Autocrine action and its underlying mechanism of nitric oxide on intracellular Ca2+ homeostasis in vascular endothelial cells

The rise in cytosolic Ca(2+) concentration (Ca(2+)(i)) in vascular endothelial cells (ECs) activates the production and release of nitric oxide (NO). NO modifies Ca(2+)(i) homeostasis in many types of nonendothelial cells. However, its effect on endothelial Ca(2+)(i) homeostasis at basal and excited states remains unclear. In the present study, to elucidate the effect of NO on basal Ca(2+)(i), inositol 1,4,5-trisphosphate-induced Ca(2+)(i) release (IICR) was blocked by expressing an antisense against type-1 inositol 1,4,5-trisphosphate receptors or by microinjecting heparin to individual ECs, and the effects of NO that was released by and diffused from adjacent IICR-intact ECs were recorded. After ATP or bradykinin stimulation, IICR-inhibited ECs showed a marked reduction of basal Ca(2+)(i), which was abolished by N(G)-monomethyl-l-arginine monoacetate pretreatment. The reduction disappeared in sparsely seeded ECs. Exogenous NO gas mimicked the effect of ATP or bradykinin to reduce basal Ca(2+)(i). Blocking plasma membrane Ca(2+)-ATPase (PMCA), but not Na(+)-Ca(2+) exchange or sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase, suppressed the reduction, indicating that the reduction resulted from a NO-dependent potentiation of PMCA. To elucidate the effect of NO on elevated Ca(2+)(i), ATP-, bradykinin-, or thapsigargin-evoked Ca(2+)(i) response in the presence and absence of NO production was compared in adjacent IICR-intact ECs. NO was found to potentiate PMCA, which, in turn, greatly attenuated agonist-evoked Ca(2+)(i) elevation. NO also potentiated Ca(2+) influx, which markedly increased the sustained phase of Ca(2+)(i) elevation and possibly NO production. NO did not affect other Ca(2+)(i)-elevating and Ca(2+)(i)-sequestrating components. Thus, NO-dependent potentiation of PMCA is crucial for Ca(2+)(i) homeostasis over a wide Ca(2+)(i) range.     

Ca2+ cycling in heart cells from ground squirrels: adaptive strategies for intracellular Ca2+ homeostasis

Heart tissues from hibernating mammals, such as ground squirrels, are able to endure hypothermia, hypoxia and other extreme insulting factors that are fatal for human and nonhibernating mammals. This study was designed to understand adaptive mechanisms involved in intracellular Ca(2+) homeostasis in cardiomyocytes from the mammalian hibernator, ground squirrel, compared to rat. Electrophysiological and confocal imaging experiments showed that the voltage-dependence of L-type Ca(2+) current (I(Ca)) was shifted to higher potentials in ventricular myocytes from ground squirrels vs. rats. The elevated threshold of I(Ca) did not compromise the Ca(2+)-induced Ca(2+) release, because a higher depolarization rate and a longer duration of action potential compensated the voltage shift of I(Ca). Both the caffeine-sensitive and caffeine-resistant components of cytosolic Ca(2+) removal were more rapid in ground squirrels. Ca(2+) sparks in ground squirrels exhibited larger amplitude/size and much lower frequency than in rats. Due to the high I(Ca) threshold, low SR Ca(2+) leak and rapid cytosolic Ca(2+) clearance, heart cells from ground squirrels exhibited better capability in maintaining intracellular Ca(2+) homeostasis than those from rats and other nonhibernating mammals. These findings not only reveal adaptive mechanisms of hibernation, but also provide novel strategies against Ca(2+) overload-related heart diseases.     

Effect of protein S-glutathionylation on Ca2+ homeostasis in cultured aortic endothelial cells

Diamide is a membrane-permeable, thiol-oxidizing agent that rapidly and reversibly oxidizes glutathione to GSSG and promotes formation of protein-glutathione mixed disulfides. In the present study, the acute effect of diamide on free cytosolic Ca2+ concentration ([Ca2+]i) was examined in fura-2-loaded bovine aortic endothelial cells. At low concentrations (50, 100 μM), diamide reversibly increased spontaneous, asynchronous Ca2+ oscillations, whereas, at higher concentrations (250, 500 μM), diamide caused an immediate synchronized Ca2+ oscillation in essentially all cells of the monolayer, followed by a time-dependent rise in basal [Ca2+]i. The effects of diamide on [Ca2+]i dynamics were independent of extracellular Ca2+. Inhibition of phospholipase C by U-73122 prevented the observed changes in [Ca2+]i. Additionally, the diamide-induced oscillations, but not the rise in basal [Ca2+]i, were blocked by inhibition of the inositol-1,4,5-trisphosphate (IP3) receptor (IP3R) by 2-aminoethyl diphenyl borate. However, diamide failed to alter the plasmalemmal distribution of a green fluorescent protein-tagged phosphatidylinositol-4,5-bisphosphate binding protein, demonstrating that diamide does not activate phospholipase C. Inhibition of glutathione reductase by N,N'-bis(2-chloroethyl)-N-nitrosourea or depletion of glutathione by l-buthionine-sulfoximine enhanced the effects of diamide, which, under these conditions, could only be reversed by addition of dithiothreitol to the wash buffer. Biochemical assays showed that both the IP3R and the plasmalemmal Ca2+-ATPase pump could be reversibly glutathionylated in response to diamide. These results demonstrate that diamide promotes Ca2+ release from IP3-sensitive internal Ca2+ stores and elevates basal [Ca2+]i in the absence of extracellular Ca2+, effects that may be related to a diamide-induced glutathionylation of the IP3R and the plasmalemmal Ca2+-ATPase Ca2+ pump, respectively.     

FK506 blocks intracellular Ca2+ oscillations in bovine adrenal glomerulosa cells

The inositol 1,4,5-trisphosphate (InsP(3)) receptor is a ligand-gated Ca(2+) channel playing an important role in the control of intracellular Ca(2+). In the study presented here, we demonstrate that angiotensin (AngII), phorbol ester (PMA), and FK506 significantly increase the level of InsP(3) receptor phosphorylation in intact bovine adrenal glomerulosa cells. With a back-phosphorylation approach, we showed that the InsP(3) receptor is a good substrate for protein kinase C (PKC) and that FK506 increases the level of PKC-mediated InsP(3) receptor phosphorylation. With a microsomal preparation from bovine adrenal cortex, we showed that PKC enhances the release of Ca(2+) induced by a submaximal dose of InsP(3). We also showed that FK506 blocks intracellular Ca(2+) oscillations in isolated adrenal glomerulosa cells by progressively increasing the intracellular Ca(2+) concentration to a high plateau level. This effect is consistent with an inhibitory role of FK506 on calcineurin dephosphorylation of the InsP(3) receptor, thus keeping the receptor in a phosphorylated, high-conductance state. Our results provide further evidence for the crucial role of the InsP(3) receptor in the regulation of intracellular Ca(2+) oscillations and show that FK506, by maintaining the phosphorylated state of the InsP(3) receptor, causes important changes in the Ca(2+) oscillatory process.     

Mitochondrial Ca2+ homeostasis in intact cells

Ca2+ is a key regulator not only of multiple cytosolic enzymes, but also of a variety of metabolic pathways occurring within the lumen of intracellular organelles. Until recently, no technique to selectively monitor the Ca2+ concentration within defined cellular compartments was available. We have recently proposed the use of molecularly engineered Ca(2+)-sensitive photoproteins to obtain such a result and demonstrated the application of this methodology to the study of mitochondrial and nuclear Ca2+ dynamics. We here describe in more detail the use of chimeric recombinant aequorin targeted to the mitochondria. The technique can be applied with equivalent results to different cell models, transiently or permanently transfected. In all the cell types we analyzed, mitochondrial Ca2+ concentration ([Ca2+]m) increases rapidly and transiently upon stimulation with agonists coupled to InsP3 generation. We confirm that the high speed of mitochondrial Ca2+ accumulation with this type of stimuli depends on the generation of local gradients of Ca2+ in the cytosol, close to the channels sensitive to InsP3. In fact, only activation of these channels, but not the simple release from internal stores, as that elicited by blocking the intracellular Ca2+ ATPases, results in a fast mitochondrial Ca2+ accumulation. We also provide evidence in favor of a microheterogeneity among mitochondria of the same cells, about 30% of them apparently sensing the microdomains of high cytosolic Ca2+ concentration ([Ca2+]c). The changes in [Ca2+]m appear sufficiently large to induce a rapid activation of mitochondrial dehydrogenases, which can be followed by monitoring the level of NAD(P)H fluorescence. A general scheme can thus be envisaged by which the triggering of a plasma membrane receptor coupled to InsP3 generation raises the Ca2+ concentration both in the cytoplasm (thereby triggering energy-consuming processes, such as cell proliferation, motility, secretion, etc.) and in the mitochondria, where it activates the metabolic activity according to the increased cell needs.     

Sphingosine 1-phosphate receptors modulate intracellular Ca2+ homeostasis

Ligation of sphingosine 1-phosphate (S1P) to a set of specific receptors named S1P receptors (S1PRs) regulates important biological processes. Although the ability of S1P to increase cytosolic Ca2+ in various cell types is well known, the role of the individual S1PRs has not been fully characterized. Here, we provide a complete analysis of S1P-dependent intracellular Ca2+ homeostasis in HeLa cells. Overexpression of S1P2, or S1P3, but not S1P1, leads to a significant increase in cytosolic and mitochondrial [Ca2+] in response to S1P challenge. Moreover, cells ectopically expressing S1P2, or S1P3 exhibited an appreciable decrease of the free Ca2+ concentration in the endoplasmic reticulum, dependent on stimulation of receptors by S1P endogenously present in the culture medium which was accompanied by a reduced susceptibility to C2-ceramide-induced cell death. These results demonstrate a differential contribution of individual S1PRs to Ca2+ homeostasis and its possible implication in the regulation of cell survival.     

A patch-clamp study of the Ca2+ mobilization from internal stores in bovine aortic endothelial cells. I. Effects of caffeine on intracellular Ca2+ stores

Summary The effects of agents known to interfere with Ca²⁺ release processes of endoplasmic reticulum were investigated in bradykinin (BK)-stimulated bovine aortic endothelial cells (BAE cells), via the activation of Ca²⁺-activated potassium channels [K(Ca²⁺) channels]. In cell-attached patch experiments, the external application of caffeine (1 mm) caused a brief activation of K(Ca²⁺) channels in Ca²⁺-free and Ca²⁺-containing external solutions. The application of BK (10 nm) during cell stimulation by caffeine (1–20 mm) invariably led to a drastic channel activation which was maintained during a recording period longer than that observed in caffeine-free conditions. In addition, the cell exposure to caffeine (20 mm) during the BK stimulation enhanced systematically the channel activation process. Since a rapid inhibition of BK-evoked channel activity was also produced by removing caffeine from the bath medium, it is proposed that the sustained single-channel response recorded in the concomittant presence of both agents was due to their synergic action on internal stores and/or the external Ca²⁺ entry pathway resulting in an increased [Ca²⁺]_i. In addition, the local anesthetic, procaine, depressed the initial BK-induced K(Ca²⁺) channel activity and completely blocked the secondary phase of the channel activation process related to the external Ca²⁺ influx into stimulated cells. In contrast, this blocking effect of procaine was not observed on the initial caffeine-elicited channel activity and could not suppress the external Ca²⁺-dependent phase of this channel activation process. Our results confirm the existence of at least two pharmacologically distinct types of Ca²⁺-release from internal stores in BAE cells: an inositol 1,4,5-triphosphate (InsP₃)-dependent and a caffeine-induced Ca²⁺-release process.The authors would like to thank Dr. A. Diarra for his contribution to the fluorescence measurements and Diane Vallerand for preparing cell cultures. These data were presented in part at the 14th Scientific Meeting of the International Society of Hypertension (Madrid, Spain, June 14–18, 1992), and have been published in abstract form in the Journal of Hypertension (1992). Dominique Thuringer is a fellow of the Heart and Stroke Foundation of Canada. Rémy Sauvé is a senior fellow from the Fonds de la Recherche en Santé du Québec. This work was supported by a grant from the Medical Research Council of Canada.     

A patch-clamp study of the Ca2+ mobilization from internal stores in bovine aortic endothelial cells. II. Effects of thapsigargin on the cellular Ca2+ homeostasis

Summary Evidence was provided, in the preceding paper (Thuringer & Sauvé, 1992), that the external Ca²⁺-dependent phase of the Ca²⁺ signals evoked by bradykinin (BK) or caffeine in bovine aortic endothelial cells (BAE), differ in their respective sensitivity to procaine. To examine whether the emptying of the InsP₃-sensitive Ca²⁺ store is the signal for activating the agonist-evoked Ca²⁺ entry, we have investigated the effects of thapsigargin (TSG), a known inhibitor of the microsomal Ca²⁺-ATPase activity in a variety of cell types, via the activity of calcium-activated potassium channels [K(Ca²⁺) channels]. In cell-attached experiments, the external application of TSG caused a sustained or oscillatory activation of K(Ca²⁺) channels depending on both the cells and doses tested. The TSG-evoked channel activity could be reversibly blocked by removing extracellular Ca²⁺, and strongly decreased by adding 10 mm procaine to the bath medium. In Ca²⁺-free external conditions, TSG did not promote an apparent Ca²⁺ discharge from internal stores but prevented in a dose- and timedependent manner the subsequent agonist-evoked channel activity related to the release of internally sequestered Ca²⁺. These results confirm that TSG and BK release Ca²⁺ from the same internal stores but with different kinetics. Because the channel response to caffeine was found to be poorly sensitive to procaine, in contrast to that evoked by BK and TSG, it may be concluded that both BK and TSG activate the same Ca²⁺ entry pathway. Therefore, the emptying of the InsP₃-sensitive Ca²⁺ store is likely to be the main signal for activating the agonist-evoked Ca²⁺ entry in BAE cells.The authors wish to thank Diane Vallerand for preparing cell cultures. These data were presented in part at the 14th Scientific Meeting of the International Society of Hypertension (Madrid, Spain, June 14–18, 1992), and have been published in abstract form in the Journal of Hypertension (1992). Dominique Thuringer is a fellow of the Heart and Stroke Foundation of Canada. Rémy Sauvé is a senior fellow from the Fonds de la Recherche en Santé du Québec. This work was supported by a grant from the Medical Research Council of Canada.     

Cytotoxic hyperthermia and Ca2+ homeostasis: the effect of heat on Ca2+ uptake by nonmitochondrial intracellular Ca2+ stores

The effect of cytotoxic hyperthermia on Ca2+ transport by intracellular, nonmitochondrial Ca2+ stores of the human colon cancer cell line, HT-29, was studied using cells permeabilized with saponin. Saponin treatment permitted equilibration of the cytosol with a defined extracellular medium consisting of an intracellular-like ionic composition, ATP and an ATP-regenerating system, and Ca2+/EGTA buffers to adjust the free [Ca2+]. Under the conditions employed, ATP-dependent Ca2+ uptake in saponin-permeabilized cells was demonstrated to be exclusively due to nonmitochondrial Ca2+ stores, e.g., endoplasmic reticulum or calciosomes. Heat treatment for 120 min at 44.5 degrees C sufficient to kill 80% of the cells inhibited ATP-dependent Ca2+ uptake by 50% in terms of rate and total Ca2+ accumulated. With cells made thermotolerant by either arsenite or heat treatment 24 h prior to challenge heating, ATP-dependent Ca2+ uptake was resistant to a second equivalent heat dose. Efflux of Ca2+ from saponin-permeabilized cells when measured at 37 degrees C was unaffected by a prior heat treatment (44.5 degrees C for 120 min).     

Early effects of metabolic inhibition on intracellular Ca2+ in toad pacemaker cells: involvement of Ca2+ stores

The early effects of metabolic inhibition on intracellular Ca(2+) concentration ([Ca(2+)](i)), Ca(2+) current, and sarcoplasmic reticulum (SR) Ca(2+) content were studied in single pacemaker cells from the sinus venosus of the cane toad. The amplitude of the spontaneous elevations of systolic [Ca(2+)](i) (Ca(2+) transients) was reduced after 5-min exposure to 2 mM NaCN from 338 +/- 30 to 189 +/- 37 nM (P < 0.005, n = 9), and the spontaneous firing rate was reduced from 27 +/- 2 to 12 +/- 4 beats/min (P < 0.002, n = 9). It has been proposed that CN(-) acts by inhibition of cytochrome P-450, resulting in a reduction of cAMP and Ca(2+) current. To test this proposal, we used clotrimazole, a cytochrome P-450 inhibitor, which also decreased the Ca(2+) transients and firing rate. CN(-) caused an insignificant fall of Ca(2+) current (23 +/- 11%) but a substantial reduction of SR Ca(2+) content (by 65 +/- 5%), whereas clotrimazole produced a larger reduction of Ca(2+) current and did not affect the SR Ca(2+) content. Thus the main effect of CN(-) does not seem to be through inhibition of cytochrome P-450. In conclusion, CN(-) appears to reduce Ca(2+) release from the SR mainly by reducing SR Ca(2+) content. A likely cause of the decreased SR content is reduced Ca(2+) uptake by the SR pump.     

Oxidative-induced membrane damage in diabetes lymphocytes: Effects on intracellular Ca2 + homeostasis

Oxidative stress is linked to several human diseases, including diabetes. However, the intracellular signal transduction pathways regulated by reactive oxygen species (ROS) remain to be established. Deleterious effects of ROS stem from interactions with various ion transport proteins such as ion channels and pumps, primarily altering Ca^{2 +} homeostasis and inducing cell dysfunction. This study characterized the Ca^{2 +} transport system in lymphocytes of patients with type-2 diabetes, evaluating the possible correlation between cell modifications and the existence of specific oxidative stress damage. Lymphocytes from type-2 diabetes patients displayed oxidative stress features (accumulation of some ROS species, membrane peroxidation, increase in protein carbonyls, increase in SOD and Catalase activity) and Ca^{2 +} dyshomeostasis (modified voltage-dependent and inositol 1,4,5-triphosphate-mediated Ca^{2 +} channel activities, decrease in Ca^{2 +} pumps activity). The data support a correlation between oxidative damage and alterations in intracellular Ca^{2 +} homeostasis, possibly due to modification of the ionic control in lymphocytes of type-2 diabetes patients.     

RNA--induced silencing of the plasma membrane Ca2+-ATPase 2 in neuronal cells: effects on Ca2+ homeostasis and cell viability

Intraneuronal calcium ([Ca(2+)](i)) regulation is altered in aging brain, possibly because of the changes in critical Ca(2+) transporters. We previously reported that the levels of the plasma membrane Ca(2+)-ATPase (PMCA) and the V(max) for enzyme activity are significantly reduced in synaptic membranes in aging rat brain. The goal of these studies was to use RNA(i) techniques to suppress expression of a major neuronal isoform, PMCA2, in neurons in culture to determine the potential functional consequences of a decrease in PMCA activity. Embryonic rat brain neurons and SH-SY5Y neuroblastoma cells were transfected with in vitro--transcribed short interfering RNA or a short hairpin RNA expressing vector, respectively, leading to 80% suppression of PMCA2 expression within 48 h. Fluorescence ratio imaging of free [Ca(2+)](i) revealed that primary neurons with reduced PMCA2 expression had higher basal [Ca(2+)](i), slower recovery from KCl-induced Ca(2+) transients, and incomplete return to pre-stimulation Ca(2+) levels. Primary neurons and SH-SY5Y cells with PMCA2 suppression both exhibited significantly greater vulnerability to the toxicity of various stresses. Our results indicate that a loss of PMCA such as occurs in aging brain likely leads to subtle disruptions in normal Ca(2+) signaling and enhanced susceptibility to stresses that can alter the regulation of Ca(2+) homeostasis.     

Acute effects of testosterone on intracellular Ca2+ kinetics in rat coronary endothelial cells are exerted via aromatization to estrogens

The objective of this work was to evaluate the effects of testosterone (T) and 17beta-estradiol (E(2)) on coronary microvascular endothelial cells (CMECs) of male and female rats. To analyze the short-term effects of such sex steroid hormones on intracellular Ca(2+) concentration ([Ca(2+)](i)) kinetics, we used the chelating agent fura-2 acetoxymethyl ester. We also explored the possibility of testosterone aromatization by using selective inhibitors of the aromatase enzyme cytochrome P-450 aromatase (P450(arom)), aminoglutethimide (4 microM), and 4-hydroxyandrostenedione (4 microM). The presence of P450(arom) was investigated by immunocytochemical and immunoblot assays using peptide-generated polyclonal antibodies raised against a 20-amino acid synthetic fragment of rat P450(arom) and by in situ hybridization to locate the aromatase mRNA in such cells. The activity of P450(arom) was demonstrated by the stereospecific loss of the tritium atom of [1beta-(3)H]androstenedione. Our results indicate that both T and E(2) induced a rapid increase in [Ca(2+)](i). The fact that the effects of E(2) and T were carried out within milliseconds suggests that they were exerted at the membrane level and not through intracellular receptors. The possibility of involvement of PLC-beta in these effects is suggested because U-73122 (a PLC inhibitor) blocked the effects of both T and E(2). Immunocytochemical assays indicated the expression of androgenic and estrogenic receptors in these cells. The effects of T were blocked by the selective aromatase inhibitors. We also demonstrated membrane association of P450(arom), expression of the ovary-specific mRNA after in situ hybridization, and E(2) formation resulting from a significant activity of P450(arom) in CMECs. There were no gender-based differences.     

Ca2+ channels and intracellular Ca2+ stores in neuronal and neuroendocrine cells

Changes in [Ca2+]i are essential in modulating a variety of cellular functions. In no other cell type does the regulation of [Ca2+]i reach the level of sophistication observed in cells of neuronal origin. Because of its physicochemical characteristics, the fluorescent Ca2+ indicator Fura-2 has become extremely popular among neuroscientists. The use of this probe, however, has generated a number of problems, in particular, extracytosolic trapping and leakage from intact cells. In the first part of this contribution we briefly discuss the practical application of Fura-2 to the study of [Ca2+]i in primary cultures of neurons and astrocytes. In the second part, we review some recent data (mainly from our laboratories) obtained in neurons and neuroendocrine cells, concerning the regulation of different types of Ca2+ channels and the role and mechanism of intracellular Ca2+ mobilization. The experimental evidence supporting the existence of a previously unrecognised organelle, the calciosome, that we hypothesize represents the functional equivalent in non-muscle cells of sarcoplasmic reticulum, will also briefly be discussed.     

Effect of calmodulin antagonists on the membrane potential and intracellular Ca2+ stores in endothelial cells

The role of calmodulin in electrical and Ca2+ signalling in intact rat aortic endothelial cells has been investigated. Calmodulin antagonists released Ca2+ from acetylcholine-sensitive and insensitive intracellular Ca2+ stores activated Ca(2+)-activated K+ channels and hyperpolarized endothelial cells. These findings indicate that calmodulin is involved in the regulation of intracellular Ca2+ stores in endothelial cells and is an essential agent for maintaining them in the Ca(2+)-loaded state.     

Myeloperoxidase-derived oxidants inhibit sarco/endoplasmic reticulum Ca2+-ATPase activity and perturb Ca2+ homeostasis in human coronary artery endothelial cells

The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) plays a critical role in Ca(2+) homeostasis via sequestration of this ion in the sarco/endoplasmic reticulum. The activity of this pump is inhibited by oxidants and impaired in aging tissues and cardiovascular disease. We have shown previously that the myeloperoxidase (MPO)-derived oxidants HOCl and HOSCN target thiols and mediate cellular dysfunction. As SERCA contains Cys residues critical to ATPase activity, we hypothesized that HOCl and HOSCN might inhibit SERCA activity, via thiol oxidation, and increase cytosolic Ca(2+) levels in human coronary artery endothelial cells (HCAEC). Exposure of sarcoplasmic reticulum vesicles to preformed or enzymatically generated HOCl and HOSCN resulted in a concentration-dependent decrease in ATPase activity; this was also inhibited by the SERCA inhibitor thapsigargin. Decomposed HOSCN and incomplete MPO enzyme systems did not decrease activity. Loss of ATPase activity occurred concurrent with oxidation of SERCA Cys residues and protein modification. Exposure of HCAEC, with or without external Ca(2+), to HOSCN or HOCl resulted in a time- and concentration-dependent increase in intracellular Ca(2+) under conditions that did not result in immediate loss of cell viability. Thapsigargin, but not inhibitors of plasma membrane or mitochondrial Ca(2+) pumps/channels, completely attenuated the increase in intracellular Ca(2+) consistent with a critical role for SERCA in maintaining endothelial cell Ca(2+) homeostasis. Angiotensin II pretreatment potentiated the effect of HOSCN at low concentrations. MPO-mediated modulation of intracellular Ca(2+) levels may exacerbate endothelial dysfunction, a key early event in atherosclerosis, and be more marked in smokers because of their higher SCN(-) levels.     

The caffeine-sensitive Ca2+ store in bovine adrenal chromaffin cells; an examination of its role in triggering secretion and Ca2+ homeostasis

The effect of caffeine on catecholamine secretion and intracellular free Ca2+ concentration [( Ca2+]i) in bovine adrenal chromaffin cells was examined using single fura-2-loaded cells and cell populations. In cell populations caffeine elicited a large (approximately 200 nM) transient rise in [Ca2+]i that was independent of external Ca2+. This rise in [Ca2+]i triggered little secretion. Single cell measurements of [Ca2+]i showed that most cells responded with a large (greater than 200 nM) rise in [Ca2+]i, whereas a minority failed to respond. The latter, whose caffeine-sensitive store was empty, buffered a Ca2+ load induced by a depolarizing stimulus more effectively than those whose store was full. The caffeine-sensitive store in bovine chromaffin cells may be involved in Ca2+ homeostasis rather than in triggering exocytosis.     

Ca2+-activated K+ channels in murine endothelial cells: block by intracellular calcium and magnesium

The intermediate (IK(Ca)) and small (SK(Ca)) conductance Ca(2+)-sensitive K(+) channels in endothelial cells (ECs) modulate vascular diameter through regulation of EC membrane potential. However, contribution of IK(Ca) and SK(Ca) channels to membrane current and potential in native endothelial cells remains unclear. In freshly isolated endothelial cells from mouse aorta dialyzed with 3 microM free [Ca(2+)](i) and 1 mM free [Mg(2+)](i), membrane currents reversed at the potassium equilibrium potential and exhibited an inward rectification at positive membrane potentials. Blockers of large-conductance, Ca(2+)-sensitive potassium (BK(Ca)) and strong inward rectifier potassium (K(ir)) channels did not affect the membrane current. However, blockers of IK(Ca) channels, charybdotoxin (ChTX), and of SK(Ca) channels, apamin (Ap), significantly reduced the whole-cell current. Although IK(Ca) and SK(Ca) channels are intrinsically voltage independent, ChTX- and Ap-sensitive currents decreased steeply with membrane potential depolarization. Removal of intracellular Mg(2+) significantly increased these currents. Moreover, concomitant reduction of the [Ca(2+)](i) to 1 microM caused an additional increase in ChTX- and Ap-sensitive currents so that the currents exhibited theoretical outward rectification. Block of IK(Ca) and SK(Ca) channels caused a significant endothelial membrane potential depolarization (approximately 11 mV) and decrease in [Ca(2+)](i) in mesenteric arteries in the absence of an agonist. These results indicate that [Ca(2+)](i) can both activate and block IK(Ca) and SK(Ca) channels in endothelial cells, and that these channels regulate the resting membrane potential and intracellular calcium in native endothelium.     

Evoked transient intracellular free Ca2+ changes and secretion in isolated bovine adrenal medullary cells

When isolated bovine adrenal medullary cells are incubated with the lipid-soluble Quin 2 acetoxymethyl ester, the ester permeates the plasma membrane and enters the cytosol, where it is hydrolysed by endogenous enzymes to yield an impermeant fluorescent indicator (Quin 2) which is sensitive to Ca2+ in the 0.1 microM range. This technique permits the average intracellular free Ca2+ level ([Ca2+]i) to be determined in a suspension of adrenal medullary cells. Unstimulated cells have a [Ca2+]i of 97 +/- 4 nM (n = 69). This level seems independent of extracellular calcium in the range 0.5-2 mM. When the extracellular calcium concentration is lowered to ca. 10(-7) M, however, [Ca2+]i decreases. A transient increase in [Ca2+]i occurs when cells are challenged with either acetylcholine or a high potassium medium. The time course of the [Ca2+]i transient rises to a maximum within seconds, and decreases to basal levels over minutes. The maximum level of [Ca2+]i associated with secretion is very variable. Hexamethonium, methyoxyverapamil, and the absence of extracellular calcium block not only the secretory response but also the [Ca2+]i transient. The action of acetylcholine leading to the Ca2+]i transient is blocked when cells are suspended in a depolarizing medium. Extracellular magnesium inhibits both the [Ca2+]i transient and the secretory response evoked by acetylcholine. Secretion is, however, more sensitive to magnesium inhibition than is calcium entry. The magnitudes of the [Ca2+]i transient and the secretory response decrease as the concentration of intracellular Quin 2 increases. Measurements of the amount of indicator titrated with calcium, as a result of an acetylcholine or potassium challenge, suggest that the increase in the apparent calcium content of the cytosol might arise from two contributing sources of calcium entry.     

Patterns of intracellular Ca2+ concentrations in fertilized bovine eggs.

Sperm-induced calcium (Ca2+) changes were examined in zona pellucida-intact, mature bovine eggs injected with the fluorescent Ca2+ indicator fura-2 dextran (fura-2 D). Fifty four percent (37/68) of the dye-injected, inseminated bovine eggs were fertilized and 43% (16/37) of the fertilized eggs exhibited Ca2+ elevations during the time of measurement. All (16/16) of the eggs with Ca2+ elevations were fertilized but none of the unfertilized eggs (0/31) showed intracellular Ca2+ elevations. Six of 13 eggs that were later examined and found to be fertilized at the time of the Ca2+ recordings did not show sperm-induced Ca2+ elevations. Fifty percent (8/16) of the eggs with Ca2+ elevations exhibited a single Ca2+ rise as a response to sperm penetration during the 60-min period in which these eggs were monitored. Twelve percent (2/16) of the eggs responded with two Ca2+ elevations spaced by 50- and 51-min intervals and 38% (6/16) of the eggs exhibited multiple elevations with intervals of 15-29 min. In the latter group, one egg was polyspermic. The mean amplitude of the sperm-induced Ca2+ elevations was 564 +/- 58 nM. Eggs with single elevations reached higher peak concentrations than eggs with multiple elevations (p < 0.05). The mean duration of the Ca2+ elevations was 166 +/- 13 sec and was similar among eggs with different Ca2+ patterns. The first elevations detected occurred at a mean of 6.6 +/- 0.5 h after insemination. Fertilization in this study was confirmed by looking at pronuclear formation 16 h post-insemination or by DNA staining immediately after the fluorescence readings. Eggs exhibiting Ca2+ elevations ranged in stage of fertilization from just penetrated to pronuclear. Injection of inositol 1,4,5 trisphosphate (5 microM in the injection pipette) into 6 bovine eggs induced an immediate Ca2+ elevation with a mean peak Ca2+ value of 700 +/- 60 nM and a mean duration of 103 +/- 21 sec. Incubation of bovine eggs with 200 microM thimerosal induced periodic Ca2+ rises. The mean number of Ca2+ elevations observed in 35 min of recordings was 3.0 +/- 0.5 (n = 9, range 1-5). The mean peak Ca2+ value of the first thimerosal-induced Ca2+ elevation was 990 +/- 210 nM. The results of this study indicate that fertilization can evoke intracellular Ca2+ elevations in bovine eggs and that the periodicity of these Ca2+ elevations is different among eggs. Furthermore, both inositol 1,4,5-trisphosphate and thimerosal were able to induce intracellular Ca2+ release in bovine eggs.