Chronic intrauterine pulmonary hypertension compromises fetal pulmonary artery smooth muscle cell O2 sensing

To test the hypothesis that chronic intrauterine pulmonary hypertension (PHTN) compromises pulmonary artery (PA) smooth muscle cell (SMC) O2 sensing, fluorescence microscopy was used to study the effect of an acute increase in Po2 on the cytosolic Ca2+ concentration ([Ca2+]i) of chronically hypoxic subconfluent monolayers of PA SMC in primary culture. PA SMCs were derived from fetal lambs with PHTN due to intrauterine ligation of the ductus arteriosus. Acute normoxia decreased [Ca2+]i in control but not PHTN PA SMC. In control PA SMC, [Ca2+]i increased after Ca2+-sensitive (KCa) and voltage-sensitive (Kv) K+ channel blockade and decreased after diltiazem treatment. In PHTN PA SMC, KCa blockade had no effect, whereas Kv blockade and diltiazem increased [Ca2+]i. Inhibition of sarcoplasmic reticulum Ca2+ ATPase activity caused a greater increase in [Ca2+]i in controls compared with PHTN PA SMC. Conversely, ryanodine caused a greater increase of [Ca2+]i in PHTN compared with control PA SMC. KCa channel mRNA is decreased and Kv channel mRNA is unchanged in PHTN PA SMC compared with controls. We conclude that PHTN compromises PA SMC O2 sensing, alters intracellular Ca2+ homeostasis, and changes the predominant ion channel that determines basal [Ca2+]i from KCa to Kv.     

Calcium channel blockers nifedipine and diltiazem inhibit Ca2+ release from intracellular stores in neutrophils.

We have undertaken a detailed study of the mechanisms of maintenance of intracellular Ca2+ homeostasis in human polymorphonuclear neutrophils (PMN) and its implications for phagocytosis and IgG Fc receptor (FcR) signaling. When PMN were incubated in Ca(2+)-free medium, cytoplasmic calcium concentration ([Ca2+]i) was markedly depressed and intracellular stores were depleted of calcium. [Ca2+]i in these depleted cells increased within 1 min when PMN were placed in medium containing Ca2+ and then decreased to a level close to the normal basal [Ca2+]i, replenishing the intracellular Ca2+ pools. LaCl3 prevented entry of Ca2+ into Ca(2+)-depleted PMN, but the calcium channel blockers nifedipine, diltiazem, and verapamil did not. Nifedipine and diltiazem but not verapamil inhibited the movement of Ca2+ from cytosol to intracellular stores. Nifedipine and diltiazem inhibited the normal increase in [Ca2+]i from aggregated IgG binding to FcR and also prevented formyl-methionyl-leucyl-phenyl-alanine (fMLP)-induced [Ca2+]i rise. Verapamil had no effect on either an fMLP- or IgG-mediated increase in [Ca2+]i. Consistent with this, nifedipine and diltiazem inhibited fMLP-stimulated phagocytosis (which is dependent on an increase in [Ca2+]i) when PMN had repleted intracellular stores. In contrast, LaCl3 inhibited fMLP-stimulated ingestion only in PMN which had intracellular store depleted. None of these compounds had any effect on phorbol dibutyrate-stimulated ingestion (which is independent of a [Ca2+]i rise). In summary, these data show that Ca2+ is in rapid equilibrium between intracellular and extracellular compartments in PMN. Exchange of cytoplasmic Ca2+ with the extracellular space is inhibited by LaCl3, while exchange of Ca2+ between the cytosol and intracellular stores is inhibited by the dihydropyridine nifedipine and the benzothiazepine diltiazem. These data suggest that these drugs, which are known to regulate some plasma membrane Ca2+ channels in excitable cells, can also regulate Ca2+ release from intracellular stores in PMN and that this regulation may have significant effects on PMN function.     

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.     

Parathyroid hormone-activated calcium channels in an osteoblast-like clonal osteosarcoma cell line. cAMP-dependent and cAMP-independent calcium channels

Changes in free cytosolic calcium were measured in UMR-106 cells in response to parathyroid hormone (PTH) stimulation. Bovine PTH-(1-34) induced an increase in [Ca2+]i with the contour of the rise in [Ca2+]i occurring in three successive phases: a rapid increase in [Ca2+]i occurring within seconds, rapid decrement in [Ca2+]i to near-resting levels within 1 min, and slow increment in [Ca2+]i. Phase one and phase three increases in [Ca2+]i were dependent on medium calcium. The phase one rise in [Ca2+]i was inhibitable by the calcium channel blockers lanthanum and verapamil. Only the phase one rise in [Ca2+]i was blocked by preincubation of the cells with the phorbol ester, phorbol 12-myristate 13-acetate. This channel was also blocked when cellular cAMP levels were increased prior to PTH stimulation. The phase two decrement of [Ca2+]i was due to the rapid inactivation of the phase one calcium channel. The phase three rise in [Ca2+]i was mediated by cellular cAMP levels. This cAMP-dependent Ca2+ channel was insensitive to pretreatment of the cells with phorbol diesters and showed low sensitivity to Ca2+ channel blockers. It is concluded that UMR-106 cells respond to PTH stimulation by the activation of a cAMP-independent Ca2+ channel. This channel rapidly inactivates. The subsequent PTH-dependent increase in cellular cAMP is followed by activation of a cAMP-dependent Ca2+ channel resulting in a slow rise in [Ca2+]i.     

Early agonist-mediated ionic events in cultured vascular smooth muscle cells. Calcium mobilization is associated with intracellular acidification

Angiotensin II, a potent vasoconstrictor peptide, increases free cytoplasmic Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC) by release of nonmitochondrial Ca2+ stores and stimulates an amiloride-sensitive Na+ influx, presumably via Na+/H+ exchange. We recently have found that the angiotensin II-mediated change in VSMC intracellular pH has two components, an early rapid acidification phase and a slower recovery phase involving Na+-dependent alkalinization. In the present study, we show that the early acidification is not mediated via Na+/H+ exchange. Instead, we propose a mechanism which involves increases in [Ca2+]i and Ca2+ efflux with a subsequent rise in intracellular H+. Agonists, in addition to angiotensin II, which increase [Ca2+]i in cultured VSMC, including platelet-derived growth factor, vasopressin, and bradykinin, induce an acidification, while agonists which fail to raise [Ca2+]i do not. The time course and magnitude of agonist-stimulated 45Ca2+ efflux correlate with the acidification response. The angiotensin II concentration-response relationship for acidification and Ca2+ mobilization are similar. Furthermore, inhibition of changes in [Ca2+]i by treatment with phorbol ester, cyclic GMP, or quin2 loading prevent agonist-mediated acidification. The effects of altering extracellular [Ca2+] and [H+] on agonist-mediated intracellular acidification and H+ efflux suggest that the acidification is due to ATP-dependent unidirectional H+ influx, perhaps via the plasma membrane Ca2+-ATPase, and not to a Ca2+/H+ antiport. This agonist-mediated acidification represents a previously undescribed ionic event in VSMC activation which may be involved in excitation-response coupling.     

Platelet-derived growth factor stimulates non-mitochondrial Ca2+ uptake and inhibits mitogen-induced Ca2+ signaling in Swiss 3T3 fibroblasts

Changes in intracellular free Ca2+ concentration [( Ca2+]i) were used to study the interaction between mitogens in Swiss 3T3 fibroblasts. Platelet-derived growth factor (PDGF) produced an increase in [Ca2+]i and markedly decreased the increases in [Ca2+]i caused by subsequent addition of bradykinin and vasopressin. If the order of the additions was reversed the [Ca2+]i response to PDGF was not inhibited by bradykinin or vasopressin. Inhibition of protein kinase C by staurosporine or chronic treatment of the cells with phorbol 12-myristate 13-acetate prevented the inhibitory effect of PDGF on the [Ca2+]i response to vasopressin but not bradykinin. PDGF did not decrease the receptor binding of bradykinin and produced only a small decrease in the receptor binding of vasopressin. PDGF decreased the rise in [Ca2+]i caused by the Ca2+ ionophores 4-bromo-A23187 and ionomycin and by a membrane perturbing ether lipid, 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine, both in the presence and absence of external Ca2+. There was no change in cell 45Ca2+ influx caused by PDGF, vasopressin, or bradykinin. 45Ca2+ efflux from cells exposed to PDGF and vasopressin mirrored the changes in [Ca2+]i caused by the agents, that is, PDGF added after vasopressin produced a further increase in 45Ca2+ efflux but vasopressin did not increase 45Ca2+ efflux after PDGF. PDGF but not vasopressin caused an increase in the uptake of 45Ca2+ into an inositol 1,4,5-trisphosphate-insensitive non-mitochondrial store in permeabilized cells. The results suggest that the decreased [Ca2+]i response to mitogens after PDGF represents an action of PDGF at a point beyond the release of intracellular Ca2+ and the influx of external Ca2+, caused by an increase in the rate of removal of cytoplasmic free Ca2+. This increased removal of cytoplasmic Ca2+ by PDGF is not due to the increased export of Ca2+ from the cell but results from increased Ca2+ uptake into non-mitochondrial stores.     

Regulation of free cytosolic Ca2+ in the peptic and parietal cells of the rabbit gastric gland

Quin 2-loaded isolated rabbit gastric glands and purified peptic cells were used to measure free cytosolic Ca2+ ([Ca2+]i) during hormone stimulation. Rabbit gastric glands are composed of peptic and parietal cells with less than 1% endocrine cells. Although both cell types responded to the same hormones, they may be distinguished in terms of the source of Ca2+ bringing about the change in [Ca2+]i. Experiments were designed to assign changes in [Ca2+]i to either the peptic or parietal cells and to attempt to maintain these distinctions in the mixed cell population of gastric glands. It was shown that the peptide cholecystokinin octapeptide induced a rapid and transient increase in [Ca2+]i of isolated peptic cells. This signal was independent of medium Ca2+ and insensitive to the Ca2+ channel blockers La3+ and nifedipine. In gastric glands, the Ca2+ outdependent increase in (Ca2+)i (the secondary transient) was slower and dose dependently blocked by La3+ and nifedipine. This allowed [Ca2+]i levels in the physiologically more intact rabbit gastric glands to be dissected and correlated with fluorescence changes of quin 2 in either cell type. The transient increase in [Ca2+]i coincided with a burst of pepsin but not acid secretion. A subsequent slower phase of pepsin secretion took place while the cells restored near resting [Ca2+]i. Using a combination of the Ca2+ ionophore A23187 and the protein kinase C activating phorbol ester 12-O-tetra-decanoylphorbol 13-acetate, the hormone response pattern of pepsin secretion could be mimicked. The intracellular Ca2+ stores of the peptic cells in the gastric gland remained depleted of Ca2+ until specific antagonists were added. The reloading of intracellular stores required medium Ca2+ although [Ca2+]i was maintained at resting level during the entire reloading period. Hence, a specialized pathway of Ca2+ reloading is postulated.     

Ca2+ clearance mechanisms in neurohypophysial terminals of the rat

The importance of intracellular calcium ([Ca2+]i) in the release of vasopressin (AVP) and oxytocin from the central nervous system neurohypopyhysial nerve terminals has been well-documented. To date, there is no clear understanding of Ca2+ clearance mechanisms and their interplay with transmembrane Ca2+ entry, intracellular [Ca2+]i transients, cytoplasmic Ca2+ stores and hence the release of AVP at the level of a single nerve terminal. Here, we studied the mechanism of Ca2+ clearance in freshly isolated nerve terminals of the rat neurohypophysis using Fura-2 Ca2+ imaging and measured the release of AVP by radioimmuno assay. An increase in the K+ concentration in the perfusion solution from 5 to 50 mM caused a rapid increase in [Ca2+]i and AVP release. Returning K+ concentration to 5 mM led to rapid restoration of both responses to basal level. The K+-evoked [Ca2+]i and AVP increase was concentration-dependent, reliable, and remained of constant amplitude and time course upon successive applications. Extracellular Ca2+ removal completely abolished the K+-evoked responses. The recovery phase was not affected upon replacement of NaCl with sucrose or drugs known to act on intracellular Ca2+ stores such as thapsigargin, cyclopiazonic acid, caffeine or a combination of caffeine and ryanodine did not affect either resting or K+-evoked [Ca2+]i or AVP release. By contrast, the plasma membrane Ca2+ pump inhibitor, La3+, markedly slowed down the recovery phase. The mitochondrial respiration uncoupler, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), slightly but significantly increased the basal [Ca2+]i, and also slowed down the recovery phase of both [Ca2+]i and release responses. In conclusion, we show in nerve terminals that (i) Ca2+ extrusion through the Ca2+ pump in the plasma membrane plays a major role in the Ca2+ clearance mechanisms of (ii) Ca2+ uptake by mitochondria also contributes to the Ca2+ clearance and (iii) neither Na+/Ca2+ exchangers nor Ca2+ stores are involved in the Ca2+ clearance or in the maintenance of basal [Ca2+]i or release of AVP.     

Receptor-mediated calcium influx in PC12 cells. ATP and bradykinin activate two independent pathways

In the neurosecretory cell line PC12 the cytosolic free Ca2+ concentration, [Ca2+]i, and membrane potential were affected by both external ATP and the nonapeptide bradykinin, BK. The latter caused a rapid and large release of Ca2+ from intracellular stores (Ca2+ redistribution) and, in the presence of external Ca2+, a long lasting, but moderate Ca2+ influx, which was insensitive to dihydropyridine blockers. On the contrary, ATP evoked a [Ca2+]i rise which rapidly inactivated. At least three different mechanisms accounted for the ATP-induced increase in [Ca2+]i: less than 20% of the total response was due to intracellular Ca2+ redistribution, consistent with a small increase in inositol 1,4,5-trisphosphate level; the rest (over 80%) was equally accounted for by ATP-activated cation channels and voltage-gated Ca2+ channels. ATP and BK (the latter after K+ channel blockade) caused plasma membrane depolarization. With both agonists the inward current was carried by both Na+ and Ca2+, although the BK-activated current appeared to be more selective for Ca2+. Channels triggered by ATP and BK differed not only in their cation selectivity, but also in modulation by both [Ca2+]i and drugs such as the phorbol ester phorbol 12-myristate 13-acetate and the new antagonist of ligand-activated Ca2+ influx, SK&F 96365.     

Effect of methylglyoxal on intracellular calcium levels and viability in renal tubular cells

Methylglyoxal (2-oxopropanal), a physiological glucose metabolite, is a highly reactive dicarbonyl compound that can induce stress in cells and cause apoptotic cell death. This study examines the early signaling effects of methylglyxal on renal cells. It was found that methylglyoxal caused a slow and sustained rise of intracellular Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner (EC50=1.8 mM). Methylglyoxal also induced a [Ca2+]i rise when extracellular Ca2+ was removed, but the magnitude was reduced by 80%. Depletion of intracellular Ca2+ stores with thapsigargin (TG), an endoplasmic reticulum (ER) Ca2+ pump inhibitor, did not affect methylglyoxal's effect. In Ca2+-free medium, the methylglyoxal-induced [Ca2+]i rise was abolished by depleting stored Ca2+ with carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler). Methylglyoxal-caused [Ca2+]i rise in the Ca2+-containing medium was not affected by modulation of protein kinase C activity, presence of voltage-gated Ca2+ channel blockers, or preincubation with thiol-containing antioxidants. U73122, an inhibitor of phospholipase C, abolished ATP (but not methylglyoxal)-induced [Ca2+]i rise. Furthermore, the [Ca2+]i-elevating effect of methylglyoxal was cell type-dependent, because methylglyoxal failed to cause [Ca2+]i rises in CHO-K1, neutrophils, or platelets. Pretreatment with methylglyoxal for 0-24 h decreased cell viability in a concentration- and time-dependent manner. Meanwhile, methylglyoxal-induced cell death involved apoptotic and necrotic events, the former being the dominant. These findings suggest that methylglyoxal induced a significant rise in [Ca2+]i in Madin-Darby canine kidney (MDCK) renal tubular cells by stimulating both extracellular Ca2+ influx and CCCP-sensitive intracellular Ca2+ release via as yet unidentified mechanisms. The cell type-specific Ca2+ signaling may play an important role in the early process of cytotoxic action of methylglyoxal.     

Mechanisms of [Ca2+]i elevation by H2O2 in islets of rats

Activity of reactive oxygen species is elevated in diabetes mellitus and has been implicated in the destruction of cellular components. The toxic effect of reactive oxygen species was investigated by testing the effect of H2O2 on [Ca2+]i in isolated islets of Langehans. H2O2 increased [Ca2+]i in a dose-dependent manner, which was irreversible at high concentrations. The maximum effect of H2O2 on [Ca2+]i was larger than those of KCl, glucose, ATP, carbachol and endothelin-1. The effect of H2O2 was only partially attenuated by removal of external Ca2+ and by the in-organic Ca2+ channel blocker nickel, but was not blocked by voltage-dependent or -independent Ca2+ channel blockers nimodipine, nicardipine, SK&F 96365, econazole and lanthanum. H2O2, disrupted [Ca2+]i homeostasis in islets by affecting both release and influx of Ca2+ and causing dysfunction of Ca2+ clearance systems and may contribute to the pathological process of diabetes.     

Chronic intrauterine pulmonary hypertension increases capacitative calcium entry in fetal pulmonary artery smooth muscle cells

Oxygen causes perinatal pulmonary dilatation. Although fetal pulmonary artery smooth muscle cells (PA SMC) normally respond to an acute increase in oxygen (O2) tension with a decrease in cytosolic calcium ([Ca2+]i), an acute increase in O2 tension has no net effect on [Ca(2+)](i) in PA SMC derived from lambs with chronic intrauterine pulmonary hypertension (PHTN). The present experimental series tests the hypothesis that an acute increase in O2 tension decreases capacitative calcium entry (CCE) in normal, but not hypertensive, fetal PA SMC. PA SMC were isolated from late-gestation fetal lambs after either ligation of the ductus arteriosus (PHTN) or sham (control) operation at 127 days gestation. PA SMC were isolated from the distal PA (>or=4th generation) and maintained under hypoxic conditions ( approximately 25 Torr) in primary culture. After fura 2 loading, apparent [Ca2+]i in PA SMC was determined as the ratio of 340- to 380-nm fluorescence intensity. Under both hypoxic and normoxic conditions, cyclopiazonic acid (CPA) increased [Ca2+]i more in PHTN than in control PA SMC. CCE was determined in PA SMC under hypoxic and normoxic conditions, after superfusion with zero extracellular Ca2+ and intracellular store depletion with CPA, followed by superfusion with Ca2+-containing solution, in the presence of the voltage-operated calcium channel blockade. CCE was increased in PHTN compared with control PA SMC under conditions of both acute and sustained normoxia. Transient receptor potential channel gene expression was greater in control compared with PHTN PA SMC. PHTN may compromise perinatal pulmonary vasodilation, in part, by modulating PA SMC CCE.     

Evidence for receptor-mediated calcium entry and refilling of intracellular calcium stores in FRTL-5 rat thyroid cells.

The aim of the present study was to investigate the relationship between agonist-induced changes in intracellular free Ca2+ ([Ca2+]i) and the refilling of intracellular Ca2+ stores in Fura 2-loaded thyroid FRTL-5 cells. Stimulating the cells with ATP induced a dose-dependent increase in ([Ca2+]i). The ATP-induced increase in [Ca2+]i was dependent on both release of sequestered intracellular Ca2+ as well as influx of extracellular Ca2+. Addition of Ni2+ prior to ATP blunted the component of the ATP-induced increase in [Ca2+]i dependent on influx of Ca2+. In cells stimulated with ATP in a Ca(2+)-free buffer, readdition of Ca2+ induced a rapid increase in [Ca2+]i; this increase was inhibited by Ni2+. In addition, the ATP-induced influx of 45Ca2+ was blocked by Ni2+. Stimulating the cells with noradrenaline (NA) also induced release of sequestered Ca2+ and an influx of extracellular Ca2+. When cells were stimulated first with NA, a subsequent addition of ATP induced a blunted increase in [Ca2+]i. If the action of NA was terminated by addition of prazosin, and ATP was then added, the increase in [Ca2+]i was restored to control levels. Addition of Ni2+ prior to prazosin inhibited the restoration of the ATP response. In the presence of extracellular Mn2+, ATP stimulated quenching of Fura 2 fluorescence. The quenching was probably due to influx of Mn2+, as it was blocked by Ni2+. The results thus suggested that stimulating release of sequestered Ca2+ in FRTL-5 cells was followed by influx of extracellular Ca2+ and rapid refilling of intracellular Ca2+ stores.     

Platelet-derived growth factor and angiotensin II cause increases in cytosolic free calcium by different mechanisms in vascular smooth muscle cells

Platelet-derived growth factor (PDGF) and angiotensin II (AII) are thought to mediate their biological effects in vascular smooth muscle cells (VSMCs) by causing alterations in cytosolic free calcium ([ Ca2+]i). In this study we examine the pathways by which PDGF and AII alter [Ca2+]i in VSMCs. Addition of PDGF resulted in a rapid, transient, concentration-dependent increase in [Ca2+]i; this rise in [Ca2+]i was blocked completely by preincubation of cells with ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) or CoCl2, by the voltage-sensitive Ca2+-channel antagonists verapamil or nifedipine, by 12-O-tetradecanoylphorbol-13-acetate (TPA), or by pertussis toxin. AII also caused an increase in [Ca2+]i; however, AII-stimulated alterations in [Ca2+]i displayed different kinetics compared with those caused by PDGF. Pretreatment of cells with 8-(diethylamine)-octyl-3,4,5-trimethyoxybenzoate hydrochloride (TMB-8), almost totally inhibited AII-induced increases in [Ca2+]i. EGTA or CoCl2 only slightly diminished AII-stimulated increases in [Ca2+]i. Nifedipine, verapamil, TPA, and pertussis toxin pretreatment were without effect on AII-induced increases in [Ca2+]i. PDGF and AII both stimulated increases in total inositol phosphate accumulation, although the one-half maximal concentration (ED50) for alterations in [Ca2+]i and phosphoinisitide hydrolysis differed by a factor of 10 for PDGF (3 X 10(-10) M for Ca2+ vs. 2.5 X 10(-9) M for phosphoinositide hydrolysis), but they were essentially identical for AII (7.5 X 10(-9) M for Ca2+ vs. 5.0 X 10(-9) M for phosphoinositide hydrolysis). PDGF stimulated mitogenesis (as measured by [3H]-thymidine incorporation into DNA) in VSMCs with an ED50 similar to that for PDGF-induced alterations in phosphoinositide hydrolysis. PDGF-stimulated mitogenesis was blocked by pretreatment of cells with voltage-sensitive Ca2+ channel blockers, TPA, or pertussis toxin. These results suggest that PDGF and AII cause alterations in [Ca2+]i in VSMCs by at least quantitatively distinct mechanisms. PDGF binding activates a pertussis-toxin-sensitive Ca2+ influx into cells via voltage-sensitive Ca2+ channels (blocked by EGTA, verapamil, and nifedipine), as well as stimulating phosphoinositide hydrolysis leading to release of Ca2+ from intracellular stores. AII-induced alterations in [Ca2+]i are mainly the result of phosphoinositide hydrolysis and consequent entry of Ca2+ into the cytoplasm from intracellular stores. Our data also suggest that changes in [Ca2+]i caused by PDGF are required for PDGF-stimulated mitogenesis.     

Effect of nortriptyline on intracellular Ca2+ handling and viability in canine renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, the effect of nortriptyline, an antidepressant, on intracellular Ca2+ concentration ([Ca2+]i) was measured by using fura-2. Nortriptyline (> 10 microM) caused a rapid increase of [Ca2+]i in a concentration-dependent manner (EC50 = 75 microM). Nortriptyline-induced [Ca2+]i increase was prevented by 40% by removal of extracellular Ca2+ but was not altered by voltage-gated Ca2+ channel blockers. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca2+]i, increase, after which the increasing effect of nortriptyline on [Ca2+], was abolished; also, pretreatment with nortriptyline reduced a large portion of thapsigargin-induced [Ca2+]i increase. U73122, an inhibitor of phospholipase C, abolished ATP (but not nortriptyline)-induced [Ca2+]i increase. Overnight incubation with 10 microM nortriptyline decreased cell viability by 16%, and 50 microM nortriptyline killed all cells. Prechelation of cytosolic Ca2+ with BAPTA did not alter nortriptyline-induced cell death. These findings suggest that nortriptyline rapidly increased [Ca2+]i in renal tubular cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release, and was cytotoxic at higher concentrations in a Ca(2+)-dissociated manner.     

The T-cell antigen receptor regulates sustained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influx and ongoing intracellular Ca2+ mobilization.

Signal transduction by the T-cell antigen receptor involves the turnover of polyphosphoinositides and an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is due initially to the release of Ca2+ from intracellular stores, but is sustained by the influx of extracellular Ca2+. To examine the regulation of sustained antigen-receptor-mediated increases in [Ca2+]i, we studied the relationships between extracellular Ca2+ influx, the mobilization of Ca2+ from intracellular stores, and the contents of inositol polyphosphates after stimulation of the antigen receptor on a human T-cell line, Jurkat. We demonstrate that sustained antigen-receptor-mediated increases in [Ca2+]i are associated with ongoing depletion of intracellular Ca2+ stores. When antigen-receptor-ligand interactions are disrupted, [Ca2+]i and inositol 1,4,5-trisphosphate return to basal values over 3 min. Under these conditions, intracellular Ca2+ stores are repleted if extracellular Ca2+ is present. There is a tight temporal relationship between the fall in [Ca2+]i, the return of inositol 1,4,5-trisphosphate to basal values, and the repletion of intracellular Ca2+ stores. Reversal of the increase in [Ca2+]i preceeds any fall in inositol tetrakisphosphate by 2 min. These studies suggest that sustained antigen-receptor-induced increases in [Ca2+]i, although dependent on extracellular Ca2+ influx, are also regulated by ongoing inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ mobilization. In addition, an elevated concentration of inositol tetrakisphosphate in itself is insufficient to sustain an increase in [Ca2+]i within Jurkat cells.     

Effects of acetylcholine, TSH and other stimulators on intracellular calcium concentration in dog thyroid cells

The intracellular free calcium concentration, [Ca2+]i, has been measured in dog thyroid cells using the fluorescent Ca2+-indicator, quin2. Acetylcholine or its non-hydrolyzable analog, carbamylcholine rapidly increased [Ca2+]i by 40 +/- 4% (mean +/- SE) over the basal level of 81 +/- 2 nM. This increase was totally abolished by atropine, a muscarinic cholinergic receptor blocker, but was not influenced by verapamil, a voltage dependent-calcium channel blocker. Depletion of extracellular Ca2+ by the addition of EGTA, diminished but did not abolish the response to carbamylcholine. These data suggest that cholinergic effectors increase [Ca2+]i by mobilization of Ca2+ from intracellular stores rather than from an influx of Ca2+. Addition of TSH, isoproterenol, phorbol ester, dibutyryl cyclic GMP or cyclic AMP did not elicit any change in [Ca2+]i suggesting that their action may not involve any mobilization of intracellular Ca2+. These data provide direct evidence that in the thyroid cell, cholinergic agents act via their receptors to cause a rapid increase in [Ca2+]i, which may mediate their metabolic effects.     

Stimulus-Induced Oscillations in Guard Cell Cytosolic Free Calcium

Ca2+ is implicated as a second messenger in the response of stomata to a range of stimuli. However, the mechanism by which stimulus-induced increases in guard cell cytosolic free Ca2+ ([Ca2+]i) are transduced into different physiological responses remains to be explained. Oscillations in [Ca2+]i may provide one way in which this can occur. We used photometric and imaging techniques to examine this hypothesis in guard cells of Commelina communis. External Ca2+ ([Ca2+]e), which causes an increase in [Ca2+]i, was used as a closing stimulus. The total increase in [Ca2+]i was directly related to the concentration of [Ca2+]e, both of which correlated closely with the degree of stomatal closure. Increases were oscillatory in nature, with the pattern of the oscillations dependent on the concentration of [Ca2+]e. At 0.1 mM, [Ca2+]e induced symmetrical oscillations. In contrast, 1.0 mM [Ca2+]e induced asymmetric oscillations. Oscillations were stimulus dependent and modulated by changing [Ca2+]e. Experiments using Ca2+ channel blockers and Mn2+-quenching studies suggested a role for Ca2+ influx during the oscillatory behavior without excluding the possible involvement of Ca2+ release from intracellular stores. These data suggest a mechanism for encoding the information required to distinguish between a number of different Ca2+-mobilizing stimuli in guard cells, using stimulus-specific patterns of oscillations in [Ca2+]i.     

Con A刺激致T淋巴细胞胞浆游离Ca~(2+)浓度升高

本文分别应用荧光Ca~(2+)指示剂Quin2和Indo-1研究了Con A刺激的T淋巴细胞[Ca~(2+)]i升高过程及其发生机制.结果表明Con A与T淋巴细胞作用可导致细胞[Ca~(2+)]i的迅速升高.这种增加的胞内游离Ca~(2+)不仅来自胞外Ca~(2+)的内流,也来源于胞内钙库的释放.其中Ca~(2+)内流与T细胞钙通道的开放有关.可被钙通道抑制剂戊脉胺抑制,细胞的去极化及钾通道阻断剂四乙胺均不能阻断Ca~(2+)的内流,提示Ca~(2+)内流不是通过电位操纵的钙通道实现的,也与拥通道的开闭无关.Ca~(2+)内流可能是通过Con A受体活化的受体操纵的钙通道而实现的.     

Endothelin isopeptides evoke Ca2+ signaling and oscillations of cytosolic free [Ca2+] in human mesangial cells

Isopeptides of the newly discovered peptide family, endothelins (ET), caused a concentration-dependent increase in intracellular free [Ca2+] ([Ca2+]i) in human glomerular mesangial cells. ET isopeptides and sarafotoxin S6b caused transient and sustained [Ca2+]i waveforms which resulted from mobilization of intracellular Ca2+ stores and from Ca2+ influx through a dihydropyridine-insensitive Ca2+ channel. Ca2+ signaling evoked by ET isopeptides underwent a marked adaptive, desensitization response. Although activation of protein kinase C attenuated ET-induced Ca2+ signaling, desensitization by ET isopeptides was independent of protein kinase C. High concentrations of ET-1 and ET-2 also caused oscillations of [Ca2+]i that partially depended on extracellular Ca2+. These results suggest that an increase in [Ca2+]i constitutes a common pathway of signal transduction for the ET peptide family.