Serratia marcescens Hemolysin (ShlA) Binds Artificial Membranes and Forms Pores in a Receptor-independent Manner

A non-discharge mutant of Paramecium tetraurelia (nd12-35 degrees C, lacking exocytotic response upon stimulation with the nonpermeable polycationic secretagogue aminoethyldextran, AED), in the pawnA genetic context (d4-500r, lacking ciliary voltage-dependent Ca2+ influx), was shown to lack (45)Ca2+ entry from outside upon AED stimulation. In contrast, cells grown at 25 degrees C behave like the wildtype. To check the functional properties in more detail, fluorochrome-loaded 35 degrees C cells were stimulated, not only with AED (EC(100) = 10(-6) M in wildtype cells), but also with 4-chloro-meta-cresol, (4CmC, 0.5 mM), a permeable activator of ryanodine receptor-type Ca2+ release channels, usually at extracellular [Ca2+] of 50 microM, and eventually with a Ca2+ chelator added. We confirm that pwA-nd12(35 degrees C) cells lack any Ca2+ influx and any exocytosis of trichocysts in response to any stimulus. As we determined by x-ray microanalysis, total calcium content in alveolar sacs (subplasmalemmal stores) known to be mobilized upon exocytosis stimulation in wild-type cells, contain about the same total calcium in 35 degrees C as in 25 degrees C cells, and Ca2+ mobilization from alveoli by AED or 4CmC is also nearly the same. Due to the absence of any AED-induced Ca2+ influx in 35 degrees C cells and normal Ca2+ release from stores found by x-ray microanalysis one can exclude a "CICR"-type mechanism (Ca2+-induced Ca2+ release) and imply that normally a store-operated Ca2+ ("SOC") influx would occur (as in 25 degrees C cells). Furthermore, 35 degrees C cells display a significantly lower basal intracellular [Ca2+], so that any increase upon stimulation may be less expressed or even remain undetected. Under these conditions, any mobilization of Ca2+ from stores cannot compensate for the lack of Ca2+ influx, particularly since normally both components have to cooperate to achieve full exocytotic response. Also striking is our finding that 35 degrees C cells are unable to perform membrane fusion, as analyzed with the Ca2+ ionophore, A23187. These findings were corroborated by cryofixation and freeze-fracture analysis of trichocyst docking sites after AED or 4CmC stimulation, which also revealed no membrane fusion. In sum, in nd12 cells increased culture temperature entails multiple defects, notably insensitivity to any Ca2+ signal, which, moreover, cannot develop properly due to a lower basal [Ca2+] level and the lack of Ca2+ influx, despite normal store activation.     

Simultaneous measurement of Ca2+ release and influx into smooth muscle cells in response to caffeine. A novel approach for calculating the fraction of current carried by calcium

Activation of ryanodine receptors on the sarcoplasmic reticulum of single smooth muscle cells from the stomach muscularis of Bufo marinus by caffeine is accompanied by a rise in cytoplasmic [Ca2+] ([Ca2+]i), and the opening of nonselective cationic plasma membrane channels. To understand how each of these pathways contributes to the rise in [Ca2+]i, one needs to separately monitor Ca2+ entry through them. Such information was obtained from simultaneous measurements of ionic currents and [Ca2+]i by the development of a novel and general method to assess the fraction of current induced by an agonist that is carried by Ca2+. Application of this method to the currents induced in these smooth muscle cells by caffeine revealed that approximately 20% of the current passing through the membrane channels activated following caffeine application is carried by Ca2+. Based on this information we found that while Ca2+ entry through these channels rises slowly, release of Ca2+ from stores, while starting at the same time, is much faster and briefer. Detailed quantitative analysis of the Ca2+ release from stores suggests that it most likely decays due to depletion of Ca2+ in those stores. When caffeine was applied twice to a cell with only a brief (30 s) interval in between, the amount of Ca2+ released from stores was markedly diminished following the second caffeine application whereas the current carried in part by Ca2+ entry across the plasma membrane was not significantly affected. These and other studies described in the preceding paper indicate that activation of the nonselective cation plasma membrane channels in response to caffeine was not caused as a consequence of emptying of internal Ca2+ stores. Rather, it is proposed that caffeine activates these membrane channels either by direct interaction or alternatively by a linkage between ryanodine receptors on the sarcoplasmic reticulum and the nonselective cation channels on the surface membrane.     

Caffeine activates a Ca(2+)-permeable, nonselective cation channel in smooth muscle cells

The effects of caffeine on cytoplasmic [Ca2+] ([Ca2+]i) and plasma membrane currents were studied in single gastric smooth muscle cells dissociated from the toad, Bufo marinus. Experiments were carried out using Fura-2 for measuring [Ca2+]i and tight-seal voltage-clamp techniques for recording membrane currents. When the membrane potential was held at -80 mV, in 15% of the cells studied caffeine increased [Ca2+]i without having any effect on membrane currents. In these cells ryanodine completely abolished any caffeine induced increase in [Ca2+]i. In the other cells caffeine caused both an increase in [Ca2+]i and activation of an 80-pS nonselective cation channel. In this group of cells ryanodine only partially blocked the increase in [Ca2+]i induced by caffeine; moreover, the change in [Ca2+]i that did occur was tightly coupled to the time course and magnitude of the cation current through these channels. In the presence of ryanodine, blockade of the 80-pS channel by GdCl3 or decreasing the driving force for Ca2+ influx through the plasma membrane by holding the membrane potential at +60 mV almost completely blocked the increase in [Ca2+]i induced by caffeine. Thus, the channel activated by caffeine appears to be permeable to Ca2+. Caffeine activated the cation channel even when [Ca2+]i was clamped to below 10 nM when the patch pipette contained 10 mM BAPTA suggesting that caffeine directly activates the channel and that it is not being activated by the increase in Ca2+ that occurs when caffeine is applied to the cell. Corroborating this suggestion were additional results showing that when the membrane was depolarized to activate voltage-gated Ca2+ channels or when Ca2+ was released from carbachol- sensitive internal Ca2+ stores, the 80-pS channel was not activated. Moreover, caffeine was able to activate the channel in the presence of ryanodine at both positive and negative potentials, both conditions preventing release of Ca2+ from stores and the former preventing its influx. In summary, in gastric smooth muscle cells caffeine transiently releases Ca2+ from a ryanodine-sensitive internal store and also increases Ca2+ influx through the plasma membrane by activating an 80- pS cation channel by a mechanism which does not seem to involve an elevation of [Ca2+]i.     

Sub-second quenched-flow/X-ray microanalysis shows rapid Ca2+ mobilization from cortical stores paralleled by Ca2+ influx during synchronous exocytosis in Paramecium cells

Though only actual local free Ca2+ concentrations, [Ca2+], rather than total Ca concentrations, [Ca], govern cellular responses, analysis of total calcium fluxes would be important to fully understand the very complex Ca2+ dynamics during cell stimulation. Using Paramecium cells we analyzed Ca2+ mobilization from cortical stores during synchronous (< or = 80 ms) exocytosis stimulation, by quenched-flow/cryofixation, freeze-substitution (modified for Ca retention) and X-ray microanalysis which registers total calcium concentrations, [Ca]. When the extracellular free calcium concentration, [Ca2+]e, is adjusted to approximately 30 nM, i.e. slightly below the normal free intracellular calcium concentration, [Ca2+]i = 65 nM, exocytosis stimulation causes release of 52% of calcium from stores within 80 ms. At higher extracellular calcium concentration, [Ca2+]e = 500 microM, Ca2+ release is counterbalanced by influx into stores within the first 80 ms, followed by decline of total calcium, [Ca], in stores to 21% of basal values within 1 s. This includes the time required for endocytosis coupling (350 ms), another Ca2+-dependent process. To confirm that Ca2+ mobilization from stores is superimposed by rapid Ca2+ influx and/or uptake into stores, we substituted Sr2+ for Ca2+ in the medium for 500 ms, followed by 80 ms stimulation. This reveals reduced Ca signals, but strong Sr signals in stores. During stimulation, Ca2+ is spilled over preformed exocytosis sites, particularly with increasing extracellular free calcium, [Ca2+]e. Cortically enriched mitochondria rapidly gain Ca signals during stimulation. Balance calculations indicate that total Ca2+ flux largely exceeds values of intracellular free calcium concentrations locally required for exocytosis (as determined previously). Our approach and some of our findings appear relevant also for some other secretory systems.     

Localization and heterogeneity of agonist-induced changes in cytosolic calcium concentration in single bovine adrenal chromaffin cells from video imaging of fura-2.

Temporal and spatial changes in the concentration of cytosolic free calcium ([Ca2+]i) in response to a variety of secretagogues have been examined in adrenal chromaffin cells using digital video imaging of fura-2-loaded cells. Depolarization of the cells with high K+ or challenge with nicotine resulted in a rapid and transient elevation of [Ca2+]i beneath the plasma membrane consistent with Ca2+ entry through channels. This was followed by a late phase in which [Ca2+]i rose within the cell interior. Agonists that act through mobilization of inositol phosphates produced an elevation in [Ca2+]i that was most marked in an internal region of the cell presumed to be the site of IP3-sensitive stores. When the same cells were challenged with nicotine or high K+, to trigger Ca2+ entry through voltage-dependent channels, the rise in [Ca2+]i was most prominent in the same localized region of the cells. These results suggest that Ca2+ entry through voltage-dependent channels results in release of Ca2+ from internal stores and that the bulk of the measured rise in [Ca2+]i is not close to the exocytotic sites on the plasma membrane. Analysis of the time courses of changes in [Ca2+]i in response to bradykinin, angiotensin II and muscarinic agonists showed that these agonists produced highly heterogeneous responses in the cell population. This heterogeneity was most marked with muscarinic agonists which in some cells elicited oscillatory changes in [Ca2+]i. Such heterogeneous changes in [Ca2+]i were relatively ineffective in eliciting catecholamine secretion from chromaffin cells. A single large Ca2+ transient, with a component of the rise in [Ca2+]i occurring beneath the plasma membrane, may be the most potent signal for secretion.     

Mechanisms of Ca2+ store depletion in single endothelial cells in a Ca(2+)-free environment.

Depletion of agonist-sensitive Ca2+ stores results in activation of capacitative Ca2+ entry (CCE) in endothelial cells. The proportion of Ca2+ stores contributing to the regulation of CCE is unknown. In fura-2/am loaded single endothelial cells freshly isolated from bovine left circumflex coronary arteries, we investigated whether a resting period in a Ca(2+)-free environment results in emptying of bradykinin-sensitive Ca2+ stores (BsS) and activation of CCE. In a Ca(2+)-free environment, depletion of BsS occurred in a time-dependent manner (59% after 10 min in Ca(2+)-free solution). This effect was prevented by inhibition of the Na(+)-Ca2+ exchange but not by a blockade of ryanodine-sensitive Ca2+ release (RsCR). In contrast to BsS, mitochondrial Ca2+ content remained unchanged in the Ca(2+)-free environment. Remarkably, activity of CCE (monitored as Mn2+ influx) did not increase after depletion of BsS in the Ca(2+)-free environment. In contrast to Mn2+ influx, the effect of re-addition of Ca2+ to elevate bulk Ca2+ concentration ([Ca2+]b) decreased with the time the cells rested in Ca(2+)-free buffer. This decrease was prevented by an inhibition of RsCR. In low Na+ conditions the effect of Ca2+ on [Ca2+]b was reduced while it did not change the time the cells rested in Ca(2+)-free solution. After a 2 min period in low Na+ conditions, ryanodine-induced Ca2+ extrusion was markedly diminished. Inhibition of RsCR re-established the effect of Ca2+ on [Ca2+]b in low Na+ conditions. Collapsing subplasmalemmal Ca2+ stores with nocodazole, increased the effect of Ca2+ on [Ca2+]b. In nocodazole-treated cells, the effect of Ca2+ on [Ca2+]b was not reduced in Ca(2+)-free environment. These data indicate that activation of CCE is not associated with the agonist-sensitive Ca2+ pools that deplete rapidly in a Ca(2+)-free environment. Subplasmalemmal ryanodine-sensitive Ca2+ stores (RsS) are emptied in Ca(2+)-free/low Na+ solution and re-sequester Ca2+ which enters the cells prior an increase in [Ca2+]b occurs. Thus, in endothelial cells there are differences in the functions of various subplasmalemmal Ca2+ stores (i.e. BsS and RsS), which include either activation of CCE or regulation of subplasmalemmal Ca2+.     

Phase-dependent contributions from Ca2+ entry and Ca2+ release to caffeine-induced [Ca2+]i oscillations in bullfrog sympathetic neurons.

Sympathetic neurons display robust [Ca2+]i oscillations in response to caffeine and mild depolarization. Oscillations occur at constant membrane potential, ruling out voltage-dependent changes in plasma membrane conductance. They are terminated by ryanodine, implicating Ca(2+)-induced Ca2+ release. Ca2+ entry is necessary for sustained oscillatory activity, but its importance varies within the oscillatory cycle: the slow interspike rise in [Ca2+]i requires Ca2+ entry, but the rapid upstroke does not, indicating that it reflects internal Ca2+ release. Sudden alterations in [Ca2+]o, [K+]o, or [caffeine]o produce immediate changes in d[Ca2+]i/dt and provide information about the relative rates of surface membrane Ca2+ transport as well as uptake and release by internal stores. Based on our results, [Ca2+]i oscillations can be explained in terms of coordinated changes in Ca2+ fluxes across surface and store membranes.     

Intracellular calcium stores are involved in growth hormone-releasing hormone signal transduction in rat somatotrophs.

In rat pituitary somatotrophs, the stimulation of growth hormone secretion by growth hormone-releasing hormone (GHRH) is a Ca(2+)-dependent event involving Ca2+ influx. The presence of calcium-induced calcium release (CICR) Ca2+ stores has been suggested in these cells. The aim of our study was to demonstrate the presence of CICR stores in rat somatotrophs and to determine their function in GHRH Ca2+ signalling. To this end we measured cytosolic free Ca2+ concentration ([Ca2+]i), using indo-1 in purified rat somatotrophs in primary culture, while altering intracellular Ca2+ stores. Ionomycin (10 ttM) or 4-bromo-A23187 (10 ItM), used to mobilise organelle-bound Ca2+, raised [Ca2+]i in the absence of extracellular Ca2+. Caffeine (5 to 50 mM), used to mobilise Ca2+ from CICR stores, transiently raised [Ca2+]i in 65% of cells tested. The response to 40 mM caffeine was abolished when Ca2+ stores were depleted, with 1 microM thapsigargin or with 10 microM ryanodine. All cells that responded to 40 mM caffeine responded to 10 nM GHRH. The [Ca2+]i response to 10 nM GHRH was reversible and repeatable. However, the second response was 38% smaller than the first. Ryanodine treatment abolished the reduction in the second [Ca2+]i response, while thapsigargin increased the reduction by 67%. We conclude that rat somatotrophs possess CICR Ca2+ stores and that they account for 30-35% of the GHRH-induced increase in [Ca2+]i, and that their partial depletion is involved in somatotroph desensitization.     

Effect of the sulfhydryl reagent thimerosal on cytosolic free Ca2+ and membrane potential of thymocytes.

The sulfhydryl reagent thimerosal at concentrations 5-100 microM has been found to induce a variety of changes in ion transport in rat thymocytes. In particular, [Ca2+]i increases about 10-fold from the basal level. The [Ca2+]i response to thimerosal displays a two-stage time course, with the main [Ca2+]i rise during the second stage. Evidence has been obtained for the depletion of intracellular Ca2+ pools in thimerosal-treated cells, however, Ca2+ mobilization from intracellular stores does not contribute significantly into [Ca2+]i rise. Thimerosal elicits permeability not only for Ca2+, but also for Mn2+ and Ni2+, which is Ca(2+)-dependent. We failed to get any evidence on thimerosal-induced inhibition of the plasma membrane Ca(2+)-ATPase. The induction of Ca2+ influx, rather than inhibition of Ca(2+)-ATPase, accounts for the disturbance of [Ca2+]i homeostasis in thimerosal-treated cells. Thimerosal also elicits changes in monovalent ion fluxes resulting in marked depolarization. The latter seems unrelated to the changes in [Ca2+]i and is suggested to be mediated both by increased permeability for Na+ and a decreased one for K+. Thimerosal significantly stimulates AA release from thymocytes. Evidence has been presented that AA metabolite(s), probably, LO product(s), may mediate the changes in the transport of mono- and divalent cations elicited by the sulfhydryl reagent. Prolonged treatment of thymocytes with thimerosal resulted in cell death.     

Ca(2+)-dependent K(+) current and exocytosis in responses to caffeine and muscarine in voltage-clamped guinea-pig adrenal chromaffin cells

We characterized changes in membrane currents and the cytosolic Ca(2+) concentration, [Ca(2+)](i), in response to caffeine, and compared them with those in response to muscarine using the perforated patch-clamp technique and fura-2 microfluorimetry in guinea-pig adrenal chromaffin cells. Catecholamine release from single voltage-clamped cells was monitored with amperometry using carbon microelectrodes. Caffeine produced a transient outward current (I(out)) at holding potentials over - 60 mV, increasing in amplitude with increasing the potentials. It also evoked a rapid increase of [Ca(2+)](i) at all potentials examined. The current-voltage relation revealed that the activation of K(+) channels was responsible for the I(out) evoked by caffeine. Both current and [Ca(2+)](i) responses were reversibly abolished by cyclopiazonic acid, an inhibitor of Ca(2+)-pump ATPase. At - 30 mV, the caffeine-induced I(out), but not [Ca(2+)](i), was partly inhibited by either charybdotoxin or apamin. In the majority of cells tested, caffeine induced a larger I(out) but a smaller [Ca(2+)](i) increase than muscarine. Caffeine and muscarine increased catecholamine release from voltage-clamped single cells concomitant with the transient increase of [Ca(2+)](i), and there was a positive correlation between them. These results indicate that caffeine activates Ca(2+)-dependent K(+) channels and catecholamine secretion due to the release of Ca(2+) from internal stores in voltage-clamped adrenal chromaffin cells of the guinea-pig. There seems to be a spatial difference between [Ca(2+)](i) increased by Ca(2+) release from caffeine-sensitive stores and that released from muscarine (inositol 1,4,5-trisphosphate)-sensitive ones.     

My favorite cell--Paramecium

A Paramecium cell has a stereotypically patterned surface, with regularly arranged cilia, dense-core secretory vesicles and subplasmalemmal calcium stores. Less strikingly, there is also a patterning of molecules; for instance, some ion channels are restricted to certain regions of the cell surface. This design may explain very effective and selective responses, such as that to Ca(2+) upon stimulation. It enables the cell to respond to a Ca(2+) signal precisely secretion (exocytosis) or by changing its ciliary activity. These responses depend on the location and/or type of signal, even though these two target structures co-exist side-by-side, and normally only limited overlap occurs between the different functions. Furthermore, the patterning of exocytotic sites and the possibility of synchronous exocytosis induction in the sub-second time range have considerably facilitated analyses, and thus led to new concepts of exocytotic membrane fusion. It has been possible to dissect complicated events like overlapping Ca(2+) fluxes produced from external sources and from internal stores. Since molecular genetic approaches have become available for Paramecium, many different gene products have been identified only some of which are known from "higher" eukaryotes. Although a variety of basic cellular functions are briefly addressed to demonstrate the uniqueness of this unicellular organism, this article focuses on exocytosis regulation.     

Video imaging of cytosolic Ca2+ in pancreatic beta-cells stimulated by glucose, carbachol, and ATP.

In order to define the differences in the distribution of cytosolic free Ca2+ ([Ca2+]i) in pancreatic beta-cells stimulated with the fuel secretagogue glucose or the Ca(2+)-mobilizing agents carbachol and ATP, we applied digital video imaging to beta-cells loaded with fura-2.83% of the cells responded to glucose with an increase in [Ca2+]i after a latency of 117 +/- 24 s (mean +/- S.E., 85 cells). Of these cells, 16% showed slow wave oscillations (frequency 0.35/min). In order to assess the relationship between membrane potential and the distribution of the [Ca2+]i rise, digital image analysis and perforated patch-clamp methods were applied simultaneously. The system used allowed sufficient temporal resolution to visualize a subplasmalemmal Ca2+ transient due to a single glucose-induced action potential. Glucose could also elicit a slow depolarization which did not cause Ca2+ influx until the appearance of the first of a train of action potentials. [Ca2+]i rose progressively during spike firing. Inhibition of Ca2+ influx by EGTA abolished the glucose-induced rise in [Ca2+]i. In contrast, the peak amplitude of the [Ca2+]i response to carbachol was not significantly different in normal or in Ca(2+)-deprived medium. Occasionally, the increase of the [Ca2+]i rise was polarized to one area of the cell different from the subplasmalemmal rise caused by glucose. The amplitude of the response and the number of responding cells were significantly increased when carbachol was applied after the addition of high glucose (11.2 mM). ATP also raised [Ca2+]i and promoted both Ca2+ mobilization and Ca2+ influx. The intracellular distribution of [Ca2+]i was homogeneous during the onset of the response. A polarity in the [Ca2+]i distribution could be detected either in the descending phase of the peak or in subsequent peaks during [Ca2+]i oscillations caused by ATP. In the absence of extracellular Ca2+, the sequential application of ATP and carbachol revealed that carbachol was still able to raise [Ca2+]i after exhaustion of the ATP response. This may be due to desensitization to the former agonist, since the response occurred in the same area of the cell. These results reveal subtle differences in [Ca2+]i distribution following membrane depolarization with glucose or the application of Ca(2+)-mobilizing agonists.     

Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels

An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that most cells exhibited spontaneous, extracellular Ca(2+)-dependent action potentials (APs). Spiking was initiated by a slow pacemaker depolarization from a baseline potential between -75 and -50 mV, and AP frequency increased with membrane depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Characterization of the inward currents in GT1 cells revealed the presence of tetrodotoxin-sensitive Na+, Ni(2+)-sensitive T-type Ca2+, and dihydropyridine-sensitive L-type Ca2+ components. The availability of Na+ and T-type Ca2+ channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in cytosolic Ca2+ concentration ([Ca2+]i), with an early, extracellular Ca(2+)-independent peak and a sustained, extracellular Ca(2+)-dependent phase. During the peak [Ca2+]i response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The GnRH-induced change in firing pattern accounted for about 50% of the elevated Ca2+ influx, the remainder being independent of spiking. Basal [Ca2+]i was also dependent on Ca2+ influx through AP-driven and voltage-insensitive pathways. Thus, in both resting and agonist-stimulated GT1 cells, membrane depolarization limits the participation of Na+ and T-type channels in firing, but facilitates AP-driven Ca2+ influx.     

Advancing age alters rapid and spontaneous refilling of caffeine-sensitive calcium stores in sympathetic superior cervical ganglion cells.

Intracellular calcium concentration ([Ca2+]i) release from smooth endoplasmic reticulum (SER) stores plays an important role in cell signaling. These stores are rapidly refilled via influx through voltage-gated calcium channels or spontaneously via store-operated calcium channels and subsequent pumping by SER Ca2+-ATPases. We measured [Ca2+]i transients in isolated fura 2-loaded superior cervical ganglion cells from 6-, 12-, 20-, and 24-mo-old Fischer 344 rats. For rapid refilling, [Ca2+]i transients were elicited by a 1) 5-s exposure to K+, 2) caffeine to release Ca2+ from SER stores, 3) K+ to refill SER Ca2+ stores, and 4) caffeine. The percent difference between the peak and rate of rise of the first and second caffeine-evoked [Ca2+]i transient significantly declined over the age range of 12-24 mo. To estimate spontaneous refilling, cells were depolarized for 5 s with 68 mM K+ (control), followed by a 10-s exposure to 10 mM caffeine "conditioning stimulus" to deplete [Ca2+]i stores. Caffeine was then rapidly applied for 5 s at defined intervals from 60 to 300 s. Integrated caffeine-evoked [Ca2+]i transients were measured and plotted as a percentage of the K+ response vs. time. The derivative of the refilling time curves significantly declined over the age range from 12-24 mo. Overall, these data suggest that the ability of superior cervical ganglion cells to sustain release of [Ca2+]i following rapid or spontaneous refilling declines with advancing age. Compromised ability to sustain calcium signaling may possibly alter the overall function of adrenergic neurons innervating the cerebrovasculature.     

Effect of shear stress on cytosolic Ca2+ of calf pulmonary artery endothelial cells.

The purpose of the present study was to determine if hemodynamic shear stress increases free cytosolic Ca2+ concentration ([Ca2+]i) of cultured pulmonary artery endothelial cells exposed to steady laminar fluid flow in a parallel plate chamber. Average [Ca2+]i was estimated by measuring cell-associated fura-2 fluorescence using microfluorimetric analysis. To determine [Ca2+]i close to the membrane surface, 86Rb+ efflux via Ca(2+)-dependent K+ channels was measured. Upon initiation of flow or upon step increases in flow, no change in [Ca2+]i was observed using fura-2. However, increases in shear stress produced a large, transient increase in 86Rb+ efflux. The shear stress-dependent increase in 86Rb+ efflux was not blocked by either tetrabutylammonium ions (20 mM) or by charybdotoxin (10 nM), two specific inhibitors of the Ca(2+)-dependent K+ channel of vascular endothelial cells. These results demonstrate that shear stress per se has little effect on either the average cytosolic [Ca2+]i as measured by fura-2 or on [Ca2+]i close to the cytoplasmic surface of the plasmalemma as measured by the activity of Ca(2+)-dependent K+ channels.     

Mobilizing store Ca(2+) in the presence of La(3+) evokes exocytosis in bovine chromaffin cells

The effect on exocytosis of La(3+), a known inhibitor of plasma membrane Ca(2+)-ATPases and Na(+)/Ca(2+) exchangers, was studied using cultured bovine adrenal chromaffin cells. At high concentrations (0.3-3 mM), La(3+) substantially increased histamine-induced catecholamine secretion. This action was mimicked by other lanthanide ions (Nd(3+), Eu(3+), Gd(3+), and Tb(3+)), but not several divalent cations. In the presence of La(3+), the secretory response to histamine became independent of extracellular Ca(2+). La(3+) enhanced secretion evoked by other agents that mobilize intracellular Ca(2+) stores (angiotensin II, bradykinin, caffeine, and thapsigargin), but not that due to passive depolarization with 20 mM K(+). La(3+) still enhanced histamine-induced secretion in the presence of the nonselective inhibitors of Ca(2+)-permeant channels SKF96365 and Cd(2+), but the enhancement was abolished by prior depletion of intracellular Ca(2+) stores with thapsigargin. La(3+) inhibited (45)Ca(2+) efflux from preloaded chromaffin cells in the presence or absence of Na(+). It also enhanced and prolonged the rise in cytosolic [Ca(2+)] measured with fura-2 during mobilization of intracellular Ca(2+) stores with histamine in Ca(2+)-free buffer. The results suggest that the efficacy of intracellular Ca(2+) stores in evoking exocytosis is enhanced dramatically by inhibiting Ca(2+) efflux from the cell.     

Calcium-dependent inactivation of L-type calcium channels in planar lipid bilayers.

Intracellular Ca2+ can inhibit the activity of voltage-gated Ca channels by modulating the rate of channel inactivation. Ca(2+)-dependent inactivation of these channels may be a common negative feedback process important for regulating Ca2+ entry under physiological and pathological conditions. This article demonstrates that the inactivation of cardiac L-type Ca channels, reconstituted into planar lipid bilayers and studied in the presence of a dihydropyridine agonist, is sensitive to Ca2+. The rates and extents of inactivation, determined from ensemble averages of unitary Ba2+ currents, decreased when the calcium concentration facing the intracellular surface of the channel ([Ca2+]i) was lowered from approximately 10 microM to 20 nM by the addition of Ca2+ chelators. The rates and extents of Ba2+ current inactivation could also be increased by subsequent addition of Ca2+ raising the [Ca2+]i to 15 microM, thus demonstrating that the Ca2+ dependence of inactivation could be reversibly regulated by changes in [Ca2+]i. In addition, reconstituted Ca channels inactivated more quickly when the inward current was carried by Ca2+ than when it was carried by Ba2+, suggesting that local increases in [Ca2+]i could activate Ca(2+)-dependent inactivation. These data support models in which Ca2+ binds to the channel itself or to closely associated regulatory proteins to control the rate of channel inactivation, and are inconsistent with purely enzymatic models for channel inactivation.     

Ryanodine Inhibits Caffeine-Evoked Ca2+ Mobilization and Catecholamine Secretion from Cultured Bovine Adrenal Chromaffin Cells

The effects of ryanodine, a selective inhibitor of the Ca(2+)-induced Ca2+ release mechanism, on caffeine-evoked changes in cytosolic Ca2+ concentration ([Ca2+]i) and catecholamine secretion were investigated using cultured bovine adrenal chromaffin cells. Caffeine (5-40 mM) caused a concentration-dependent transient rise in [Ca2+]i and catecholamine secretion in Ca2+/Mg(2+)-free medium containing 0.2 mM EGTA. Ryanodine (5 x 10(-5) M) alone had no effect on either [Ca2+]i or catecholamine secretion. Although the application of ryanodine plus caffeine caused the same increase in both [Ca2+]i and catecholamine secretion as those induced by caffeine alone, ryanodine (4 x 10(-7) - 5 x 10(-5) M) irreversibly prevented the increase in both [Ca2+]i and catecholamine secretion resulting from subsequent caffeine application over a range of concentrations. The secretory response to caffeine was markedly enhanced by replacement of Na+ with sucrose in Ca2+/Mg(2+)-free medium, and this enhanced response was also blocked by ryanodine. Caffeine was found to decrease the susceptibility of the secretory apparatus to Ca2+ in digitonin-permeabilized cells. These results indicate that caffeine mobilizes Ca2+ from intracellular stores, the function of which is irreversibly blocked by ryanodine, resulting in the increase in catecholamine secretion in the bovine adrenal chromaffin cell.     

Regulatory mechanisms of the acrosome reaction revealed by multiview microscopy of single starfish sperm

The acrosome reaction in many animals is a coupled reaction involving an exocytotic step and a dramatic change in cell shape. It has been proposed that these morphological changes are regulated by intracellular ions such as Ca2+ and H+. We report here simultaneous visualization, under a multiview microscope, of intracellular free Ca2+ concentration ([Ca2+]i), intracellular pH (pHi), and morphological changes in a single starfish sperm (Asterina pectinifera). [Ca2+]i and pHi were monitored with the fluorescent probes indo-1 and SNARF-1, respectively. The acrosome reaction was induced with ionomycin. After the introduction of ionomycin in the medium, [Ca2+]i increased gradually and reached a plateau in approximately 30 s. The fusion of the acrosomal vacuole took place abruptly before the plateau, during the rising phase. Although the speed of the [Ca2+]i increase varied among the many sperm tested, exocytosis in all cases occurred at the same [Ca2+]i of approximately 2 microM (estimated using the dissociation constant of indo-1 for Ca2+ of 1.1 microM). This result suggests that the exocytotic mechanism in starfish sperm responds to [Ca2+]i rapidly, with a reaction time of the order of one second or less. Unlike the change in [Ca2+]i, an abrupt increase in pHi was observed immediately after exocytosis, suggesting the presence of a proton mobilizing system that is triggered by exocytosis. The rapid increase in pHi coincided with the formation of the acrosomal rod and the beginning of vigorous movement of the flagellum, both of which have been proposed to be pHi dependent. The exocytotic event itself was visualized with the fluorescent membrane probe RH292. The membrane of the acrosomal vacuole, concealed from the external medium in an unreacted sperm, was seen to fuse with the plasma membrane.     

Role of Ca(2+)-ATPase in spontaneous oscillations of cytosolic free Ca2+ in GH3 rat pituitary cells.

The relative contribution of voltage-sensitive Ca2+ channels, Ca(2+)-ATPases, and Ca2+ release from intracellular stores to spontaneous oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) observed in secretory cells is not well characterized owing to a lack of specific inhibitors for a novel thapsigargin (Tg)-insensitive Ca(2+)-ATPase expressed in these cells. We show that spontaneous [Ca2+]i oscillations in GH3 cells were unaffected by Ca2+ depletion in inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores by the treatment of Tg, but could be initiated by application of caffeine. Moreover, we demonstrate for the first time that these spontaneous [Ca2+]i oscillations were highly temperature dependent. Decreasing the temperature from 22 to 17 degrees C resulted in an increase in the frequency, a reduction in the amplitude, and large inhibition of [Ca2+]i oscillations. Furthermore, the rate of ATP-dependent 45Ca2+ uptake into GH3-derived microsomes was greatly reduced at 17 degrees C. The effect of decreased temperatures on extracellular Ca2+ influx was minor because the frequency and amplitude of spontaneous action potentials, which activate L-type Ca2+ channels, was relatively unchanged at 17 degrees C. These results suggest that in GH3 secretory cells, Ca2+ influx via L-type Ca2+ channels initiates spontaneous [Ca2+]i oscillations, which are then maintained by the combined activity of Ca(2+)-ATPase and Ca(2+)-induced Ca2+ release from Tg/IP3-insensitive intracellular stores.