Characterisation of the serotonin efflux induced by cytosolic Ca2+ and Na+ concentration increase in human platelets.

BACKGROUND/AIM: The present study aimed at elucidating the mechanism(s) of serotonin (5-HT) efflux induced by thapsigargin from human platelets in the absence of extra-cellular Ca2+. METHODS: Efflux of pre-loaded radiolabeled serotonin was generally determined by filtration techniques. Cytosolic concentrations of Ca2+, Na+ and H+ were measured with appropriate fluorescent probes. RESULTS: 5-HT efflux from control or reserpine-treated platelets--where reserpine prevents 5-HT transport into the dense granules--was proportional to thapsigargin evoked cytosolic [Ca2+]c increase. Accordingly factors as prostacyclin, aspirin and calyculin which reduced [Ca2+]c-increase also inhibited the 5-HT efflux. Thapsigargin, which also caused a remarkable increase in cytosolic [Na+]c, promoted less 5-HT release, in parallel to lower [Na+]c and [Ca2+]c increase, when added to platelet suspensions containing low [Na+]. The Na+/H+ exchanger monensin increased the [Na+]c and induced 5-HT efflux without affecting the Ca2+ level. The 5-HT efflux induced by both [Ca2+] or [Na+]c increase did not depend on pH or membrane potential changes, whereas it decreased in the absence of extra-cellular K+, and increased in the absence of Cl- or Na+. CONCLUSION: Increases in [Ca2+]c and [Na+]c independently induce serotonin efflux through the outward directed plasma membrane serotonin transporter SERT. This event might be physiologically important at the level of capillaries or narrowed arteries where platelets are subjected to high shear stress which causes [Ca2+]c increase followed by 5-HT release which might exert vasodilatation.     

Elevated cytosolic Ca2+ activates phospholipase D in human platelets

We have examined the activation of phospholipase D in human platelets treated with alpha-thrombin. When incubated with 1-O-[9,10-3H2]hexadecyl-2-lysophosphatidylcholine (PtdCho) and 1-alkyl-[32P]lysoPtdCho for 2 h, platelets formed 3H/32P-labeled PtdCho in a ratio of 11:1. After incubation of such labeled platelets with alpha-thrombin for 5 min, increased accumulation of 3H/32P-labeled phosphatidic acid (PtdOH) was detected in the same ratio, indicating the action of phospholipase D. The Ca2+ ionophore A23187 and alpha-thrombin each stimulated the formation of labeled PtdOH as above in a time- and concentration-dependent manner, with only minor changes in labeled diglyceride. A23187 was able to cause increases in labeled PtdOH comparable to those observed with alpha-thrombin. beta-Phorbol 12,13-dibutyrate, an activator of protein kinase C, only slightly stimulated the accumulation of labeled PtOH. The protein kinase C inhibitor, staurosporine, totally blocked these changes but only slightly inhibited the increases in labeled PtdOH promoted by alpha-thrombin. These results suggest that an increase in intracellular Ca2+, rather than protein kinase C activity, is a major factor regulating phospholipase D in platelets exposed to alpha-thrombin. We have also examined the relative contributions of phospholipase D and diglyceride kinase (following phospholipase C action) to PtdOH accumulation in [32P]Pi-labeled platelets by comparing the 32P-specific radioactivities of PtdOH, PtdCho, and metabolic gamma-ATP in control and alpha-thrombin-exposed platelets. Based on these determinations, we conclude that 13 and 87% of incremental PtdOH in human platelets exposed to alpha-thrombin arises via phospholipase D acting on PtdCho and phospholipase C/diglyceride kinase, respectively.     

Inhibition by antioxidants of agonist evoked cytosolic Ca++ increase, ATP secretion and aggregation of aspirinated human platelets

The synthetic antioxidants butylated hydroxytoluene (BHT), nordihydroguaiaretic acid and the one-electron donor 1,1'-dimethylferrocene, inhibit cytosolic Ca++ increase, shape change, aggregation and ATP secretion in aspirinated washed human platelets stimulated by thrombin, vasopressin and platelet-activating factor. The antioxidants also inhibit cytosolic Ca++ increase originating from intracellular stores in the presence of EGTA. The effect of phorbol ester (TPA), which promotes platelet aggregation and secretion without raising the cytosolic Ca++, is also antioxidant-sensitive. Since agonist activation of aspirinated platelets does not involve cyclooxygenase or lipoxygenase metabolites, it is suggested that other yet unknown free radical-dependent pathways are involved in the mechanism of platelet activation, both in the protein kinase C-independent events leading to the cytosolic Ca++ increase, and in those, largely protein kinase C-dependent, leading to aggregation and ATP secretion.     

Ca2+ mobilization primes protein kinase C in human platelets. Ca2+ and phorbol esters stimulate platelet aggregation and secretion synergistically through protein kinase C.

Low concentrations of Ca2+-mobilizing agonists such as vasopressin, platelet-activating factor, ADP, the endoperoxide analogue U44069 and the Ca2+ ionophore A23187 enhance the binding of [3H]phorbol 12,13-dibutyrate (PdBu) to intact human platelets. This effect is prevented by preincubation of platelets with prostacyclin (except for A23187). Adrenaline, which does not increase Ca2+ in the platelet cytosol, does not enhance the binding of [3H]PdBu to platelets. In addition, all platelet agonists except adrenaline potentiate the phosphorylation of the substrate of protein kinase C (40 kDa protein) induced by PdBu. Potentiation of protein kinase C activation is associated with increased platelet aggregation and secretion. Stimulus-induced myosin light-chain phosphorylation and shape change are not significantly affected, but formation of phosphatidic acid is decreased in the presence of PdBu. The results may indicate that low concentrations of agonists induce in intact platelets the translocation of protein kinase C to the plasma membrane by eliciting mobilization of Ca2+, and thereby place the enzyme in a strategic position for activation by phorbol ester. Such activation enhances platelet aggregation and secretion, but at the same time suppresses activation of phospholipase C. Therefore, at least part of the synergism evoked by Ca2+ and phorbol ester is mediated through a single pathway which involves protein kinase C. It is likely that the priming of protein kinase C by prior Ca2+ mobilization occurs physiologically in activated platelets.     

Human platelets use a cytosolic Ca2+ nanodomain to activate Ca2+-dependent shape change independently of platelet aggregation

Human platelets use a rise in cytosolic Ca²⁺ concentration to activate all stages of thrombus formation, however, how they are able to decode cytosolic Ca²⁺ signals to trigger each of these independently is unknown. Other cells create local Ca²⁺ signals to activate Ca²⁺-sensitive effectors specifically localised to these subcellular regions. However, no previous study has demonstrated that agonist-stimulated human platelets can generate a local cytosolic Ca²⁺ signal. Platelets possess a structure called the membrane complex (MC) where the main intracellular calcium store, the dense tubular system (DTS), is coupled tightly to an invaginated portion of the plasma membrane called the open canalicular system (OCS). Here we hypothesised that human platelets use a Ca²⁺ nanodomain created within the MC to control the earliest phases of platelet activation. Dimethyl-BAPTA-loaded human platelets were stimulated with thrombin in the absence of extracellular Ca²⁺ to isolate a cytosolic Ca²⁺ nanodomain created by Ca²⁺ release from the DTS. In the absence of any detectable rise in global cytosolic Ca²⁺ concentration, thrombin stimulation triggered Na⁺/Ca²⁺ exchanger (NCX)-dependent Ca²⁺ removal into the extracellular space, as well as Ca²⁺-dependent shape change in the absence of platelet aggregation. The NCX-mediated Ca²⁺ removal was dependent on the normal localisation of the DTS, and immunofluorescent staining of NCX3 demonstrated its localisation to the OCS, consistent with this Ca²⁺ nanodomain being formed within the MC. These results demonstrated that human platelets possess a functional Ca²⁺ nanodomain contained within the MC that can control shape change independently of platelet aggregation.     

Modulation by external Ca2+ and nicardipine of Ca2+ influx and cytosolic concentration in human erythrocytes

Variations of Ca2+ influx (evaluated by the initial rate of 45Ca2+ uptake) and cytosolic free Ca2+ concentration ([Ca2+]i, measured with fura-2) were investigated in human erythrocytes. When external Ca2+ concentration ([Ca2+]o) rose from 1 to 2 mM, the initial rate of Ca2+ influx nearly doubled whereas [Ca2+]i increased only by 15%. Nicardipine dose-dependently decreased both initial rate of Ca2+ influx and [Ca2+]i (up to 53 and 18%. respectively at 10(-6) M). The less marked changes in [Ca2+]i than in Ca2+ influx indicate a partial adjustment of the Ca2+ extruding-pump activity to of Ca2+ influx. In vivo administration of nicardipine reduced [Ca2+]i only when its initial value exceeded 80 nM and prevented the rise in [Ca2+]i induced by the increase in [Ca2+]o. Our results indicate that nicardipine may reduce Ca2+ influx in human erythrocytes and participate in the control of [Ca2+]i when elevated.     

Synergistic regulation of cytosolic Ca2+ concentration by somatostatin and alpha 1-adrenergic agonists in mouse astrocytes.

The effects of somatostatin and alpha 1-adrenergic receptor agonists on cytosolic Ca2+ in striatal astrocytes from the embryonic mouse in primary culture have been investigated by microfluorimetry. Methoxamine or somatostatin induced a transitory increase in cytosolic Ca2+, but their combined addition led to a sustained increase in cytosolic Ca2+ which seems to be due to a Ca2+ influx since it was not observed in the absence of external Ca2+. Voltage-independent Ca2+ channels contribute to this process. Indeed, voltage-operated calcium channels are not involved since neither dihydropyridines nor La3+ were effective in suppressing the sustained cytosolic Ca2+ elevation. Moreover, depolarization by 50 mM KCl, which was ineffective alone, suppressed the effect of somatostatin observed in the presence of the alpha 1 agonist, methoxamine. The implication of arachidonic acid in the observed potentiation is suggested by the following observations: 1) arachidonic acid induced a sustained elevation of cytosolic Ca2+ similar to that evoked by the co-application of methoxamine and somatostatin; 2) the addition of ETYA, an inactive and non-metabolizable analogue of arachidonic acid suppressed the calcium plateau produced by the agonists. In addition, direct activation of PKC by an exogeneous diacylglycerol analogue allowed somatostatin alone to evoke a sustained elevation of cytosolic Ca2+. Therefore, methoxamine through the successive activation of PLC and PKC could allow a lipase, probably PLA2, to be stimulated by somatostatin. Since arachidonic acid has already been shown to trigger the opening of K+ channels and the formation of inositol phosphates, somatostatin, through the arachidonic acid-mediated hyperpolarization could increase the Ca2+ driving force and thus improve Ca2+ influx through the inositol phosphate gated channels.     

Biphasic regulation of mitochondrial Ca2+ uptake by cytosolic Ca2+ concentration

A rise in cytosolic Ca(2+) concentration is used as a key activation signal in virtually all animal cells, where it triggers a range of responses including neurotransmitter release, muscle contraction, and cell growth and proliferation [1]. During intracellular Ca(2+) signaling, mitochondria rapidly take up significant amounts of Ca(2+) from the cytosol, and this stimulates energy production, alters the spatial and temporal profile of the intracellular Ca(2+) signal, and triggers cell death [2-10]. Mitochondrial Ca(2+) uptake occurs via a ruthenium-red-sensitive uniporter channel found in the inner membrane [11]. In spite of its critical importance, little is known about how the uniporter is regulated. Here, we report that the mitochondrial Ca(2+) uniporter is gated by cytosolic Ca(2+). Ca(2+) uptake into mitochondria is a Ca(2+)-activated process with a requirement for functional calmodulin. However, cytosolic Ca(2+) subsequently inactivates the uniporter, preventing further Ca(2+) uptake. The uptake pathway and the inactivation process have relatively low Ca(2+) affinities of approximately 10-20 microM. However, numerous mitochondria are within 20-100 nm of the endoplasmic reticulum, thereby enabling rapid and efficient transmission of Ca(2+) release into adjacent mitochondria by InsP(3) receptors on the endoplasmic reticulum. Hence, biphasic control of mitochondrial Ca(2+) uptake by Ca(2+) provides a novel basis for complex physiological patterns of intracellular Ca(2+) signaling.     

Simultaneous flow cytometric measurements of thrombin-induced cytosolic pH and Ca2+ fluxes in human platelets

Human platelets exhibit an extremely rapid increase in cytoplasmic Ca2+ concentrations ((Ca2+]in) and a dose-dependent cytoplasmic pH change ((pH]in) upon thrombin stimulation. A cytoplasmic alkalinization, maximal by 60 s, is preceded by a very rapid acidification, which is masked by the alkalinization when saturating thrombin doses are used. Using the pH probe 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein we report here the kinetics of simultaneous cytoplasmic pH and Ca2+ changes in thrombin-stimulated platelets, measured in single cells by flow cytometry. This permits analysis of the responding subpopulation. Maximal thrombin stimulation (greater than or equal to 4.5 nM) induces a dose-dependent increase in pHin from approximately 7.0 to 7.30 and a maximal [Ca2+]in transient of up to 800 nM. The Ca2+ transient coincides temporally with the rapid initial acidification, while the alkalinization is maximal considerably later. The Ca2+ transients occur maximally in each responding cell, but occur only in a subpopulation of the platelets at subsaturating (less than 4.5 nM) thrombin doses; in contrast, the dose-dependent cytoplasmic acidification appears to occur uniformly in all platelets. The rapid increase in [Ca2+]in is not dependent on the alkalinization, and the former occurs maximally in amiloride treated, Na+/H+ exchange inhibited human platelets. These results indicate that the acidification and the rise in [Ca2+]in may be interrelated, whereas the cytoplasmic alkalinization (maximal considerably later than either the acidification or the [Ca2+]in rise) may be independent of these earlier, temporally correlated increases in H+ and Ca2+ concentrations.     

血小板激活时胞质游离钙浓度与两种颗粒数变化的相关性

用电镜形态计量法检测血小板α颗粒（αＧ）和致密颗粒（ｄＧ）的数密度,用钙荧光指示剂Ｆｕｒａ２检测血小板胞质游离Ｃａ＾２＋浓度（「Ｃａ＾２＋」ｉ）,观察到在钙离子导体Ａ２３１８７作用下,血小板「Ｃａ＾２＋」ｉ明显升高。凝血酶与ＡＤＰ也都分别引起「Ｃａ＾２＋」ｉ升高,且有浓度依赖性,选用三种激动剂的不同量以反映血小板不同程度激活时,测定「Ｃａ＾２＋」与颗粒数密度,分析两者间的相关性,发现αＧ和ｄＧ的数     

Two thrombin-activated Ca2+ channels in human platelets.

The regulation of extracellular Ca2+ entry into fura-2-loaded human platelets was examined following stimulation with thrombin. In the presence of external Ca2+, stimulation of platelets with thrombin resulted in a rapid increase, followed by a plateau, in intracellular Ca2+ concentration ([Ca2+]i). Pretreatment with wortmannin, a specific inhibitor of myosin light chain kinase, suppressed only the plateau phase and had no effect on the initial rapid increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thrombin induced a transient and relatively small increase in [Ca2+]i caused by Ca2+ release from internal stores. When Ca2+ was added subsequently to the Ca(2+)-free medium within 10 min after thrombin activation, a marked increase in [Ca2+]i was seen, reflecting thrombin-stimulated external Ca2+ entry. With the Ca(2+)-free medium, wortmannin did not affect either the Ca2+ mobilization from the internal stores or the rapid external Ca2+ entry at early time points (within 5 s) after thrombin stimulation, whereas it significantly inhibited Ca2+ entry when Ca2+ was added later (at 3 min). Wortmannin inhibition of this late Ca2+ entry and that of 20-kDa myosin light chain phosphorylation after thrombin stimulation were dose- and preincubation time-dependent and correlated well with each other. These results suggest that two different channels are responsible for Ca2+ entry in human platelets at the early and late phases of thrombin stimulation and that the channel responsible for the late phase of Ca2+ entry may be activated by a mechanism involving myosin light chain kinase.     

Single islet beta-cell stimulation by nutrients: relationship between pyridine nucleotides, cytosolic Ca2+ and secretion.

It is generally believed that the initiation of insulin secretion by nutrient stimuli necessitates the generation of metabolic coupling factors, leading to membrane depolarization and the gating of voltage-sensitive Ca2+ channels. To establish this sequence of events, the kinetics of endogenous fluorescence of reduced pyridine nucleotides [NAD(P)H], reflecting nutrient metabolism, were compared to those of cytosolic calcium ([Ca2+]i) rises in single cultured rat islet beta-cells. In preliminary experiments, the loss of quinacrine fluorescence from prelabelled cells was used as an indicator of secretion. This dye is concentrated in the acidic insulin-containing secretory granules. Both glucose and 2-ketoisocaproate (KIC) raised [Ca2+]i in a dose-dependent manner. There was marked cellular heterogeneity in the [Ca2+]i response patterns. The two nutrient stimuli also increased NAD(P)H fluorescence, again showing cell-to-cell variations. In combined experiments, where the two parameters were measured in the same cell, the elevation of the NAD(P)H fluorescence preceded the rise in [Ca2+]i, confirming the statistical evaluation performed on separate cells. The application of two consecutive glucose challenges revealed coordinated changes in [Ca2+]i and NAD(P)H fluorescence. Finally, quinacrine secretion was stimulated by two nutrients with onset times similar to those recorded for [Ca2+]i elevations. These results clearly demonstrate that increased metabolism occurs during the lag period preceding Ca2+ influx via voltage-sensitive Ca2+ channels, a prerequisite for the triggering of insulin secretion by nutrient stimuli.     

Stimulus-secretion coupling in bovine parathyroid cells. Dissociation between secretion and net changes in cytosolic Ca2+

The relationship between the concentration of cytosolic free Ca2+ ([Ca2+]i) and secretion of parathyroid hormone (PTH) was investigated in isolated bovine parathyroid cells using the fluorescent Ca2+ indicator, quin 2. Increasing the concentration of extracellular Ca2+ from 0.5 to 2.0 mM caused a 3-fold increase in [Ca2+]i (from 183 +/- 4 to 568 +/- 21 nM) which was associated with a 2-4-fold decrease in secretion of PTH. Decreasing extracellular Ca2+ to about 1 microM caused a corresponding fall in [Ca2+]i to 60-90 nM. Extracellular Ca2+-induced changes in [Ca2+]i were not affected by omission of extracellular Na+. Depolarizing concentrations of K+ (30 mM) depressed [Ca2+]i at all concentrations of extracellular Ca examined, and this was associated with increased secretion of PTH. Ionomycin (0.1 or 1 microM) increased [Ca2+]i at extracellular Ca2+ concentrations of 0.5, 1.0, and 2.0 mM, but inhibited secretion of PTH only at Ca concentrations near the "Ca2+ set point" (1.25 microM). In contrast, dopamine, norepinephrine (10 microM each), and Li+ (20 mM) potentiated secretion of PTH without causing any detectable change in [Ca2+]i. The results obtained with these latter secretagogues provide evidence for a mechanism of secretion which is independent of net changes in [Ca2+]i. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) did not alter [Ca2+]i or secretion of PTH at low (0.5 mM) extracellular Ca2+ concentrations. At 2.0 mM extracellular Ca2+, however, TPA (20 nM or 1 microM) depressed [Ca2+]i and potentiated secretion of PTH. The addition of TPA prior to raising the extracellular Ca2+ concentration reduced the subsequent increase in [Ca2+]i. The results show that the effects of TPA on secretion in the parathyroid cell are not readily dissociated from changes in [Ca2+]i and suggest that some TPA-sensitive process, perhaps involving protein kinase C, may be involved in those mechanisms that regulate [Ca2+]i in response to changes in extracellular Ca2+.     

Tributyltin increases cytosolic free Ca2+ concentration in thymocytes by mobilizing intracellular Ca2+, activating a Ca2+ entry pathway, and inhibiting Ca2+ efflux.

The immunotoxic environmental pollutant tri-n-butyltin (TBT) kills thymocytes by apoptosis through a mechanism that requires an increase in intracellular Ca2+ concentration. The addition of TBT (EC50 = 2 microM) to fura-2-loaded rat thymocytes resulted in a rapid and sustained increase in the cytosolic free Ca2+ concentration ([Ca2+]i) to greater than 1 microM. In nominally Ca(2+)-free medium, TBT slightly but consistently increased thymocyte [Ca2+]i by about 0.11 microM. The subsequent restoration of CaCl2 to the medium resulted in a sustained overshoot in [Ca2+]i; similarly, the addition of MnCl2 produced a rapid decrease in the intracellular fura-2 fluorescence in thymocytes exposed to TBT. The rates of Ca2+ and Mn2+ entry stimulated by TBT were essentially identical to the rates stimulated by 2,5-di-(tert.-butyl)-1,4-benzohydroquinone (tBuBHQ), which has previously been shown to empty the agonist-sensitive endoplasmic reticular Ca2+ store and to stimulate subsequent Ca2+ influx by a capacitative mechanism. The addition of excess [ethylenebis(oxyethylenenitrilo)]tetraacetic acid to thymocytes produced a rapid return to basal [Ca2+]i after tBuBHQ treatment but a similar rapid return to basal [Ca2+]i was not observed after TBT treatment. In addition, TBT produced a marked inhibition of both Ca2+ efflux from the cells and the plasma membrane Ca(2+)-ATPase activity. Also, TBT treatment resulted in a rapid decrease in thymocyte ATP level. Taken together, our results show that TBT increases [Ca2+]i in thymocytes by the combination of intracellular Ca2+ mobilization, stimulation of Ca2+ entry, and inhibition of the Ca2+ efflux process. Furthermore, the ability of TBT to apparently mobilize the tBuBHQ-sensitive intracellular Ca2+ store followed by Ca2+ and Mn2+ entry suggests that the TBT-induced [Ca2+]i increase involves a capacitative type of Ca2+ entry.     

Rapid kinetics of agonist-evoked changes in cytosolic free Ca2+ concentration in fura-2-loaded human neutrophils.

The initial kinetics of agonist-evoked rises in the cytosolic Ca2+ concentration [Ca2+]i were investigated in fura-2-loaded human neutrophils by stopped-flow fluorimetry. The rises in [Ca2+]i evoked by chemotactic peptide (fMet-Leu-Phe), platelet-activating factor and ADP all lagged behind agonist addition by 1-1.3 s. Lag times were not significantly different in the presence and in the absence of external Ca2+. Stimulation of the cells in the presence of extracellular Mn2+ resulted in a quench of fluorescence with a similar lag time to [Ca2+]i rise. The delay in onset of the rise in [Ca2+]i evoked by fMet-Leu-Phe was dependent on concentration, becoming longer at lower concentrations of agonist. These results indicate that both the agonist-evoked discharge of the intracellular Ca2+ stores and the generation of bivalent-cation influx lag behind agonist-receptor binding in neutrophils. Both pathways thus appear to be mediated by indirect mechanisms, rather than by a directly coupled process such as a receptor-operated channel. The temporal coincidence of the onset of store discharge with the commencement of bivalent-cation influx suggests that the two events may be causally linked.     

Dual effect of nitric oxide on cytosolic Ca2+ concentration and insulin secretion in rat pancreatic beta-cells

Inisolated rat pancreatic -cells, the nitric oxide (NO) donor NOC-7 at1 µM reduced the amplitude of the oscillations of cytosolicCa²⁺ concentration ([Ca²⁺]_c)induced by 11.1 mM glucose, and at 10 µM terminated them. In thepresence of N^G-nitro-L-arginine(L-NNA), however, NOC-7 at 0.5 and 1 µM increased theamplitude of the [Ca²⁺]_c oscillations,although the NO donor at 10 µM still suppressed them. Aqueous NOsolution also had a dual effect on the[Ca²⁺]_c oscillations. The soluble guanylatecyclase inhibitor LY-83583 and the cGMP-dependent protein kinaseinhibitor KT5823 inhibited the stimulatory effect of NO, and8-bromo-cGMP increased the amplitude of the[Ca²⁺]_c oscillations. Patch-clamp analyses inthe perforated configuration showed that 8-bromo-cGMP inhibited wholecell ATP-sensitive K⁺ currents in the isolated ratpancreatic -cells, suggesting that the inhibition by cGMP ofATP-sensitive K⁺ channels is, at least in part, responsiblefor the stimulatory effect of NO on the[Ca²⁺]_c oscillations. In the presence ofL-NNA, the glucose-induced insulin secretion from isolatedislets was facilitated by 0.5 µM NOC-7, whereas it was suppressed by10 µM NOC-7. These results suggest that NO facilitatesglucose-induced [Ca²⁺]_c oscillations of-cells and insulin secretion at low concentrations, which effectsare mediated by cGMP, whereas NO inhibits them in a cGMP-independentmanner at high concentrations.

     

Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells.

A rapid rise in the level of cytosolic free calcium ([Ca2+]i) is believed to be one of several early triggering signals in the activation of T lymphocytes by antigen. Although Ca2+ release from intracellular stores and its contribution to Ca2+ signaling in many cell types is well documented, relatively little is known regarding the role and mechanism of Ca2+ entry across the plasma membrane. We have investigated mitogen-triggered Ca2+ signaling in individual cells of the human T-leukemia-derived line, Jurkat, using fura-2 imaging and patch-clamp recording techniques. Phytohemagglutinin (PHA), a mitogenic lectin, induces repetitive [Ca2+]i oscillations in these cells peaking at micromolar levels with a period of 90-120 s. The oscillations depend critically upon Ca2+ influx across the plasma membrane, as they are rapidly terminated by removal of extracellular Ca2+, addition of Ca(2+)-channel blockers such as Ni2+ or Cd2+, or membrane depolarization. Whole-cell and perforated-patch recording methods were combined with fura-2 measurements to identify the mitogen-activated Ca2+ conductance involved in this response. A small, highly selective Ca2+ conductance becomes activated spontaneously in whole-cell recordings and in response to PHA in perforated-patch experiments. This conductance has properties consistent with a role in T-cell activation, including activation by PHA, lack of voltage-dependent gating, inhibition by Ni2+ or Cd2+, and regulation by intracellular Ca2+. Moreover, a tight temporal correlation between oscillations of Ca2+ conductance and [Ca2+]i suggests a role for the membrane Ca2+ conductance in generating [Ca2+]i oscillations in activated T cells.     

Ca2+ signaling in human fetoplacental vasculature: effect of CGRP on umbilical vein smooth muscle cytosolic Ca2+ concentration

CGRP is a potent vasodilator with increased levels in fetoplacental circulation during late pregnancy. We have recently demonstrated that acute CGRP exposure to fetoplacental vessels in vitro induced vascular relaxation, but the signaling pathway of CGRP in fetoplacental vasculature remains unclear. We hypothesized that CGRP relaxes fetoplacental vasculature via regulating smooth muscle cytosolic Ca2+ concentrations. In the present study, by using human umbilical vein smooth muscle (HUVS) cells (HUVS-112D), we examined CGRP receptors, cAMP generation, and changes in cellular Ca2+ concentrations on CGRP treatment. These cells express mRNA for CGRP receptor components, calcitonin receptor-like receptor, and receptor activity-modifying protein-1. Direct saturation binding for 125I-labeled CGRP to HUVS cells and Scatchard analysis indicate specificity of the receptors for CGRP [dissociation constant (K(D)) = 67 nM, maximum binding capcity (Bmax) = 2.7 pmol/million cells]. Exposure of HUVS cells to CGRP leads to a dose-dependent increase in intracellular cAMP accumulation, and this increase is prevented by CGRP antagonist CGRP(8-37). Using fura-2-loaded HUVS cells, we monitored the effects of CGRP on intracellular Ca2+ concentration ([Ca2+]i). In the presence of extracellular Ca2+, bradykinin (10(-6) M), a fetoplacental vasoconstrictor, increases HUVS cells [Ca2+]i concentration. CGRP (10(-8) M) abolishes bradykinin-induced [Ca2+]i elevation. When the cells were pretreated with glibenclamide, an ATP-sensitive potassium channel blocker, the CGRP actions on bradykinin-induced Ca2+ influx were profoundly inhibited. In the absence of extracellular Ca2+, CGRP (10(-8) M) attenuated the increase of [Ca2+]i induced by a sarcoplasmic reticulum Ca2+ pump ATPase inhibitor thapsigargin (10(-5) M). Furthermore, Rp-cAMPS, a cAMP-dependent protein kinase A inhibitor, blocks CGRP actions on thapsigargin-induced Ca2+ release from sarcoplasmic reticulum. Our results suggested that CGRP relaxes human fetoplacental vessels by not only inhibiting the influx of extracellular Ca2+ but also attenuating the release of intracellular Ca2+ from the sarcoplasmic reticulum, and these actions might be attributed to CGRP-induced intracellular cAMP accumulation.