Mobilization Of Intracellular Stores Research Articles

Calcium signaling in human airway goblet cells following purinergic activation

Despite the general importance of Ca(2+) signaling in signal transduction, and of goblet cell mucin hypersecretion in inflammatory pulmonary diseases, measurement of airway goblet cell intracellular Ca(2+) (Ca(i)(2+)) has not been reported. In this article, we describe the results of experiments measuring Ca(i)(2+) in primary cultures of human bronchial goblet cells after stimulation with the purinergic agonist adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) and phorbol 12-myristate 13-acetate (PMA). Ca(2+) signaling in human goblet cells after purinergic stimulation follows the classic paradigm of a Ca(i)(2+) transient from a basal activity of 110 nM to a peak response of 260.1 +/- 41.2 nM within 2 min, followed by a long superbasal plateau (155.3 +/- 0.2 nM) between 10 and 15 min. The rise in Ca(i)(2+) appears to result from a mobilization of intracellular stores, because the transient was nearly abolished by inhibition of PLC with the phosphatidylinositol-specific PLC inhibitor U-73122, and it was not affected significantly by removal of extracellular Ca(2+). Loading goblet cells with BAPTA inhibited the ATPgammaS-induced Ca(2+) transient by 86.0 +/- 13.1%, relative to control. Finally, in contrast to the massive effects of high doses of PMA (300 nM) on mucin secretion from goblet cells, phorbol ester stimulated a small (27.1 +/- 7% of the ATPgammaS control peak), brief rise in Ca(i)(2+). This diminutive signal likely denotes a local Ca(2+) gradient, which may be associated with the mucin granule exocytotic process.

Evidence for Multiple Calcium Response Mechanisms in Mammalian Olfactory Receptor Neurons

Olfactory receptor neurons employ a diversity of signaling mechanisms for transducing and encoding odorant information. The simultaneous activation of subsets of receptor neurons provides a complex pattern of activation in the olfactory bulb that allows for the rapid discrimination of odorant mixtures. While some transduction elements are conserved among many species, some species-specificity occurs in certain features that may relate to their particular physiology and ecological niche. However, studies of olfactory transduction have been limited to a relatively small number of vertebrate and invertebrate species. To better understand the diversity and evolution of olfactory transduction mechanisms, we studied stimulus-elicited calcium fluxes in olfactory neurons from a previously unstudied mammalian species, the domestic cat. Isolated cells from cat olfactory epithelium were stimulated with odorant mixtures and biochemical agents, and cell responses were measured with calcium imaging techniques. Odorants elicited either increases or decreases in intracellular calcium; odorant-induced calcium increases were mediated either by calcium fluxes through the cell membrane or by mobilization of intracellular stores. Individual cells could employ multiple signaling mechanisms to mediate responses to different odorants. The physiological features of these olfactory neurons suggest greater complexity than previously recognized in the role of peripheral neurons in encoding complex odor stimuli. The investigation of novel and unstudied species is important for understanding the mechanisms of odorant signaling that apply to the olfactory system in general and suggests both broadly conserved and species-specific evolutionary adaptations.

Stretch activation of Ca2+ transients in pancreatic beta cells by mobilization of intracellular stores.

Nonadrenergic, noncholinergic neurons have been proposed to synchronize pulsatile insulin release from the islets in the pancreas by triggering transient increases of the cytoplasmic Ca2+ concentration ([Ca2+]i) in beta-cells via an inositol trisphoshate-dependent mechanism. To test whether pancreatic beta-cells respond to stretch activation with similar types of transients and whether these Ca signals propagate to other beta-cells in the presence and absence of cell contacts. Single cells and small aggregates were prepared from beta-cell-rich islets from mice. After 2-5 days of culture, [Ca2+]i was measured with digital imaging and the indicator fura-2 during superfusion with a medium containing 20 mmol/L glucose and 50 micromol/L methoxyverapamil. Membrane stretch was induced by osmotic swelling or focal touch stimulation. Lowering the medium osmolarity with 100-102 mOSM/L by removal of sucrose or by dilution resulted in a 2-3-fold increase in the number of transients during an initial 5-minute period. Sucrose omission was stimulatory also after isosmolar replacement with readily penetrating urea. The intracellular Ca2+-ATPase inhibitor thapsigargin suppressed both the spontaneously occurring transients and those initiated by volume expansion. Touch stimuli induced [Ca2+]i transients, which rapidly propagated to cells within the same aggregate or lacking contact. The observations support the idea that beta-cells both receive and regenerate extracellular signals triggering [Ca2+]i transients. Touch stimulation is a useful tool for investigating the propagation of [Ca2+]i signals between pancreatic beta-cells lacking physical contact.

Impact of atrial fibrillation on the clinical course of hypertrophic cardiomyopathy

The heart's extraordinary metabolic flexibility allows it to adapt to normal changes in physiology in order to preserve its function. Alterations in the metabolic profile of the heart have also been attributed to pathological conditions such as ischemia and hypertrophy; however, research during the past decade has established that cardiac metabolic adaptations can precede the onset of pathologies. It is therefore critical to understand how changes in cardiac substrate availability and use trigger events that ultimately result in heart dysfunction. This review examines the mechanisms by which the heart obtains fuels from the circulation or from mobilization of intracellular stores. We next describe experimental models that exhibit either an increase in glucose use or a decrease in FA oxidation, and how these aberrant conditions affect cardiac metabolism and function. Finally, we highlight the importance of alternative, relatively under-investigated strategies for the treatment of heart failure. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.

Regulation of Ca 2+ signals in a parotid cell line Par-C5

The Ca 2+ signaling system in an established immortalized rat parotid acinar cell line, Par-C5, was examined using the Ca 2+-sensitive fluorescent indicator fura-2 and by measuring inositol 1,4,5-trisphosphate (IP 3) formation. Agonist-induced increase in intracellular Ca 2+ ([Ca 2+] i) by mobilization of intracellular stores and influx across the cell membrane was stimulated by acetylcholine (ACh) and ATP, whereas noradrenaline-(NA)-induced a small [Ca 2+] i increase mediated primarily by release from intracellular Ca 2+ stores. [Ca 2+] i increase by ACh and ATP was meditated through the phosphoinositide signal pathway since both agonists significantly increased 1,4,5-IP 3 formation and Ca 2+ mobilization was abolished by the phospholipase C inhibitor U73122. In Ca 2+-free medium, ACh or ATP discharged the IP 3-sensitive Ca 2+ store and essentially abolished subsequent [Ca 2+] i response to thapsigargin (TG). Exposure to ionomycin and monensin after TG induced a further mobilization of Ca 2+, suggesting IP 3-insensitive stores are present. Furthermore, depletion of IP 3-sensitive Ca 2+ stores by TG, ACh and ATP enhanced plasmalemmal Ca 2+-entry pathways. Exposure to tumor necrosis factor-α (TNF-α), a cytokine associated with lymphocyte invasion of salivary epithelial cells in autoimmune disorders, significantly reduced ACh-stimulated Ca 2+ mobilization. TNF-α inhibitory effect on Ca 2+ mobilization was not directly due to an interaction on muscarinic receptors since ACh-induced 1,4,5-IP 3 formation was not altered. These results in the Par-C5 cell line indicate 1) [Ca 2+] i is regulated by muscarinic and P2Y-nucleotide receptors and partly by α 1-adrenergic receptors; 2) IP 3-sensitive and -insensitive Ca 2+ stores exist; 3) Ca 2+ influx activated by ACh, ATP or TG is mediated by the store-operated Ca 2+ entry pathway; and 4) muscarinic agonist-stimulated Ca 2+ mobilization is altered by the cytokine TNF-α.

Vasopressin signaling pathways in vascular smooth muscle.

Arginine vasopressin (AVP) exhibits both acute and long-term effects on vascular smooth muscle cells (VSMC). Acutely, AVP regulates vascular tone and stimulates contraction. Longer term exposure of VSMC to AVP in the absence of other mitogenic agents results in cell hypertrophy without increases in cell number, and increased expression of a number of muscle-specific genes including the smooth muscle form of alpha-actin (SM-alpha-actin). These responses can be distinguished from the proliferative responses seen with growth factors such as platelet-derived growth factor (PDGF), which increase DNA synthesis and cell number and suppress SM-alpha-actin expression. In cultured VSMC, all the effects of AVP are mediated through the V1a receptor which signals through G-proteins. This review examines post-receptor signaling events mediated by AVP in VSMC. AVP rapidly increases intracellular Ca2+ via mobilization of intracellular stores and entry of extracellular Ca2+ via specific cation channels. This pathway, via activation of myosin light chain kinase, is critical for the early contractile response. Increased intracellular Ca2+ also leads to increased arachidonic acid release and eicosanoid production through the action of phospholipase A2. The activation of protein kinases by AVP is examined, focusing on members of the mitogen-activated protein kinase family. These enzymes are likely to play an important role in promoting growth of VSMC as well as modulating their state of differentiation through transcriptional control of muscle-specific gene expression. Recent studies suggesting a role for c-Jun amino terminal kinases in the regulation of smooth muscle-alpha-actin expression are described.

Stimulation of Intracellular Ca2+ Levels in Human Neutrophils by Soluble Immune Complexes

Soluble immune complexes bind to unprimed neutrophils and generate intracellular Ca2+ transients but fail to activate the NADPH oxidase. Following priming of the neutrophils with either tumor necrosis factor alpha or granulocyte-macrophage colony-stimulating factor, stimulation of the cells with the soluble immune complexes leads to an enhanced Ca2+ signal and significant secretion of reactive oxidants. The enhanced Ca2+ signal observed in primed neutrophils results from the influx of Ca2+ from the external environment and is partly sensitive to tyrosine kinase inhibitors. This is in contrast to the Ca2+ signal observed in unprimed neutrophils, which arises from the mobilization of intracellular stores. When the surface expression of FcgammaRIIIb on primed neutrophils was decreased either through incubation with Pronase or phosphoinositide-specific phospholipase C, the extra enhanced Ca2+ mobilization seen in primed cells was significantly lowered, while the initial rise in intracellular Ca2+ was unaffected. Depletion of FcgammaRIIIb had no significant effect on the Ca2+ transients in unprimed neutrophils. Cross-linking FcgammaRII, but not FcgammaRIIIb, induced increases in intracellular Ca2+ in unprimed neutrophils, while cross-linking either of these receptors increased Ca2+ levels in primed neutrophils. The FcgammaRII-dependent intracellular Ca2+ rise in primed cells was unaffected by incubation in Ca2+-free medium, whereas the FcgammaRIIIb-dependent transient was significantly decreased when Ca2+ influx was prevented in Ca2+-free medium supplemented with EGTA. Cross-linking either FcgammaRII or FcgammaRIIIb in primed or unprimed cells failed to stimulate substantial levels of inositol 1,4,5-trisphosphate production. These results indicate that following stimulation of primed neutrophils with soluble immune complexes the enhanced Ca2+ mobilization observed is the result of a functional activation of the glycosylphosphatidylinositol-linked FcgammaRIIIb.

κ-Opioid receptor expression defines a phenotypically distinct subpopulation of astroglia: relationship to Ca 2+ mobilization, development, and the antiproliferative effect of opioids

To assess the role of κ-opioid receptors in astrocyte development, the effect of κ-agonists on the growth of astroglia derived from 1–2-day-old mouse cerebra was examined in vitro. κ-Opioid receptor expression was assessed immunocytochemically (using KA8 and KOR1 antibodies), as well as functionally by examining the effect of κ-receptor activation on intracellular calcium ([Ca 2+] i) homeostasis and DNA synthesis. On days 6–7, as many as 50% of the astrocytes displayed κ-receptor (KA8) immunoreactivity or exhibited increases in [Ca 2+] i in response to κ-agonist treatment (U69,593 or U50,488H). Exposure to U69,593 (100 nM) for 72 h caused a significant reduction in number and proportion of glial fibrillary acidic protein-immunoreactive astrocytes incorporating bromodeoxyuridine (BrdU) that could be prevented by co-administering the κ-antagonist, nor-binaltorphimine (300 nM). In contrast, on day 14, only 5 or 14%, respectively, of the astrocytes were κ-opioid receptor (KA8) immunoreactive or displayed functional increases in [Ca 2+] i. Furthermore, U69,593 (100 nM) treatment failed to inhibit BrdU incorporation at 9 days in vitro. Experimental manipulations showed that κ-receptor activation increases astroglial [Ca 2+] i both through influx via L-type channels and through mobilization of intracellular stores (which is an important Ca 2+ signaling pathway in cell division). Collectively, these results indicate that a subpopulation of developing astrocytes express κ-opioid receptors in vitro, and suggest that the activation of κ-receptors mobilizes [Ca 2+] i and inhibits cell proliferation. Moreover, the proportion of astrocytes expressing κ-receptors was greatest during a period of rapid cell growth suggesting that they are preferentially expressed by proliferating astrocytes.

Spatial and temporal aspects of Ca 2+ oscillations in Xenopus laevis melanotrope cells

Spatio-temporal aspects of Ca 2+ signaling in melanotrope cells of Xenopus laevis have been studied with confocal laser-scanning microscopy. In the whole-frame scanning mode, two major intracellular Ca 2+ compartments, the cytoplasm and the nucleus, were visualized. The basal [Ca 2+] in the nucleus appeared to be lower than that in the cytoplasm and Ca 2+ oscillations seemed to arise synchronously in both compartments. The N-type channel blocker ω-conotoxin eliminated oscillations in both regions, indicating a strong coupling between the two compartments with respect to Ca 2+ dynamics. Line-scanning mode, which gives higher time resolution, revealed that the rise phase of a Ca 2+ oscillation is not a continuous process but consists of 3 or 4 discrete steps. Each step can be seen as a Ca 2+-wave starting at the cell membrane and going through the cytoplasm at a speed of 33.3 ± 4.3 μm/s. Before the Ca 2+-wave enters the nucleus, a delay of 120.0 ± 24.1 ms occurred. In the nucleus, the speed of a wave was 80.0 ± 3.0 μm/s. Treatment with the Ca 2+-ATPase inhibitor thapsigargin (1 μM) almost completely eliminated the apparent difference in the basal [Ca 2+] in the cytoplasm and the nucleus, reduced the delay of a Ca 2+-wave before entering the nucleus to 79.8 ± 8.7 ms, and diminished the nuclear wave speed to 35.0 ± 4.9 μ/s. These results indicate that a cytoplasmic thapsigargin-sensitive ATPase near the nuclear envelope is involved in buffering Ca 2+ before the Ca 2+ wave enters the nucleus. After sensitizing IP 3 receptors by thimerosal (10 μM) the speed of the cytoplasmic Ca 2+-wave was increased to 70.3 ± 3.6 μ/s, suggesting that IP 3 receptors may be involved in the propagation of the cytoplasmic Ca 2+ wave. Our results indicate that in melanotropes the generation and propagation of Ca 2+ oscillations is a complex event involving influx of Ca 2+ through N-type Ca 2+ channels, propagation of the cytoplasmic Ca 2+ wave through mobilization of intracellular stores and a regulated Ca 2+ entry into the nucleus. We propose that Ca 2+-binding proteins may act as a Ca 2+ store for propagation of the wave in the nucleus.

Characterization of P2 purinergic receptors on human erythro leukemia cells.

We have investigated the nature of the nucleotide receptors on human erythro leukemia (HEL) cells, a cell line with some megakaryocytic properties, using a combination of pharmacological, photoaffinity labeling, and molecular biological techniques. Fura-2 loaded HEL cells responded to 2-methylthio ATP, ATP, 2-methylthio ADP, ADP and UTP with an increase in intracellular calcium. 2 Methylthio ADP was the most potent agonist. When external calcium was chelated with EDTA, calcium responses were observed indicating the mobilization of intracellular stores. These responses showed evidence of both homologous and heterologous receptor desensitization. In photoaffinity labeling experiments, beta-[32P]-AzPET-ADP was incorporated into three protein species with mobilities corresponding to M(r) approximately 55 kDa (doublet) and approximately 43 kDa. Labeling of approximately 55 kDa proteins was specifically inhibited by ADP, while that of the approximately 43 kDa was inhibited specifically by UTP. Nucleotide sequence analysis of the positive clones obtained by screening the HEL cell cDNA library with mouse P2U cDNA revealed that the P2U receptor from HEL cells is identical to the previously cloned human P2U receptor. These experiments suggest that the HEL cells contain a P2Y purinoceptor responding to ADP, in addition to a P2U receptor and possibly also a third P2 purinoceptor with a unique agonist profile.

Rat Oligodendrocytes Express Muscarinic Receptors Coupled to Phosphoinositide Hydrolysis and Adenylyl Cyclase

Muscarinic receptors expressed by rat oligodendrocyte primary cultures were examined by measuring changes in second messengers following exposure to carbachol, an acetylcholine analog, and by polymerase chain reaction. Inositol phosphate levels were measured in [3H]myo-inositol-labelled young oligodendrocyte cultures following stimulation with carbachol. Atropine, a specific muscarinic antagonist, prevented the carbachol-induced accumulation of inositol phosphates. The formation of inositol trisphosphate was concentration- and time-dependent, with the peak at 100 microM carbachol and 10 min. Carbachol increased intracellular calcium levels, which were dependent both on the mobilization of intracellular stores and influx of extracellular calcium. In initial experiments with more selective antagonists, the mobilization of intracellular calcium was preferentially inhibited by pirenzepine, a selective M1 antagonist, but not methoctramine, a selective M2 antagonist, suggesting M1 muscarinic receptor involvement. A role for protein kinase C in the regulation of carbachol-stimulated inositol phosphate formation and intracellular calcium mobilization was demonstrated, as acute pretreatment with phorbol-12,13-myristate acetate abolished the formation of both second messengers. Pretreatment with 100 microM carbachol abolished the 40% increase in the cyclic AMP accumulation stimulated by isoproterenol, a specific beta-adrenergic agonist. In turn, the inhibition was alleviated by pretreatment with atropine, suggesting muscarinic receptor involvement. Polymerase chain reaction carried out with specific m1 and m2 muscarinic receptor oligonucleotide primers, confirmed that these cells express, at least, the two muscarinic receptor subtypes. Without excluding the expression of other subtypes, these results suggest that developing oligodendrocytes express m1 (M1) and m2 (M2) muscarinic receptors capable of mediating phosphoinositide hydrolysis, mobilization of intracellular calcium and the attenuation of beta-adrenergic stimulation of cyclic AMP formation.

Stretching increases calcium influx and efflux in cultured pulmonary arterial smooth muscle cells.

To determine the effect of a single static stretch on calcium fluxes in cultured pulmonary arterial smooth muscle cells (PASMC), calcium influx and efflux were evaluated in PASMC on a collagen-coated silicone membrane using 45Ca2+ as a tracer. A single 20% linear stretch of the silicone membrane of 1 min in duration increased calcium uptake by 71%. This effect was partially inhibited by verapamil or gadolinium, but was not altered by staurosporine, pertussis toxin, or removal of extracellular sodium. Stretch-stimulated calcium uptake attenuated over time, such that uptake during the last minute of a 5-min sustained stretch was 46% of that during the first minute of stretch. A single 20% stretch sustained for 6 min caused a 47% increase in calcium efflux, the magnitude of which was linearly related to the degree of cell stretch. Gadolinium and removal of extracellular calcium each partially inhibited stretch-induced calcium efflux. We conclude that a single static stretch of PASMC causes increases in both calcium influx and efflux. Stretch-stimulated calcium influx does not require sodium influx and is mediated in part by a pathway sensitive to both gadolinium and verapamil. Stretch-stimulated calcium efflux is due to both calcium influx via a gadolinium-sensitive pathway and mobilization of intracellular stores. Because calcium is a key cellular second messenger, these effects of stretch on cellular calcium handling may play a role in the regulation of vascular smooth muscle cell phenotype and function.

Endothelin mobilizes Ca2+ from a caffeine- and ryanodine-insensitive intracellular pool in rat atrial cells.

Endothelin-1 is a powerful inotropic peptide for the rat atrium. Its action can develop in the absence of L-type Ca2+ channel activity provided that the external Ca2(+)-concentration has been raised to supraphysiological concentrations. Endothelin stimulates phosphatidylinositol hydrolysis in new born rat atrial cells via a mechanism that is insensitive to pertussis toxin. The diacylglycerol/protein kinase C signaling pathway cannot account for the contractile action of endothelin but its activation by phorbol esters induces a partial desensitization of phospholipase C activity. Endothelin-1 and the related peptides, endothelin-2, endothelin-3, and sarafotoxin S6b, raise intracellular Ca2+ levels in rat atrial cells. The actions of endothelin-1, endothelin-2, and sarafotoxin on [Ca2+]i are mutually exclusive, suggesting that they act at the same receptor site. The rise in [Ca2+]i induced by endothelins results both from the mobilization of intracellular stores and from Ca2+ entry through the sarcolemma via a pathway that is not voltage-dependent L-type Ca2+ channels. The Ca2+ store that is mobilized in response to endothelin retains its Ca2+ content when cells were incubated for long periods of time in a 50 nM Ca2+ solution. It is insensitive to caffeine and ryanodine. These two properties distinguish it from the sarcoplasmic reticulum. Contraction experiments in which the pacing rate has been altered to favor Ca2+ accumulation into terminal cisternae of the sarcoplasmic reticulum also suggest that the Ca2+ load of the sarcoplasmic reticulum is increased in endothelin treated rat atria.

Endothelin-induced increases in vascular smooth muscle Ca2+ do not depend on dihydropyridine-sensitive Ca2+ channels.

Endothelin is a potent mammalian vasoconstrictive peptide with structural homology to cation channel-binding insect toxins. We tested the proposal that this peptide directly activates dihydropyridine-sensitive Ca2+ channels in cultured vascular smooth muscle (VSM) cells. First, we found that cell Ca2+ can be altered in VSM by activation of voltage-operated Ca2+ channels. KCl-induced depolarization and the dihydropyridine Ca2+ channel agonist (-) Bay K 8644 (10 microM) both raised cell Ca2+ more than twofold; the effect of KCl was blocked by the inhibitory enantiomer, (+) Bay K 8644 (40 microM). Similar responses were observed in Chinese hamster ovary (CHO) cells. Synthetic endothelin (4 x 10(-8) M) raised Ca2+ in VSM but not CHO cells from 100 +/- 17 to 561 +/- 34 nM within 12 s. Ca2+ subsequently fell to basal levels after 30 min. Half maximal Ca2+ response was at 4 x 10(-9) M endothelin. Unlike endothelin, thrombin raised Ca2+ in both VSM and CHO cells. The Ca2+ responses to endothelin and thrombin were not affected by nicardipine (1 microM), (+) Bay K 8644, or Ca2+-free solutions. Lastly, both hormones caused release of inositol phosphates in VSM cells. However, the response to thrombin was more than 10-fold larger and was more rapid than the response to endothelin; the thrombin response was sensitive to pertussis toxin, while the response to endothelin was not. Thus endothelin, like thrombin, raises cell Ca2+ in VSM by mobilization of intracellular stores and not by activation of dihydropyridine-sensitive Ca2+ channels. However, their receptors are distinct and they exhibit important differences in signal transduction.

Inhibition by nicardipine on endothelin-mediated inositol phosphate formation and Ca 2+ mobilization in smooth muscle cell

We have investigated the effects of endothelin on phosphoinositide metabolism and Ca 2+ mobilization in cultured A10 cells. Endothelin stimulated a significant increase in inositol phosphate formation in a time- and dose-dependent manner. IP 3 was significantly elevated by 30 sec and reached a 2.0-fold above control at 1 min. The EC 50 for endothelin was 0.5 nM. The initiation of inositol phosphate formation was independent of extracellular Ca 2+, and the Ca 2+ ionophore, A23187, did not stimulate IP 3 formation. However, the sustained elevation of inositol phosphates was partially inhibited by incubating cells in buffer lacking Ca 2+ or in buffer containing nicardipine. Endothelin mobilized both intracellular and extracellular Ca 2+ reaching a peak intracellular concentration of 350 ± 11 nM by 1 min when cells were bathed with Ca 2+-complete buffer. Intracellular Ca 2+ remained 2-fold above baseline for at least 15 min. In contrast, when cells were exposed to endothelin in Ca 2+-free buffer, the peak value of {Ca 2+} i was 195 ± 20 nM and returned to baseline by 2 min. Nicardipine completely blocked the influx of extracellular Ca 2+ but did not interfer with the mobilization of intracellular stores. We conclude that endothelin produces a rapid and sustained elevation in inositol phosphate formation. The rapid production of IP 3 is consistent with the time course for mobilization of intracellular Ca 2+. Elevated cytosolic Ca 2+ levels are maintained by the influx of extracellular Ca 2+ through a nicardipine-sensitive Ca 2+ channel and are involved in the sustained formation of inositol phosphates. These data provide an explanation for the sustained, nicardipine-inhibitable contraction of coronary artery strips induced by endothelin.

The recovery of human polymorphonuclear leucocytes from sublytic complement attack is mediated by changes in intracellular free calcium.

Using polymorphonuclear leucocyte-erythrocyte ghost hybrids entrapping the calcium-activated photoprotein obelin, we have demonstrated that sublytic amounts of the complement membrane attack complex induce a rapid but transient increase in intracellular free calcium ion concentration ([Ca2+]i). This increase in [Ca2+]i occurs prior to, and is required for, rapid removal of membrane attack complexes from the cell surface. The increase in [Ca2+]i is not only due to increased influx from outside the cell, but also results from mobilization of intracellular stores. The possible mechanism of mobilization of calcium, and the importance of an increase in [Ca2+]i as a mediator of recovery processes in nucleated cells, are discussed.

The influence of CGP 28392, A 1,4-dihydropyridine, on human platelet calcium and cyclic AMP metabolism

Use of Ca 2+ ionophores (e.g. ionophore A 23187) and Ca 2+ channel antagonists (e.g. dihydropyridines, phenylalkamines) has provided some insight into the molecular events involved in platelet activation. Recent structural modification of dihydropyridines has generated a novel class of compounds (e.g. CGP 28393) which apparently exert opposing functional effects. We report that CGP 28392 induced an elevation in free intracellular calcium concentrations ([Ca 2+] i) via both calcium influx and mobilization of intracellular stores. Treatment of washed human platelets with this Ca 2+ agonist resulted in typical Ca 2+-linked activation phenomena such as shape change and phosphorylation of M r 47 000 and M r 20 000 proteins. CGP 28392 also negatively influenced platelet cyclic AMP metabolism, and appears to exert this effect as a consequence of elevated [Ca 2+] i. The data suggest that the mechanisms of action of CGP 28392 involve dihydropyridine-susceptible structures on the cell membrane and intracellular Ca 2+ binding sites.