Desynchronising effect of the endothelium on intracellular Ca2+ concentration dynamics in vascular smooth muscle cells of rat mesenteric arteries

The kinetics of the intracellular Ca2+ concentration ([Ca2+]i) of vascular smooth muscle cells (VSMCs) in rat small mesenteric arteries was investigated by confocal laser scanning microscopy using the fluorescent Ca2+ indicator fluo-3 AM. One micromole noradrenaline (NA) induced randomly distributed transient elevations of [Ca2+]i in several single VSMCs which were weakly temporally coupled. Higher NA concentrations of 3 or 10 microM, however, induced strongly synchronised [Ca2+]i oscillations in VSMCs. In preparations with intact endothelium, the synchronisation of [Ca2+]i signals was attenuated by acetylcholine (ACh) but augmented by the NO synthase antagonist L-NAME, pointing to a desynchronising effect of the endothelium even under basal conditions. In preparations with or without intact endothelium sodium nitroprusside (SNP) as well as the gap-junction uncoupler heptanol reversibly desynchronised the [Ca2+]i transients. The effect of ACh but not that of SNP was influenced by L-NAME. Propagated intracellular [Ca2+]i waves had a velocity of 25 microm/s. The phase shift of [Ca2+]i oscillations between single VSMCs were maximally 2s and independent of the distance of up to 90 microm between individual cells. Therefore, we consider intercellular [Ca2+]i waves to be too slow to account for the synchronisation of [Ca2+]i oscillations.We conclude that the coupling of [Ca2+]i signals in vascular smooth muscle cells is not constant but highly regulated by NA and by endothelium derived NO. Oscillations of vessel contraction at high sympathetic tone may be induced by synchronisation of [Ca2+]i transients of distinct VSMCs whereas endothelium derived NO inhibits vasomotion by desynchronising [Ca2+]i transients of single VSMCs.     

Visualization of neural control of intracellular Ca2+ concentration in single vascular smooth muscle cells in situ.

The intermittent rise in intracellular Ca2+ concentration ([Ca2+]i oscillation) has been observed in many types of isolated cells, yet it has not been demonstrated whether it plays an essential role during nerve stimulation in situ. We used confocal microscopy to study Ca2+ transients in individual smooth muscle cells in situ within the wall of small arteries stimulated with perivascular sympathetic nerves or noradrenaline. We show here that the sympathetic adrenergic regulation of arterial smooth muscle cells involves the oscillation of [Ca2+]i that propagates within the cell in the form of a wave. Ca2+ release from intracellular stores plays a key role in the oscillation because it is blocked after the store depletion by ryanodine treatment. Ca2+ influx through the plasma membrane sustains the oscillation by replenishing the Ca2+ stores. These results demonstrate the involvement of [Ca2+]i oscillations in the neural regulation of effector cells within the integrated system.     

Propagation of cytoplasmic Ca2+ oscillations in clusters of pancreatic beta-cells exposed to glucose

Digital image analysis was employed for resolving the temporal and spatial variations of the cytoplasmic Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells loaded with the Ca(2+)-indicator Fura-2. Glucose-stimulated individual beta-cells exhibited large amplitude oscillations of [Ca2+]i with a mean frequency of 0.33 min-1. When Ca2+ diffusion was restricted by increasing the Ca2+ buffering capacity, the sugar-induced rise of [Ca2+]i preferentially affected the peripheral cytoplasm. When glucagon was present glucose also caused less prominent oscillations with about a 10-fold higher frequency superimposed on an elevated [Ca2+]i. In small clusters of 6-14 cells the average frequency of the large amplitude oscillations increased to 0.60 min-1. The clusters were found to contain micro-domains of electrically coupled cells with synchronized oscillations. After increasing the glucose concentration, adjacent domains became functionally coupled. The oscillations originated from different cells in the cluster. Also the fast glucagon-dependent oscillations were synchronized between cells and had different origins. The results indicate that coupling of beta-cells leads to an increased frequency of the large amplitude oscillations, and that the oscillatory characteristics are determined collectively among electrically coupled beta-cells rather than by particular pacemaker cells. In the light of these data it is necessary to reconsider the previous ideas that glucose-induced oscillations of membrane potential and [Ca2+]i require coupling between many beta-cells, and that the peak [Ca2+]i values reached during oscillations should increase with the size of the coupled cluster.     

Endothelial coordination of cerebral vasomotion via myoendothelial gap junctions containing connexins 37 and 40

Control of cerebral vasculature differs from that of systemic vessels outside the blood-brain barrier. The hypothesis that the endothelium modulates vasomotion via direct myoendothelial coupling was investigated in a small vessel of the cerebral circulation. In the primary branch of the rat basilar artery, membrane potential, diameter, and calcium dynamics associated with vasomotion were examined using selective inhibitors of endothelial function in intact and endothelium-denuded arteries. Vessel anatomy, protein, and mRNA expression were studied using conventional electron microscopy high-resolution ultrastructural and confocal immunohistochemistry and quantitative PCR. Membrane potential oscillations were present in both endothelial cells and smooth muscle cells (SMCs), and these preceded rhythmical contractions during which adjacent SMC intracellular calcium concentration ([Ca(2+)](i)) waves were synchronized. Endothelium removal abolished vasomotion and desynchronized adjacent smooth muscle cell [Ca(2+)](i) waves. N(G)-nitro-l-arginine methyl ester (10 microM) did not mimic this effect, and dibutyryl cGMP (300 muM) failed to resynchronize [Ca(2+)](i) waves in endothelium-denuded arteries. Combined charybdotoxin and apamin abolished vasomotion and depolarized and constricted vessels, even in absence of endothelium. Separately, (37,43)Gap27 and (40)Gap27 abolished vasomotion. Extensive myoendothelial gap junctions (3 per endothelial cell) composed of connexins 37 and 40 connected the endothelial cell and SMC layers. Synchronized vasomotion in rat basilar artery is endothelium dependent, with [Ca(2+)](i) waves generated within SMCs being coordinated by electrical coupling via myoendothelial gap junctions.     

Synchronized oscillations in cytoplasmic free calcium concentration in confluent bradykinin-stimulated bovine pulmonary artery endothelial cell monolayers

Bradykinin-evoked rises in [Ca2+]i were measured in fura-2-loaded bovine pulmonary artery endothelial cell monolayers by dual wavelength excitation fluorimetry. In monolayers seeded thinly and grown to confluence, bradykinin, in the presence of external Ca2+, evoked a rise in [Ca2+]i composed of an initial peak and subsequent oscillating plateau. In the absence of external Ca2+, bradykinin evoked a rise in [Ca2+]i which then returned to the basal value without oscillating. In monolayers seeded near confluent density, the bradykinin-evoked peak in [Ca2+]i was followed by a steady plateau which showed no oscillation. The addition of the phorbol ester, phorbol 12,13-dibutyrate, to a monolayer during bradykinin-evoked oscillations abolished the oscillations and lowered [Ca2+]i partway back toward the basal level. The addition of the protein kinase C inhibitor, H7, did not abolish oscillatory activity, although the frequency of oscillation was reduced. These results indicate that synchronized oscillatory activity can occur in endothelial cell monolayers. It is suggested that these oscillations are dependent on intercellular coupling developed when the cells are grown to confluence and that the mechanism responsible for generating oscillations in [Ca2+]i requires extracellular Ca2+ and involves protein kinase C.     

Regulation of intracellular Ca2+ oscillation in AR42J cells.

Recordings of [Ca2+]i in single AR42J cells loaded with Fura 2 were used to study regulation of [Ca2+]i oscillation. Continuous stimulation with the cholecystokinin analogue, (t-butyloxycarbonyl-Tyr-(SO3)-norleucine-Gly-Trp-Nle-Asp-2-phenylethyl ester) or carbachol evoked long lasting oscillation in [Ca2+]i. Removal of CCK-JMV-180 after brief stimulation did not abruptly stop the oscillation. Rather, removal of CCK-JMV-180 resulted in time-dependent reduction in amplitude with little change in frequency of oscillation. The patterns of [Ca2+]i oscillation were affected by activation of protein kinase C and protein kinase A. However, down-regulation of protein kinase C activity did not prevent stimulation of [Ca2+]i oscillation. Hence, we conclude that an active protein kinase C pathway is not crucial for [Ca2+]i oscillation in this cell line. Variation in extracellular Ca2+ concentration (Ca2+out) was used to further characterize the oscillation. Reducing Ca2+out to approximately 10 microM resulted in a time dependent inhibition of [Ca2+]i oscillation. Subsequent step increases in Ca2+out up to 2-3 mM resulted in increased amplitude and frequency of oscillation. Further increase in Ca2+out or an increase in plasma membrane permeability to Ca2+, brought about by an increase in pHo, resulted in increased amplitude, decreased frequency, and modified shape of the [Ca2+]i spikes. These observations point to the existence of regulatory mechanisms controlling the duration of Ca2+ release and entry during [Ca2+]i oscillation.     

Role of membrane potential in vasomotion of isolated pressurized rat arteries

Vasomotion, the phenomenon of vessel diameter oscillation, regulates blood flow and resistance. The main parameters implicated in vasomotion are particularly the membrane potential and the cytosolic free calcium in smooth muscle cells. In this study, these parameters were measured in rat perfused-pressurized mesenteric artery segments. The application of norepinephrine (NE) caused rhythmic diameter contractions and membrane potential oscillations (amplitude; 5.3 +/- 0.3 mV, frequency; 0.09 +/- 0.01 Hz). Verapamil (1 microM) abolished this vasomotion. During vasomotion, 10(-5) M ouabain (Na(+)-K(+) ATPase inhibitor) decreased the amplitude of the electrical oscillations but not their frequency (amplitude; 3.7 +/- 0.3 mV, frequency; 0.08 +/- 0.002 Hz). Although a high concentration of ouabain (10(-3) M) (which exhibits non-specific effects) abolished both electrical membrane potential oscillations and vasomotion, we conclude that the Na+-K+ ATPase could not be implicated in the generation of the membrane potential oscillations. We conclude that in rat perfused-pressurized mesenteric artery, the slow wave membrane type of potential oscillation by rhythmically gating voltage-dependent calcium channels, is responsible for the oscillation of intracellular calcium and thus vasomotion.     

Signaling between intracellular Ca2+ stores and depletion-activated Ca2+ channels generates [Ca2+]i oscillations in T lymphocytes

Stimulation through the antigen receptor (TCR) of T lymphocytes triggers cytosolic calcium ([Ca2+]i) oscillations that are critically dependent on Ca2+ entry across the plasma membrane. We have investigated the roles of Ca2+ influx and depletion of intracellular Ca2+ stores in the oscillation mechanism, using single-cell Ca2+ imaging techniques and agents that deplete the stores. Thapsigargin (TG; 5-25 nM), cyclopiazonic acid (CPA; 5-20 microM), and tert- butylhydroquinone (tBHQ; 80-200 microM), inhibitors of endoplasmic reticulum Ca(2+)-ATPases, as well as the Ca2+ ionophore ionomycin (5-40 nM), elicit [Ca2+]i oscillations in human T cells. The oscillation frequency is approximately 5 mHz (for ATPase inhibitors) to approximately 10 mHz (for ionomycin) at 22-24 degrees C. The [Ca2+]i oscillations resemble those evoked by TCR ligation in terms of their shape, amplitude, and an absolute dependence on Ca2+ influx. Ca(2+)- ATPase inhibitors and ionomycin induce oscillations only within a narrow range of drug concentrations that are expected to cause partial depletion of intracellular stores. Ca(2+)-induced Ca2+ release does not appear to be significantly involved, as rapid removal of extracellular Ca2+ elicits the same rate of [Ca2+]i decline during the rising and falling phases of the oscillation cycle. Both transmembrane Ca2+ influx and the content of ionomycin-releasable Ca2+ pools fluctuate in oscillating cells. From these data, we propose a model in which [Ca2+]i oscillations in T cells result from the interaction between intracellular Ca2+ stores and depletion-activated Ca2+ channels in the plasma membrane.     

Spontaneous and agonist-induced calcium oscillations in pituitary gonadotrophs

Basal and receptor-regulated changes in cytoplasmic calcium concentration ([Ca2+]i) were monitored by fluorescence analysis in individual rat pituitary gonadotrophs loaded with the calcium-sensitive dye indo-1. Most gonadotrophs exhibited low amplitude spontaneous oscillations in basal [Ca2+]i that were interspersed by quiescent periods and abolished by removal of extracellular Ca2+ or addition of calcium channel blockers. Such random fluctuations in [Ca2+]i, which reflect the operation of a plasma membrane oscillator, were not coupled to basal gonadotropin secretion. The physiological agonist GnRH induced high amplitude [Ca2+]i oscillations; when a threshold [Ca2+]i level was reached, a cytoplasmic oscillator began to generate extremely regular Ca2+ transients. The time required to reach the threshold [Ca2+]i level was inversely correlated with agonist dose; the frequency, but not the amplitude, of agonist-induced Ca2+ spiking increased with agonist concentration. The duration of the latent period decreased and the frequency of Ca2+ spiking increased with the increase in ambient temperature. At high GnRH concentrations, the calcium transients merged into biphasic responses similar to those observed in cell suspensions at all GnRH concentrations. The presence of spontaneous fluctuations in basal [Ca2+]i did not significantly change the patterns of agonist-induced [Ca2+]i responses. Also, removal of extracellular Ca2+ did not interfere with the frequency or amplitude of Ca2+ spikes, but caused the loss of the plateau phase. Blockade of intracellular Ca(2+)-ATPase pumps by thapsigargin was usually accompanied by a subthreshold increase in [Ca2+]i. In such cells the agonist-induced oscillatory pattern was transformed into the biphasic response. In about 10% of the cells, however, high thapsigargin concentrations induced coarse [Ca2+]i oscillations; subsequent stimulation of such cells with GnRH was ineffective. The cytoplasmic oscillatory and biphasic responses may represent a mechanism for differential activation of Ca(2+)-dependent enzymes and their dependent cellular processes, including hormone secretion. The membrane oscillator is probably responsible for refilling of agonist-sensitive pools during and after agonist stimulation.     

Cytosolic free calcium spiking affected by intracellular pH change

The characteristics underlying cytosolic free calcium oscillation were evaluated by superfused dual wave-length microspectrofluorometry of fura-2-loaded single acinar cells from rat pancreas. Application of a physiological concentration of cholecystokinin octapeptide (CCK) (20 pM) induced a small basal increase in cytosolic free calcium concentration ([Ca2+]i) averaging 34 nM above the prestimulation level (69 nM) with superimposed repetitive Ca2+ spike oscillation. The oscillation amplitude averaged 121 nM above the basal increase in [Ca2+]i and occurred at a frequency of one pulse every 49 s. Although extracellular Ca2+ was required for maintenance of high frequency and amplitude of the spikes with increase in basal [Ca2+]i, the primary source utilized for oscillation was intracellular. The threshold of the peak [Ca2+]i amplitude for causing synchronized and same-sized oscillations was less than 300 nM. The [Ca2+]i oscillation was sensitive to intracellular pH (pHi) change. This is shown by the fact that the large pHi shift toward acidification (delta pHi decrease, 0.95) led to a basal increase in [Ca2+]i to the spike peak level with inhibiting Ca2+ oscillation. The pHi shift toward alkalinization (delta pHi increase, 0.33) led to a basal decrease in [Ca2+]i to the prestimulation level, possibly due to reuptake of Ca2+ into the Ca2+ stores, with inhibiting Ca2+ oscillation. Whereas extracellular pH (pHo) change had only minimal effects on Ca2+ oscillation (and/or Ca2+ release from intracellular stores), the extra-Ca2+ entry process, which was induced by higher concentrations of CCK, was totally inhibited by decreasing pHo from 7.4 to 6.5. Thus the major regulatory sites by which H+ affects Ca2+ oscillation are accessible from the intracellular space.     

Calcium homeostasis in bovine somatotrophs: calcium oscillations and calcium regulation by growth hormone-releasing hormone and somatostatin.

The free intracellular calcium ion concentration ([Ca2+]i) was measured in single cells of a population containing 65-80% somatotrophs, using the fluorescent Ca(2+)-indicator Fura-2 and digital imaging microscopy. Spontaneous oscillations in [Ca2+]i ranging in frequency up to 1.5 oscillations per minute were observed in 30% of somatotrophs. These Ca2+ oscillations were blocked by the Ca2+ channel blocker CoCl2 and were thus proposed to be the result of influx of Ca2+ into the cell, possibly as the result of spontaneous electrical activity. GHRH (10-100 nM) increased [Ca2+]i in 61% of the cells studied, although the amplitude and dynamics of the response varied from cell to cell. Typically [Ca2+]i rose from 170 +/- 26 nM to 321 +/- 44 nM (n = 13) in response to a challenge with 66 nM GHRH. GHRH also increased the frequency of Ca2+ oscillations in a number of cells, and some previously quiescent cells showed Ca2+ oscillations following addition of GHRH. Forskolin, which raises cAMP levels in bovine anterior pituitary cells, also stimulated a sustained rise in [Ca2+]i in 10 out of 14 cells tested. Somatostatin (SS) (10-80 nM) rapidly reduced basal [Ca2+]i, blocked Ca2+ oscillations, and blocked the [Ca2+]i response to GHRH. The Ca2+ channel blocker CoCl2 (4 mM) had similar actions on [Ca2+]i to those of SS. These results suggest that GHRH and SS may regulate GH release by modulating Ca2+ entry into the cell through the cell membrane. The [Ca2+]i oscillations seen in a proportion of the somatotrophs were modulated in frequency by GHRH and SS, and are probably generated by influx of Ca2+ through channels in the cell membrane. Thus GH secretion may be regulated by changes in the mean level of [Ca2+]i, which in turn, may be influenced by the frequency of [Ca2+]i oscillations in bovine somatotrophs.     

Calcium oscillations in endothelial cells

Several different types of endothelial cells are now known to respond to agonist stimulation with oscillations of cytosolic free [Ca2+] ([Ca2+]i). The oscillations can be repetitive [Ca2+]i spikes or sinusoidal-like oscillations according to the type of endothelial cell. Several properties of these oscillations are described including the effect of removal of extracellular Ca2+ and of changes in membrane potential, and the spatial heterogeneity of the oscillations. Results obtained with human umbilical vein endothelial cells are assessed in relation to a model for [Ca2+]i oscillations that involves Ca(2+)-induced Ca2+ release. In some preparations the oscillations are synchronized in neighbouring cells, whereas in other preparations they are not. The degree of synchrony may have functional implications and this is discussed with respect to control of blood flow and transmural permeability. A third functional implication of oscillations, their possible effect on desensitization, is also discussed.     

Intracellular calcium oscillations regulate ciliary beat frequency of airway epithelial cells

The effect of ATP-induced Ca2+ oscillations on ciliary activity was examined in airway epithelial cells by simultaneously measuring the ciliary beat frequency (CBF) and the intracellular Ca2+ concentration ([Ca2+]i) near the base of the cilia. Exposure to extracellular ATP (ATPo) induces a rapid and large increase in both [Ca2+]i and CBF, followed by oscillations in [Ca2+]i and a sustained elevation in CBF. After each Ca2+ oscillation, the [Ca2+]i returned to near basal values. By contrast, the CBF remained elevated during these Ca2+ oscillations, although each Ca2+ oscillation induced small variations in CBF. During Ca2+ oscillations, increases in CBF closely followed the rising phase of increases in [Ca2+]i, but declines in CBF lagged behind declines in [Ca2+]i. Higher frequency Ca2+ oscillations reduced variations in CBF, producing a stable and sustained elevation in CBF. The maximal CBF was induced by Ca2+ oscillations and was 15% greater than the CBF induced by the substantially larger initial [Ca2+]i increase. These data demonstrate that the rate of CBF is not directly dependent on the absolute [Ca2+]i, but is dependent on the differential changes in [Ca2+]i and suggest that CBF in airway epithelial cells is regulated by frequency-modulated Ca2+ signaling.     

Spatial and temporal organization of calcium signalling in hepatocytes.

Treatment of hepatocytes with agonists which act via the second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), results in increases of cytosolic free Ca2+ [( Ca2+]i) which are manifest as a series of discrete [Ca2+]i transients or oscillations. With increasing agonist dose [Ca2+]i oscillation frequency increases and the initial latent period decreases, but the amplitude of the [Ca2+]i oscillations remains constant. Studies of these [Ca2+]i oscillations at the subcellular level have indicated that the [Ca2+]i changes do not occur synchronously throughout the cell, but initiate at a specific subcellular domain, adjacent to a region of the plasma membrane, and then propagate through the cell as a [Ca2+]i wave. For a given ceil, the locus of [Ca2+]i wave initiation is constant for every oscillation in a series and is also identical when the cell is sequentially stimulated with different agonists or when the phospholipase C-linked G protein is activated directly using AIF4-. The kinetics of the [Ca2+]i waves indicate that a Ca(2+)-activated mechanism is involved in propagating the oscillatory [Ca2+]i increases throughout the cell, and the data appear to be most consistent with a process of Ca(2+)-induced Ca2+ release. It is proposed that the ability to propagate [Ca2+]i oscillations into regions of the cell distal to the region in which the signal transduction apparatus is localized could serve an important function in allowing all parts of the cell to respond to the stimulus.     

Ca2+-sparks constitute elementary building blocks for global Ca2+-signals in myocytes of retinal arterioles

Spontaneous Ca2+-events were imaged in myocytes within intact retinal arterioles (diameter <40 microm) freshly isolated from rat eyes. Ca2+-sparks were often observed to spread across the width of these small cells, and could summate to produce prolonged Ca2+-oscillations and contraction. Application of cyclopiazonic acid (20 microM) transiently increased spark frequency and oscillation amplitude, but inhibited both sparks and oscillations within 60s. Both ryanodine (100 microM) and tetracaine (100 microM) reduced the frequency of sparks and oscillations, while tetracaine also reduced oscillation amplitude. None of these interventions affected spark amplitude. Nifedipine, which blocks store filling independently of any action on L-type Ca2+-channels in these cells, reduced the frequency and amplitude of both sparks and oscillations. Removal of external [Ca2+] (1mM EGTA) also reduced the frequency of sparks and oscillations but these reductions were slower in onset than those in the presence of tetracaine or cyclopiazonic acid. Cyclopiazonic acid, nifedipine and low external [Ca2+] all reduced SR loading, as indicated by the amplitude of caffeine evoked Ca2+-transients. This study demonstrates for the first time that spontaneous Ca2+-events in small arterioles of the eye result from activation of ryanodine receptors in the SR and suggests that this activation is not tightly coupled to Ca2+-influx. The data also supports a model in which Ca2+-sparks act as building blocks for more prolonged, global Ca2+-signals.     

Membrane potential and Na(+)-K+ pump activity modulate resting and bradykinin-stimulated changes in cytosolic free calcium in cultured endothelial cells from bovine atria

The effects of membrane potential on resting and bradykinin-stimulated changes in [Ca2+]i were measured in fura-2 loaded cultured endothelial cells from bovine atria by spectrofluorimetry. The basal and bradykinin-stimulated release of endothelium-derived relaxing factor, monitored by bioassay methods, were dependent on extracellular Ca2+. Similarly, the plateau phase of the biphasic [Ca2+]i response to bradykinin stimulation exhibited a dependence on extracellular Ca2+, whereas the initial transient [Ca2+]i peak was refractory to the removal of extracellular Ca2+. The effect of membrane depolarization on the plateau phase of the bradykinin-induced change in [Ca2+]i was determined by varying [K+]o. The resting membrane potential measured under current clamp conditions was positively correlated with the extracellular [K+] (52 mV change/10-fold change in [K+]o). The observed decrease in resting and bradykinin-stimulated changes in [Ca2+]i upon depolarization is consistent with an ion transport mechanism where the influx is linearly related to the electrochemical gradient for Ca2+ entry (Em - ECa). The inhibition of bradykinin-stimulated Ca2+ entry by isotonic K+ was not due to the absence of extracellular Na+ since Li+ substitution did not inhibit the agonist-induced Ca2+ entry. In K(+)-free solutions and in the presence of ouabain, bradykinin evoked synchronized oscillations in [Ca2+]i in confluent endothelial cell monolayers. These [Ca2+]i oscillations between the plateau and resting [Ca2+]i levels were dependent on extracellular Ca2+ and K+ concentrations. Although the mechanism(s) underlying [Ca2+]i oscillations in vascular endothelial cells is unclear, these results suggest a role of the membrane conductance.     

Effects of caffeine, tetracaine, and ryanodine on calcium-dependent oscillations in sheep cardiac Purkinje fibers

Membrane current and tension were measured in voltage-clamped sheep cardiac Purkinje fibers. Elevating the intracellular calcium concentration ([Ca2+]i) results in oscillations of membrane current and tension both at rest and during stimulation. During stimulation, an oscillatory transient inward current and an after contraction follow repolarization. We have examined the effects on the oscillations of changing the extracellular calcium concentration ([Ca2+]o) and of adding various drugs. In agreement with previous work, high concentrations of drugs that affect the sarcoplasmic reticulum, namely caffeine (10-20 mM), tetracaine (1 mM), and ryanodine (10 microM), abolish the oscillations. However, at lower concentrations, these three drugs have different effects on the oscillations. Caffeine (1-2 mM) decreases the oscillation amplitude but increases the frequency. Tetracaine (100-500 microM) has little effect on the magnitude of the oscillations but decreases their frequency. Ryanodine, at all concentrations used (0.1-10 microM), eventually abolishes the oscillations but, in doing so, decreases the magnitude, leaving the frequency unaffected. When [Ca2+]o was changed in order to vary [Ca2+]i, both the frequency and the magnitude of the oscillations always changed in the same direction. This suggests that these three drugs have effects in addition to just changing [Ca2+]i.     

Effect of inositol trisphosphate and calcium on oscillating elevations of intracellular calcium in Xenopus oocytes

Stimulation of many nonexcitable cells by Ca2(+)-mobilizing receptor agonists causes oscillating elevations of the intracellular free Ca2+ concentration ((Ca2+]i), rather than a continuous increase. It has been proposed that the frequency at which [Ca2+]i oscillates determines the biological response. Because the occurrence of [Ca2+] oscillations is observed together with endogenous inositol polyphosphate (InsPs) production or following InsPs application, we injected Xenopus laevis oocytes with InsPs and monitored Ca2(+)-activated Cl- currents as an assay of [Ca2+]i. Microinjection of the poorly metabolizable inositol trisphosphate (InsP3) derivatives inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) and inositol 1,4,5-trisphosphorothioate (Ins(1,4,5) P3S3) induced [Ca2+]i oscillations. The frequency at which [Ca2+]i oscillated increased with the injected dose, indicating that the frequency-generating mechanism lies distal to InsP3 production and that generation of oscillations does not require either oscillation of InsP3 levels or InsP3 metabolism. Injections of high doses of Ins(1,4,5)P3 or Ins(2,4,5)P3 inhibited ongoing oscillations, whereas Ca2+ injections decreased the amplitude of Ins(2,4,5)P3-induced oscillations without altering their frequency. Injections of the Ins(1,4,5)P3 metabolite inositol 1,3,4,5-tetrakisphosphate also caused oscillations whose frequency was related to the injected dose, although inositol tetrakisphosphate injection induced an increase in the cellular level of Ins(1,4,5)P3. The results suggest a multicomponent oscillatory system that includes the InsP3 target as well as a Ca2(+)-sensitive step that modulates amplitude.     

Endothelial cell Ca2+ increases are independent of membrane potential in pressurized rat mesenteric arteries

In rat mesenteric arteries, the ability of ACh to evoke hyperpolarization of smooth muscle cells and consummate dilatation relies on an increase in endothelial cell cytosolic free [Ca2+] and activation of Ca2+-activated K+ channels (KCa). The time course of average and spatially organized rises in endothelial cell [Ca2+]i and concomitant effects on membrane potential were investigated in individual cells of pressurized arteries and isolated sheets of native cells stimulated with ACh. In both cases, ACh stimulated a sustained and oscillating rise in endothelial cell [Ca2+]i. Overall, the oscillations remained asynchronous between cells, yet occasionally localized intercellular coordination became evident. In pressurized arteries, repetitive waves of Ca2+ moved longitudinally across endothelial cells, and depended on Ca2+-store refilling. The rise in endothelial cell Ca2+ was associated with sustained hyperpolarization of endothelial cells in both preparations. This hyperpolarization was also evident when recording from smooth muscle cells in pressurized arteries, and from resting membrane potential, selective inhibition of small-conductance K Ca (SK Ca) with apamin (50 nM) was sufficient to inhibit this response. In the presence of phenylephrine-tone, both apamin and the selective inhibitor of intermediate conductance K Ca (IK Ca) TRAM-34 (1 microM) were required to inhibit the non-nitric oxide-mediated dilatation to ACh. When hyperpolarization of endothelial cells was fully prevented either with inhibitors of K Ca or in KCl (35 mM)-depolarized cells, both the time course and frequency of oscillations in endothelial cell [Ca2+]i to ACh were unaffected. Together, these data show that although a rise in endothelial cell [Ca2+]i stimulates hyperpolarization, depletion of intracellular stores with ACh stimulates Ca2+-influx which is not significantly influenced by the increase in cellular electrochemical gradient for Ca2+ caused by that hyperpolarization.     

Effects of antioxidants on calcium signal induced by cholecystokinin in mouse pancreatic acinar cells

Digital imaging fluorescence microscopy was used to study the effect of two antioxidants, N-acetyl-cysteine (NAC) and glutathione, on the cytosolic free calcium concentration ([Ca2+]i) induced by cholecystokinin-octapeptide (CCK-8) of mouse pancreatic acinar cells. When acinar cells were preincubated with either NAC or glutathione, subsequent stimulation with CCK-8 in the presence of each antioxidant had no significant effect on the typical pattern of [Ca2+]i transient evoked by the gastrointestinal hormone. However, application of NAC to acinar cells pretreated for 60 min with the same antioxidant, strongly blocked the oscillatory pattern initiated by CCK-8, inhibiting both amplitude and frequency of calcium oscillations. By contrast, glutathione had no effect on the oscillatory pattern evoked by CCK-8. The present results allow us to speculate that during [Ca2+]i oscillation there is a production of oxidants that facilitate oscillations by enhancing release of calcium from internal stores.