory light chain (MLC) phosphorylation induced by endothelin-1. Physiological increases in [Ca2+]i induced by hyperkalemia had no effect on contractile force. Loading BAPTA, a Ca2+ chelator, in stellate cells completely blocked endothelin-1-induced increases in [Ca2+]i but had no effect on endothelin-1-stimulated force generation or MLC phosphorylation. In contrast, Y-27632, a selective rho-associated kinase inhibitor, inhibited endothelin-1-stimulated force generation by at least 70% and MLC phosphorylation by at least 80%. Taken together, these observations indicate that changes in [Ca2+]i are neither necessary nor sufficient for contractile force generation by rat stellate cells. Our results challenge the current model of contractile regulation in hepatic stellate cells and have important implications for our understanding of hepatic pathophysiology.     

Excitation-contraction coupling in cardiac muscle of lobster (Homarus americanus); the role of the sarcolemma and sarcoplasmic reticulum

The T-tubules and sarcoplasmic reticulum (SR) serving excitation-contraction (EC) coupling in lobster (Homarus americanus) cardiac muscle are similar to those in mammalian myocardium. Tetanic contraction is elicited by a burst of action potentials from the cardiac ganglion. In this study we evaluated the roles of the sarcolemma and SR in EC coupling of the ostial valve muscle (orbicularis ostii m. or OOM) of lobster heart. The OOM was mounted in a bath with saline on a microscope stage; force was measured by strain gauge. [Ca2+]i was measured using iontophoretically micro-injected fura-2 salt. Peak [Ca+]i, peak tetanic force and time to peak [Ca2+]i increased with that of stimulus train duration (TD), to a maximum at a TD of 500 ms. Force increased with [Ca2+]. Cd2+ reduced force by 90%; ryanodine and caffeine reduced tetanic [Ca2+]i transients by 80% and 70%, and force by 90% and 80%, respectively. Ryanodine, caffeine and cyclopiazonic acid slowed the decline of [Ca2+]i and force during relaxation. Relaxation required [Na+]o. The rate of decline of [Ca2+]i appeared to be a sigmoidal function of the [Ca2+]i and increased for any [Ca2+]i with TD. Inactivity slowed relaxation of force; stimulation accelerated relaxation. These data suggest important contributions of Ca2+ transport both across the sarcolemma and across the SR membrane during EC-coupling of lobster cardiac muscle, while average cytosolic [Ca2+]i regulates the rate of [Ca2+]i elimination during relaxation.     

Photolytic release of MgADP reduces rigor force in smooth muscle

Photolytic release of MgADP (25-300 microM) from caged ADP in permeabilized tonic (rabbit femoral artery-Rfa) and phasic (rabbit bladder-Rbl) smooth muscle in high-tension rigor state, in the absence of Ca(2+), caused an exponential decline (approximately 1.5% in Rfa and approximately 6% in Rbl) of rigor force, with the rate proportional to the liberated [MgADP]. The apparent second-order rate constant of MgADP binding was estimated as approximately 1.0 x 10(6) M(-1) s(-1) for both smooth muscles. In control experiments, designed to test the specificity of MgADP, photolysis of caged ADP in the absence of Mg(2+) did not decrease rigor force in either smooth muscle, but rigor force decreased after photolytic release of Mg(2+) in the presence of ADP. The effects of photolysis of caged ADP were similar in smooth muscles containing thiophosphorylated or non-phosphorylated regulatory myosin light chains. Stretching or releasing (within range of 0.1-1.2% of initial Ca(2+)-activated force) did not affect the rate or relative amplitude of the force decrease. The effect of additions of MgADP to rigor cross-bridges could result from rotation of the lever arm of smooth muscle myosin, but this need not imply that ADP-release is a significant force-producing step of the physiological cross-bridge cycle.     

Kinetics of cytosolic Ca2+ concentration after photolytic release of 1-D-myo-inositol 1,4-bisphosphate 5-phosphorothioate from a caged derivative in guinea pig hepatocytes.

The influence of 1-D-myo-inositol 1,4,5-trisphosphate (InsP3) breakdown by InsP3 5-phosphatase in determining the time course of Ca2+ release from intracellular stores was investigated with flash photolytic release of a stable InsP3 derivative, 5-thio-InsP3, from a photolabile caged precursor. The potency and Ca(2+)-releasing properties of the biologically active D isomers of 5-thio-InsP3 and InsP3 itself were compared by photolytic release in guinea pig hepatocytes. After a light flash, cytosolic free calcium concentration ([Ca2+]i) showed an initial delay before rising quickly to a peak and declining more slowly to resting levels, with time course and amplitude generally similar to those seen with photolytic release of InsP3. Differences were a three- to eightfold lower potency of 5-thio-InsP3 in producing Ca2+ release, much longer delays between photolytic release and Ca2+ efflux with low concentrations of 5-thio-InsP3 than with InsP3, and persistent reactivation of Ca2+ release, producing periodic fluctuations of cytosolic [Ca2+]i with high concentrations of 5-thio-InsP3 but not InsP3 itself. The lower potency of 5-thio-InsP3 may be a result of a lower affinity for closed receptor/channels or a lower open probability of liganded receptor/channels. The longer delays with 5-thio-InsP3 at low concentration suggest that metabolism of InsP3 by 5-phosphatase may reduce the concentration sufficiently to prevent receptor activation and may have a similar effect on InsP3 concentration during hormonal activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic regulation of intracellular calcium signals through calcium release channels

After the seminal work of Ebashi and coworkers which established the essential role of the intracellular Ca2+ concentration ([Ca2+]i) in the regulation of skeletal muscle contraction, we have witnessed an explosive elongation of the list of cell functions that are controlled by the [Ca2+]i. In numerous instances, release of intracellular Ca2+ stores plays important roles in Ca2+ signalling which displays significant variation in spatio-temporal pattern. There are two families of Ca2+ release channels, ryanodine receptors and inositol 1,4,5-trisphosphate (IP3) receptors. These Ca2+ release channels are structurally and functionally similar. In particular, the activity of both types of channels is regulated by the [Ca2+]i. The [Ca2+]i dependence of the Ca2+ release channel activity provides both types of channels with properties of a Ca2+ signal amplifier. This function of the ryanodine receptor is important in striated muscle excitation-contraction coupling, whereas that of the IP3 receptor seems to be the basis of the generation of Ca2+ waves. Thus the wide variety of Ca2+ signalling patterns seem to be critically dependent on the [Ca2+]i dependence of the Ca2+ release channels.     

Cell calcium and its regulation in smooth muscle

Two novel methods used to study smooth muscles-electron probe X-ray microanalysis and Ca2+-sensitive indicators (which are used for resolving, respectively, the spatial distribution and temporal distribution of calcium)-are briefly reviewed and the major findings obtained are summarized. In smooth muscle the sarcoplasmic reticulum is the major intracellular source of Ca2+; mitochondria do not play a significant role in the physiological regulation of [Ca2+]i. Under pathological conditions mitochondria can reversibly accumulate large amounts of calcium. Resting [Ca2+]i generally ranges from 80 to 200 nM, and is lower in phasic than in tonic smooth muscles. Removal of extracellular Ca2+ and Ca2+ entry blockers can reduce [Ca2+]i, but the effects of beta-adrenergic agents are variable. Increases in [Ca2+]i are triggered by electrical stimulation, depolarization with high K+, and excitatory agonists. Stretch, after a delay of several seconds, can cause an increase in [Ca2+]i in some smooth muscles. There is also a delay of approximately 200-400 ms between the initiation of the rise of Ca2+ and contraction that follows spontaneous action potentials or electrical stimulation. Agonist-induced Ca2+ release, a major mechanism of pharmacomechanical coupling, has been demonstrated in smooth muscles depolarized with high K; evidence suggests that it is mediated by G proteins that couple receptors to phospholipase C. Ca2+ release can be triggered directly in permeabilized smooth muscle with inositol 1,4,5-trisphosphate. Even though Ca2+ is the major physiological regulator of contraction, Ca2+ sensitivity of the regulatory-contractile apparatus differs in different (phasic and tonic) smooth muscles, and can be modulated in a given smooth muscle. The force [Ca2+]i ratio is higher during agonist-stimulated than during high K+-induced contractions, owing to agonist-induced increases in Ca2+ sensitivity mediated by G proteins. In some phasic smooth muscles (guinea pig ileum), the time course of the initial myosin light chain phosphorylation is extremely rapid and returns to basal levels while force remains elevated. In these smooth muscles there is also a marked decrease in the Ca2+ sensitivity of the regulatory-contractile apparatus during maintained depolarization in Ca2+-free or low Ca2+ solutions. It has been suggested that regulation of myosin light chain phosphatase plays a major role in the modulation of the Ca2+ sensitivity manifested as either potentiation or desensitization to [Ca2+]i.     

Regulation of sarcoplasmic reticulum Ca2+ reuptake in porcine airway smooth muscle

Regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in airway smooth muscle (ASM) during agonist stimulation involves sarcoplasmic reticulum (SR) Ca(2+) release and reuptake. The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is key to replenishment of SR Ca(2+) stores. We examined regulation of SERCA in porcine ASM: our hypothesis was that the regulatory protein phospholamban (PLN) and the calmodulin (CaM)-CaM kinase (CaMKII) pathway (both of which are known to regulate SERCA in cardiac muscle) play a role. In porcine ASM microsomes, we examined the expression and extent of PLN phosphorylation after pharmacological inhibition of CaM (with W-7) vs. CaMKII (with KN-62/KN-93) and found that PLN is phosphorylated by CaMKII. In parallel experiments using enzymatically dissociated single ASM cells loaded with the Ca(2+) indicator fluo 3 and imaged using fluorescence microscopy, we measured the effects of PLN small interfering RNA, W-7, and KN-62 on [Ca(2+)](i) responses to ACh and direct SR stimulation. PLN small interfering RNA slowed the rate of fall of [Ca(2+)](i) transients to 1 microM ACh, as did W-7 and KN-62. The two inhibitors additionally slowed reuptake in the absence of PLN. In other cells, preexposure to W-7 or KN-62 did not prevent initiation of ACh-induced [Ca(2+)](i) oscillations (which were previously shown to result from repetitive SR Ca(2+) release/reuptake). However, when ACh-induced [Ca(2+)](i) oscillations reached steady state, subsequent exposure to W7 or KN-62 decreased oscillation frequency and amplitude and slowed the fall time of [Ca(2+)](i) transients, suggesting SERCA inhibition. Exposure to W-7 completely abolished ongoing ACh-induced [Ca(2+)](i) oscillations in some cells. Preexposure to W-7 or KN-62 did not affect caffeine-induced SR Ca(2+) release, indicating that ryanodine receptor channels were not directly inhibited. These data indicate that, in porcine ASM, the CaM-CaMKII pathway regulates SR Ca(2+) reuptake, potentially through altered PLN phosphorylation.     

Phosphate release and force generation in cardiac myocytes investigated with caged phosphate and caged calcium.

The phosphate (P(i)) dissociation step of the cross-bridge cycle was investigated in skinned rat ventricular myocytes to examine its role in force generation and Ca(2+) regulation in cardiac muscle. Pulse photolysis of caged P(i) (alpha-carboxyl-2-nitrobenzyl phosphate) produced up to 3 mM P(i) within the filament lattice, resulting in an approximately exponential decline in steady-state tension. The apparent rate constant, k (rho i), increased linearly with total P(i) concentration (initial plus photoreleased), giving an apparent second-order rate constant for P(i) binding of 3100 M(-1) s(-1), which is intermediate in value between fast and slow skeletal muscles. A decrease in the level of Ca(2+) activation to 20% of maximum tension reduced k (rho i) by twofold and increased the relative amplitude by threefold, consistent with modulation of P(i) release by Ca2+. A three-state model, with separate but coupled transitions for force generation and P(i) dissociation, and a Ca(2+)-sensitive forward rate constant for force generation, was compatible with the data. There was no evidence for a slow phase of tension decline observed previously in fast skeletal fibers at low Ca(2+), suggesting differences in cooperative mechanisms in cardiac and skeletal muscle. In separate experiments, tension development was initiated from a relaxed state by photolysis of caged Ca(2+). The apparent rate constant, k(Ca), was accelerated in the presence of high P(i) consistent with close coupling between force generation and P(i) dissociation, even when force development was initiated from a relaxed state. k(Ca) was also dependent on the level of Ca(2+) activation. However, significant quantitative differences between k (rho i) and k(Ca), including different sensitivities to Ca(2+) and P(i) indicate that caged Ca(2+) tension transients are influenced by additional Ca(2+)-dependent but P i-independent steps that occur before P(i) release. Data from both types of measurements suggest that kinetic transitions associated with P(i) dissociation are modulated by the Ca(2+) regulatory system and partially limit the physiological rate of tension development in cardiac muscle.     

The effect of phosphate and calcium on force generation in glycerinated rabbit skeletal muscle fibers. A steady-state and transient kinetic study

The effect of varying concentrations of Pi and Ca2+ on isometric force and on the rate of force development in skinned rabbit psoas muscle fibers has been investigated. Steady-state results show that the three parameters that define the force-pCa relation (Po, pK, and n) all vary linearly with log [Pi]. As [Pi] increases, Po and pK decrease while n increases. The kinetics of force generation in isometrically contracting fibers were studied by laser flash photolysis of caged phosphate. The observed rate of the resulting tension transient, kPi, is 23.5 +/- 1.7 s-1 at 10 degrees C, 0.7 mM Pi, and is independent of [Ca2+] over the range pCa 4.5-7.2. By contrast, kTR, the rate of tension redevelopment following a period of isotonic shortening, is sensitive to [Ca2+] and is slower than kPi (kTR = 13.6 +/- 0.2 s-1 at pCa 4.5, 0.7 mM Pi). The results show that [Ca2+] does not directly affect the Pi release or force-generating steps of the cross-bridge cycle and show that the observed rate of force development depends on how the measurement is made. The data can be interpreted in terms of a model in which strong cross-bridges activate the thin filament, this activation being modulated by Ca2+ binding to troponin.     

Agonist-stimulated oscillations and cycling of intracellular free calcium in individual cultured muscle cells

Receptor regulation of [Ca2+]i was monitored in individual BC3H-1 muscle cells with intracellularly trapped fura-2 using digital imaging analysis techniques. Activation of alpha 1-adrenergic or H1-histaminergic receptors resulted in multiple bursts, or oscillations, of elevated [Ca2+]i with an average interval frequency of approximately 1.8 min-1. The duration of oscillatory behavior was generally more prolonged in response to phenylephrine than in response to histamine. Additionally, a larger fraction of the cells responded with [Ca2+]i oscillations to phenylephrine (approximately 90%) than to histamine (approximately 60%), although the majority of cells produced oscillations in response to both agonists. In most cells, the receptor-mediated [Ca2+]i oscillations continued for several minutes in the absence of extracellular Ca2+, although the amplitude of the individual peaks gradually decreased. The activation of [Ca2+]i oscillations by H1-receptors was more dependent upon extracellular Ca2+ than those elicited by alpha 1-receptors, reflecting the greater dependency of the histaminergic response on Ca2+ influx. Readdition of Ca2+ to the incubation buffer resulted in the resumption of the [Ca2+]i oscillations. These results indicate that considerable cycling of Ca2+ between the cytoplasm and the endoplasmic reticulum must occur. Receptor-mediated [Ca2+]i oscillations were much more prevalent in subconfluent cells than in confluent cells, possibly due to increased coupling of the cells at higher densities. The cells were capable of responding independently of one another, since sister cells displayed unique temporal responses immediately following cell division. Thus, the linkage of receptor occupancy to [Ca2+]i elevation is a functionally unique property for each individual cell and can be influenced by epigenetic factors.     

Spontaneous and stimulated transients in cytoplasmic free Ca(2+) in normal human osteoblast-like cells: aspects of their regulation.

We characterize two patterns of transients in cytoplasmic free calcium ([Ca2+]i) in normal human osteoblast-like cells (hOB cells). Firstly, spontaneous oscillations in [Ca2+]i were found to be common. The [Ca2+]i oscillations were completely inhibited by thapsigargin, indicating that Ca2+ fluxes between intracellular Ca2+ pools and the cytosol contributed to the generation of the [Ca2+]i oscillations. Removing extracellular Ca2+ either attenuated or completely inhibited spontaneous [Ca2+]i oscillations. Gadolinium, an inhibitor of stretch activated cation channels (SA-cat channels), reduced the frequency of [Ca2+]i oscillations. Hence, entry of calcium from the extracellular space, possibly through SA-cat channels also seemed to be of importance in the regulation of these [Ca2+]i oscillations. The role of the observed spontaneous [Ca2+]i oscillations in hOB cell function is not clear. Secondly, a decrease in pericellular osmolality, which causes the plasma membrane to stretch, transiently increased [Ca2+]i in hOB cells. This effect was also observed in a Ca2+ free extracellular environment, suggesting that osmotic stimuli release Ca2+ from intracellular pools. This finding indicates a possible signaling pathway by which mechanical strain can promote anabolic effects on the human skeleton.     

Ca(2+)-oscillations and Ca(2+)-waves in mammalian cardiac and vascular smooth muscle cells

In this article, we review briefly the available theories and data on [Ca2+]i-waves and [Ca2+]i-oscillations in mammalian cardiac and vascular smooth muscles. In addition to our review, we also report: (i) the existence and characterization of rapid agonist-induced [Ca2+]i-waves in cultured vascular smooth muscle cells (A7r5 cells); and (ii a new method for studying rapid [Ca2+]i-waves in mammalian cardiac ventricular cells. In mammalian cardiac muscle several types of Ca(2+)-release from sarcoplasmic reticulum (SR) are known to occur and might be involved in Ca(2+)-waves and Ca(2+)-oscillations: (a) Ca(2+)-induced release of Ca2+, of the type thought to be important in normal excitation-contraction coupling; (b) spontaneous, cyclic release of Ca2+ related to a Ca(2+)-overload of the SR; and (c) Ins(1,4,5)P3-induced Ca(2+)-release. The available data support the idea that [Ca2+]i-waves in heart propagate by a mechanism somewhat different than that involved in normal excitation-contraction coupling (a, above), perhaps involving spontaneous release of Ca2+ from an overloaded SR (b, above). In mammalian vascular smooth muscle, our data support the idea that agonist-receptor interaction (vasopressin, in this case) initiates [Ca2+]i-waves that then propagate via some form of Ca(2+)-induced release of Ca2+, perhaps in a manner similar to that proposed by Berridge and Irvine [1].     

Agonist of dihydropyridine receptors, BayK8644 depresses excitation-contraction coupling in myocytes of guinea pig heart.

BayK8644(-)(BayK), an agonist of L-type Ca2+ channels has been recently shown to impair excitation-contraction coupling in cardiac myocytes by increasing Ca2+ leak from the sarcoplasmic reticulum (SR) and by decreasing the gain factor of calcium induced release of calcium. It has been proposed that BayK affects the properties of ryanodine receptors (RyRs) of SR by binding to the sarcolemmal dihydropyridine receptors (DHPRs). This would suggest that the linkage between these receptors is more direct than currently sought. However, it has been recently found that BayK may also directly affect the RyRs increasing their open probability. In this paper we tested the effect of BayK on excitation-contraction coupling in single ventricular myocytes of guinea-pig heart superfused with 5 mM Ni2+ which blocks the L-type Ca2+ current and Na+/Ca2+ exchange. We have previously shown that it is possible to activate in these cells nearly normal Ca2+ transients and contractions despite total inhibition of ICa. This eliminated the effect of ICa increased by BayK on excitation contraction coupling thus simplifying the studied system. 0.5 microM BayK increased the diastolic [Ca2+]i and decreased the diastolic length in stimulated or rested cells superfused with Ni2+, decreased by approximately 50% amplitude of Ca2+ transients and contractions and decreased by approximately 70% the responses of cells to rapid superfusion of 15mM caffeine used as an indirect index of the SR Ca2+ content. The effects on diastolic length and [Ca2+]i in rested cells were not affected by 20 microM nifedipine. We conclude that under our experimental conditions the dominating mechanism of suppression of excitation-contraction coupling by BayK was depletion of the SR Ca2+ by the direct effect on the RyRs.     

Kinetics of cardiac thin-filament activation probed by fluorescence polarization of rhodamine-labeled troponin C in skinned guinea pig trabeculae

A genetically engineered cardiac TnC mutant labeled at Cys-84 with tetramethylrhodamine-5-iodoacetamide dihydroiodide was passively exchanged for the endogenous form in skinned guinea pig trabeculae. The extent of exchange averaged nearly 70%, quantified by protein microarray of individual trabeculae. The uniformity of its distribution was verified by confocal microscopy. Fluorescence polarization, giving probe angle and its dispersion relative to the fiber long axis, was monitored simultaneously with isometric tension. Probe angle reflects underlying cTnC orientation. In steady-state experiments, rigor cross-bridges and Ca2+ with vanadate to inhibit cross-bridge formation produce a similar change in probe orientation as that observed with cycling cross-bridges (no Vi). Changes in probe angle were found at [Ca2+] well below those required to generate tension. Cross-bridges increased the Ca2+ dependence of angle change (cooperativity). Strong cross-bridge formation enhanced Ca2+ sensitivity and was required for full change in probe position. At submaximal [Ca2+], the thin filament regulatory system may act in a coordinated fashion, with the probe orientation of Ca2+-bound cTnC significantly affected by Ca2+ binding at neighboring regulatory units. The time course of the probe angle change and tension after photolytic release [Ca2+] by laser photolysis of NP-EGTA was Ca2+ sensitive and biphasic: a rapid component approximately 10 times faster than that of tension and a slower rate similar to that of tension. The fast component likely represents steps closely associated with Ca2+ binding to site II of cTnC, whereas the slow component may arise from cross-bridge feedback. These results suggest that the thin filament activation rate does not limit the tension time course in cardiac muscle.     

Effects of Na+/Ca2+ exchange induced by SR Ca2+ release on action potentials and afterdepolarizations in guinea pig ventricular myocytes

In cardiac cells, evoked Ca2+ releases or spontaneous Ca2+ waves activate the inward Na+/Ca2+ exchange current (INaCa), which may modulate membrane excitability and arrhythmogenesis. In this study, we examined changes in membrane potential due to INaCa elicited by sarcoplasmic reticulum (SR) Ca2+ release in guinea pig ventricular myocytes using whole cell current clamp, fluorescence, and confocal microscopy. Inhibition of INaCa by Na+-free, Li+-containing Tyrode solution reversibly abbreviated the action potential duration at 90% repolarization (APD90) by 50% and caused SR Ca2+ overload. APD90 was similarly abbreviated in myocytes exposed to the Na+/Ca2+ exchange inhibitor KB-R7943 (5 microM) or after inhibition of SR Ca2+ release with ryanodine (20 microM). In the absence of extracellular Na+, spontaneous SR Ca2+ releases caused minimal changes in resting membrane potential. After the myocytes were returned to Na+-containing solution, the potentiated intracellular Ca2+ concentration ([Ca2+]i) transients dramatically prolonged APD90 and [Ca2+]i oscillations caused delayed and early afterdepolarizations (DADs and EADs). Laser-flash photolysis of caged Ca2+ mimicked the effects of spontaneous [Ca2+]i oscillations, confirming that APD prolongation, DADs, and EADs could be ascribed to intracellular Ca2+ release. These results suggest that Na+/Ca2+ exchange is a major physiological determinant of APD and that INaCa activation by spontaneous SR Ca2+ release/oscillations, depending on the timing, can account for both DADs and EADs during SR Ca2+ overload.     

Correlation between electrical activity and intracellular Ca2+ oscillations in GH3 rat anterior pituitary cells

Simultaneous measurements of electrical activity and intracellular Ca(2+) levels were performed in perforated-patch current-clamped individual GH3 cells. Both in cells showing brief (<100 ms) and long action potentials (APs), we found a good correlation between the averaged intracellular Ca2+ concentration ([Ca2+]i) and AP frequency, but not between the mean [Ca2+]i and AP duration. Nevertheless, the magnitude of spontaneous Ca2+ oscillations was highly dependent on the size and duration of the APs. The decay of the Ca2+ transients was not slowed when the size of the oscillations was varied either spontaneously or after elongation of the AP with the K+ channel blocker tetraethyl ammonium. Furthermore, the recovery from Ca2+ loads similar to those induced by the APs was slightly retarded after treatment of the cells with intracellular store Ca2+-ATPase inhibitors. Among previous results showing that caffeine-induced [Ca2+]i increases are secondary to electrical activity enhancements in GH3 cells, these data indicate that the Ca2+ entry triggered via APs is the primary determinant of the [Ca2+]i variations, and that Ca2+-induced Ca2+ release has a minor contribution to Ca2+ oscillations recorded during spontaneous activity. They also point to modulation of electrical activity patterns as a crucial factor regulating spontaneous [Ca2+]i signalling, and hence pituitary cell functions in response to physiological secretagogues.     

High temporal resolution video imaging of intracellular calcium

We have developed a system for imaging intracellular free calcium ion concentration ([Ca2+]i) at the highest rate possible with conventional video equipment. The system is intended to facilitate quantitative study of rapid changes in [Ca2+]i in cells that move. It utilizes intensified video cameras with nearly ideal properties and digital image processing to produce two images that can be ratioed without artifacts. Two dichroic mirrors direct images of cellular Indo-1 fluorescence at two different wavelengths to two synchronized video cameras, each consisting of a fast micro-channel plate image intensifier optically coupled with a tapered fiber optic bundle to a CCD image sensor. The critical technical issues in this dual-image system are: (1) minimization and correction of the small geometric and other types of differences in the images provided by the two cameras; and (2) the signal-to-noise ratio that can be achieved in single frames. We have used this system to obtain images of [Ca2+]i at 16.7 ms intervals in voltage-clamped single cardiac cells perfused internally with Indo-1 (pentapotassium salt). The images indicate that, except for the nuclear regions, [Ca2+]i is uniform during normal excitation-contraction coupling. In contrast, changes in [Ca2+]i propagate in rapid 'waves' during the spontaneous release of Ca2+ that accompanies certain 'Ca2(+)-overload conditions.'     

Uptake and release of Ca2+ by the endoplasmic reticulum contribute to the oscillations of the cytosolic Ca2+ concentration triggered by Ca2+ influx in the electrically excitable pancreatic B-cell.

The role of intracellular Ca2+ pools in oscillations of the cytosolic Ca2+ concentration ([Ca2+]c) triggered by Ca2+ influx was investigated in mouse pancreatic B-cells. [Ca2+]c oscillations occurring spontaneously during glucose stimulation or repetitively induced by pulses of high K+ (in the presence of diazoxide) were characterized by a descending phase in two components. A rapid decrease in [Ca2+]c coincided with closure of voltage-dependent Ca2+ channels and was followed by a slower phase independent of Ca2+ influx. Blocking the SERCA pump with thapsigargin or cyclopiazonic acid accelerated the rising phase of [Ca2+]c oscillations and increased their amplitude, which suggests that the endoplasmic reticulum (ER) rapidly takes up Ca2+. It also suppressed the slow [Ca2+]c recovery phase, which indicates that this phase corresponds to the slow release of Ca2+ that was taken up by the ER during the upstroke of the [Ca2+]c transient. Glucose promoted the buffering capacity of the ER and amplified the slow [Ca2+]c recovery phase. The slow phase induced by high K+ pulses was not affected by modulators of Ca2+- or inositol 1,4,5-trisphosphate-induced Ca2+ release, did not involve a depolarization-induced Ca2+ release, and was also observed at the end of a rapid rise in [Ca2+]c triggered from caged Ca2+. It is attributed to passive leakage of Ca2+ from the ER. We suggest that the ER displays oscillations of the Ca2+ concentration ([Ca2+]ER) concomitant and parallel to [Ca2+]c. The observation that thapsigargin depolarizes the membrane of B-cells supports the proposal that the degree of Ca2+ filling of the ER modulates the membrane potential. Therefore, [Ca2+]ER oscillations occurring during glucose stimulation are likely to influence the bursting behavior of B-cells and eventually [Ca2+]c oscillations.     

Spontaneous and chemoattractant-induced oscillations of cytosolic free calcium in single adherent human neutrophils

Studies with fluorescent Ca2+ indicators in large populations of neutrophils in suspension reveal a stable base line followed by a rapid agonist-induced elevation of cytosolic free calcium, [Ca2+]i, concomitant with other parameters of cellular activation. To study the role of adhesion in cell activation, we monitored [Ca2+]i in single neutrophils adhered to albumin-coated or fibronectin-coated glass coverslips before and after stimulation with the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Human neutrophils loaded with 2 microM fura 2/AM were allowed to adhere to coverslips for 15-20 min at 37 degrees C. [Ca2+]i was monitored with a dual excitation microfluorimeter with a time resolution of 200 ms. Statistical analysis was performed using an algorithm allowing to detect significant [Ca2+]i peaks. 54% of the cells showed spontaneous [Ca2+]i oscillations. The amplitude of these [Ca2+]i peaks averaged 77 +/- 10 nM above basal levels (mean value of 110 +/- 20 nM), and their mean duration was 28 +/- 5 s; periods of [Ca2+]i bursts could last up to 15 min. In "silent" cells exhibiting a stable [Ca2+]i base line without spontaneous oscillations, low concentrations of fMLP (10(-10)-10(-9) M) could induce sustained [Ca2+]i oscillations. By contrast, higher agonist concentrations (10(-6) M) induced a single [Ca2+]i transient followed by a stable base line. 47% of the cells showing spontaneous [Ca2+]i oscillations did not respond to fMLP. Spontaneous [Ca2+]i oscillations depended on the continuous presence of extracellular Ca2+. Therefore: (i) spontaneous oscillations of [Ca2+]i occur in neutrophils adherent to various substrata; (ii) these oscillations do not preclude and can be dissociated from the response to fMLP; (iii) neutrophil functions might be controlled by [Ca2+]i oscillations rather than by sustained alterations of [Ca2+]i.