Multiple signaling pathways of histamine H2 receptors. Identification of an H2 receptor-dependent Ca2+ mobilization pathway in human HL-60 promyelocytic leukemia cells

In order to analyze the complex activities of histamine H2 receptor activation on neutrophils, human HL-60 promyelocytic leukemia cells were differentiated into neutrophils by incubation with dimethyl sufoxide, loaded with the Ca2+-sensitive indicator dyes, indo-1 or fura-2, and the levels of intracellular Ca2+ ([Ca2+]i) measured in a fluorescent-activated cell sorter and fluorimeter, respectively. Histamine increased [Ca2+]i in a dose-dependent manner with a half-maximal concentration (EC50) of approximately 10(-6) to 10(-5) M, which exhibited H2 receptor specificity. Prostaglandin E2 and isoproterenol also induced [Ca2+]i mobilization in HL-60 cells, whereas the cell permeable form of cAMP and forskolin failed to increase [Ca2+]i. Since H2-receptor mediated [Ca2+]i mobilization was not inhibited by reducing the concentration of extracellular Ca2+ nor by the addition of Ca2+ channel antagonists, LaCl3 and nifedipine, [Ca2+]i mobilization is due to the release of Ca2+ from intracellular stores. Furthermore, both 10(-4) M histamine and 10(-6) M fMet-Leu-Phe increased the levels of 1,4,5-inositol trisphosphate. However, histamine-induced mobilization of [Ca2+]i was inhibited by cholera toxin but not by pertussis toxin, whereas the action of fMet-Leu-Phe was inhibited by pertussis toxin but not by cholera toxin. These data suggest that H2 receptors on HL-60 cells are coupled to two different cholera toxin-sensitive G-proteins and activate adenylate cyclase and phospholipase C simultaneously.     

Platelet-activating factor stimulates multiple signaling pathways in cultured rat mesangial cells.

We have previously reported that platelet-activating factor (PAF) elevates cytosolic free calcium concentration ([Ca2+]i) in fura-2-loaded glomerular mesangial cells. To confirm that this increase in [Ca2+]i is a result of receptor-mediated activation of phospholipase C, we investigated hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) in PAF-treated mesangial cells. PAF (10(-7) M) stimulated a rapid and transient formation of inositol trisphosphate. In concomitant experiments, PAF stimulated a biphasic accumulation of 3H-arachidonate-labeled 1,2-diacylglycerol (DAG). The secondary elevation in DAG was coincident with a rise in 3H-phosphorylcholine (PC) and 3H-phosphorylethanolamine (PE) suggesting that PAF stimulates delayed phospholipase activities which hydrolyze alternate phospholipids besides the polyphosphoinositides. This PAF-stimulated elevation in 3H-water soluble phosphorylbases was seen at 5 min but not at 15 sec suggesting that the initial rise in DAG as well as the initial elevation in [Ca2+]i are due primarily to PtdIns-4,5-P2 hydrolysis. PAF also stimulated PGE2 as well as 3H-arachidonic acid and 3H-lyso phosphatidylcholine (PtdCho) formation. We suggest that arachidonate released specifically from PtdCho via phospholipase A2 is a source of this PAF-elevated PGE2. It has been postulated that anti-inflammatory prostaglandins may antagonize the contractile and proinflammatory effects of PAF via activation of adenylate cyclase. Surprisingly, exogenous PAF reduced basal and receptor-mediated cAMP concentration indicating that PAF-stimulated transmembrane signaling pathways may oppose receptor-mediated activation of adenylyl cyclase. We have taken advantage of the different sensitivities of phospholipases A2 and C(s) to PMA, EGTA, and pertussis toxin to dissociate phospholipase A2 and C activities. Acute PMA-treatment enhanced PAF-stimulated PGE2 formation, reduced PAF-induced elevations in [Ca2+]i and had no effect upon PAF-stimulated 3H-PE. We have also demonstrated that phospholipase A2, but not PtdIns-specific phospholipase C, was sensitive to external calcium concentration. The role of a GTP-binding protein to couple PAF-receptors to the PtdIns-specific phospholipase C was confirmed as GTP gamma S synergistically elevated PAF-stimulated inositol phosphate formation. We also demonstrated that pertussis toxin ADP-ribosylates a single protein of an apparent 42 kD mass and that PAF pretreatment reduced subsequent ADP-ribosylation in a time-dependent manner. However, pertussis toxin had no effect upon phospholipase C-generated water soluble phosphorylbases or inositol phosphates. In contrast, PAF-stimulated phospholipase A2 and PAF-inhibited adenylyl cyclase activities were sensitive to pertussis toxin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cellular responses to stimulation of the M5 muscarinic acetylcholine receptor as seen in murine L cells

The membrane signaling properties of the neuronal type-5 muscarinic acetylcholine receptor (M5 AChR) as expressed in murine L cells were studied. Recipient Ltk- cells responded to ATP acting through a P2-purinergic receptor by increasing phosphoinositide hydrolysis 2-fold but were unresponsive to 17 receptor agonists that are stimulatory in other cells. L cells expressing the M5 AChR responded to carbachol (CCh) with an approximately 20-fold increase in phospholipase C activity, mobilization of Ca2+ from endogenous stores, causing a transient peak increase in the intracellular concentration of Ca2+ ([Ca2+]i), influx of extracellular Ca2+, causing a sustained increase in [Ca2+]i dependent on extracellular Ca2+, and release of [3H]arachidonic acid from prelabeled cells, without altering resting or prostaglandin E1-elevated intracellular cAMP levels. None of the effects of the M5 AChR were inhibited by pertussis toxin. The regulation of L cell [Ca2+]i was studied further. ATP had the same effects as CCh and the two agonists acted on a shared intracellular pool of Ca2+. The peak and sustained [Ca2+]i increases were reduced by cholera toxin and forskolin, neither of which altered significantly phosphoinositide hydrolysis. This is consistent with interference with the action of inositol 1,4,5-trisphosphate (IP3) through cAMP-mediated phosphorylation and suggests a continued involvement of IP3 during the sustained phase of [Ca+]i increases. The temporal pattern of the sustained [Ca2+]i increase differed whether elicited by CCh or ATP, and was enhanced in pertussis toxin-treated cells. This is consistent with existence of a kinetic control of the sustained [Ca2+]i change by a receptor-G protein-dependent mechanism independent of the IP3 effector site(s) (e.g. pulsatile activation of phospholipase C and/or pulsatile activation of a receptor/G protein-operated plasma membrane Ca2+ channel). Thus, the non-excitable L cell may be a good model for studying [Ca2+]i regulations, as may occur in other nonexcitable cells of which established cell lines do not exist, and for studying of receptors that as yet cannot be studied in their natural environment.     

Studies on molecular regulation of phagocytosis in neutrophils. Con A-mediated ingestion and associated respiratory burst independent of phosphoinositide turnover, rise in [Ca2+]i, and arachidonic acid release

The role of the activation of phosphoinositide turnover and of the increase in cytosolic free calcium, [Ca2+]i, in the phagocytosis and associated activation of the respiratory burst was investigated. We report the results obtained on the phagocytosis of yeast cells mediated by Con A in normal and in Ca2+-depleted human neutrophils. In normal neutrophils the phagocytosis was associated with a respiratory burst, a stimulation in the formation of [3H] inositol phosphates and [32P]phosphatidic acid, the release of [3H]arachidonic acid, and a rise in [Ca2+]i. Ca2+-depleted neutrophils are able to perform the phagocytosis of yeast cells mediated by Con A and to activate the respiratory burst without stimulation of [3H]inositol phosphates and [32P]phosphatidic acid formation, [3H]arachidonic acid release, and rise in [Ca2+]i. In both normal and Ca2+-depleted neutrophils the phagocytosis and the associated respiratory burst, 1) were inhibited by cytochalasin B; 2) were insensitive to H-7, an inhibitor of protein kinase C; and 3) did not involve GTP-binding protein sensitive to pertussis toxin. These findings indicate that the activation of phosphoinositide turnover, the liberation of arachidonic acid, the rise in [Ca2+]i, and the activity of protein kinase C are not necessarily required for ingestion of Con A-opsonized particles and for associated activation of the NADPH oxidase, the enzyme responsible for the respiratory burst. The molecular mechanisms of these phosphoinositide and Ca2+-independent responses are discussed.     

L-glutamate and acetylcholine mobilise Ca2+ from the same intracellular pool in cerebellar granule cells using transduction mechanisms with different Ca2+ sensitivities.

Ca2+ mobilisation induced by L-glutamate (Glu) and acetylcholine (ACh) has been studied in cultured cerebellar granule cells using digital fluorescence microscopy. The ability of Glu-receptor activation to mobilise Ca2+ was decreased when [Ca2+]o was lowered to 10 microM (from 1.8 mM). It was enhanced when [Ca2+]i was raised using 25 mM external K+ or by N-methyl-D-aspartate (NMDA), which selectively activates a distinct Glu-receptor subtype. The enhancement was dependent on entry of external Ca2+. In contrast, the ability of ACh receptor activation to mobilise Ca2+ was not affected by these conditions. Furthermore, pretreatment with pertussis toxin inhibited Ca2+ mobilisation in response to Glu-receptor activation without affecting mobilisation in response to ACh. However, activation of both receptors mobilised Ca2+ from a common, thapsigargin-sensitive pool. We conclude that there are differences in the Ca2+ mobilization pathways for the two receptor systems in cerebellar granule cells. The Ca(2+)-sensitivity of this Ca2+ mobilizing Glu receptor may have implications for its function in neuronal synaptogenesis and plasticity.     

Rac-1-mediated O2- secretion requires Ca2+ influx in neutrophil-like HL-60 cells

Neutrophil-like HL-60 cells reacted to N -formyl- l -Methionyl- l -Leucyl- l -P henylalanine (f MLP) with a rise in the intracellular calcium concentration ([Ca2]i), NADPH oxidase activation, and increased superoxide anion (O2-) production. [Ca2+]i mobilization and superoxide production were largely dependent on extracellular calcium (Ca2+]e) and a capacitative calcium entry. The monomeric G-protein, Rac-1, regulates NADPH oxidase activity. We tested the effect of removal of Ca2+]e on Rac-1 plasma membrane sequestration and activation of NADPH oxidase using immunodetection and a double labelling fluorescent method. Results showed that Rac-1 activation is mediated via a pertussis toxin (PTX)-sensitive heteromeric G-protein pathway, and that Rac-1 membrane sequestration was preceded by [Ca2+]i mobilization following entry of Ca2+ e. Therefore, we propose that O2- production is dependent on activation of PTX-sensitive G-proteins and sequestration of Rac-1 in the plasma membrane, following entry of Ca2+ e.     

Receptor-stimulated phospholipase A2 activation is coupled to influx of external calcium and not to mobilization of intracellular calcium in C62B glioma cells

C62B rat glioma cells respond to muscarinic cholinergic stimulation with transient inositol phosphate formation and phospholipase A2-dependent arachidonic acid liberation. Since phospholipase A2 is a Ca2+-sensitive enzyme, we have examined the role of the agonist-stimulated Ca2+ response in production of the arachidonate signal. The fluorescent indicator fura-2 was used to monitor changes in cytoplasmic Ca2+ levels ([Ca2+]i) of C62B cells following acetylcholine treatment. In the presence of extracellular Ca2+, acetylcholine induces a biphasic [Ca2+]i response consisting of an initial transient peak that precedes arachidonate liberation and a sustained elevation that outlasts the phospholipase A2 response. The initial [Ca2+]i peak is not altered by the absence of external Ca2+ and therefore reflects intracellular Ca2+ mobilization. The sustained elevation phase is dependent on the influx of external Ca2+; it is lost in Ca2+-free medium and restored on the addition of Ca2+. Pretreating cells with phorbol dibutyrate substantially inhibits acetylcholine-stimulated inositol phosphate formation and the peak [Ca2+]i response without affecting the sustained elevation in [Ca2+]i. This suggests that the release of internal Ca2+ stores by inositol 1,4,5-trisphosphate can be blocked without interfering with Ca2+ influx. Pretreatment with phorbol also fails to affect acetylcholine-stimulated arachidonate liberation, demonstrating that phospholipase A2 activation does not require normal intracellular Ca2+ release. Stimulated arachidonate accumulation is totally inhibited in Ca2+-free medium and restored by the subsequent addition of Ca2+. Pretreatment with verapamil, a voltage-dependent Ca2+ channel inhibitor, also blocks both the sustained [Ca2+]i elevation and arachidonate liberation without altering peak intracellular Ca2+ release. We conclude that the influx of extracellular Ca2+ is tightly coupled to phospholipase A2 activation, whereas large changes in [Ca2+]i due to mobilization of internal Ca2+ stores are neither sufficient nor necessary for acetylcholine-stimulated phospholipase A2 activation.     

Characterization of Prostaglandin E2-Induced Ca2+ Mobilization in Single Bovine Adrenal Chromaffin Cells by Digital Image Microscopy

We recently reported that prostaglandin E2 (PGE2) stimulates phosphoinositide metabolism accompanied by an increase in intracellular free Ca2+ concentration ([Ca2+]i) in cultured bovine adrenal chromaffin cells. In the present study, temporal and spatial changes in [Ca2+]i induced by PGE2 in fura-2-loaded individual cells were investigated by digital image microscopy and were compared with those induced by nicotine and histamine. Image analysis of single cells revealed that responses to PGE2 showed asynchrony with the onset of [Ca2+]i changes. After a lag time of 10-30 s, PGE2-induced [Ca2+]i changes took a similar prolonged time course in almost all cells: a rapid rise followed by a slower decline to the basal level over 5 min. Few cells exhibited oscillations in [Ca2+]i. In contrast, nicotine and histamine induced rapid and transient [Ca2+]i changes, and these [Ca2+]i changes were characteristic of each stimulant. Whereas pretreatment of the cells with pertussis toxin (100 ng/ml, 6 h) did not block the response to any of these stimulants, treatment with 12-O-tetradecanoylphorbol 13-acetate (100 nM, 10 min) completely abolished [Ca2+]i changes elicited by PGE2 and histamine. In a Ca2(+)-free medium containing 3 mM EGTA, or in medium to which La3+ was added, the [Ca2+]i response to nicotine disappeared, but that to histamine was not affected significantly. Under the same conditions, the percentage of the cells that responded to PGE2 was reduced to 37% and the prolonged [Ca2+]i changes induced by PGE2 became transient in responding cells, suggesting that the maintained [Ca2+]i increase seen in normal medium is the result of a PGE2-stimulated entry of extracellular Ca2+. Whereas the organic Ca2(+)-channel blocker nicardipine inhibited [Ca2+]i changes by all stimulants at 10 microM, these [Ca2+]i changes were not affected by any of the organic Ca2(+)-channel blockers, i.e., verapamil, diltiazem, nifedipine, and nicardipine, at 1 microM, a concentration high enough to inhibit voltage-sensitive Ca2+ channels. These results demonstrate that PGE2 may promote Ca2+ entry with concomitant release of Ca2+ from intracellular stores and that the mechanism(s) triggered by PGE2 is apparently different from that by histamine or nicotine.     

Lan-1: A Human Neuroblastoma Cell Line With M1 and M3 Muscarinic Receptor Subtypes Coupled to Intracellular Ca2+ Elevation and Lacking Ca2+ Channels Activated by Membrane Depolarization

The LAN-1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1-1,000 microM) caused a dose-dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to approximately 70% but completely abolished the plateau phase. The muscarinic-activated Ca2+ channel was gadolinium (Gd3+) blockade and nimodipine and omega-conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh-induced [Ca2+]i elevation was concentration-dependently inhibited by pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitive intracellular store. In addition, ryanodine did not prevent CCh-induced [Ca2+]i mobilization, and, finally, LAN-1 cells appear to lack caffeine-sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 microM), or after thapsigargin.     

fMet-Leu-Phe-induced activation of phospholipase D in human neutrophils. Dependence on changes in cytosolic free Ca2+ concentration and relation with respiratory burst activation.

In this study, we have investigated the Ca2+ requirements for the activation of phospholipase D by the tripeptide fMet-Leu-Phe (fMLP) in human neutrophils. EGTA inhibited the activation of phospholipase D (PLD) by 55% (n = 4). When the initial transient rise in [Ca2+]i was prevented by loading the cells with limited amounts of the Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), PLD activation was inhibited by 92% (n = 4). In the presence of both chelators, PLD activation was only 4% of control. In electropermeabilized neutrophils, too, the activation of PLD after the addition of fMLP strongly depends on the Ca2+ concentration, being almost absent with 100 nM free Ca2+ present and reaching maximum activation with a free [Ca2+] of 500 nM. We subsequently investigated the relationship between PLD activation and the activation of the respiratory burst. In neutrophils loaded with BAPTA/AM (10 microM), in which PLD activation was almost absent, a respiratory burst could be induced by fMLP, albeit with a much longer lag time. A respiratory burst could also be elicited by fMLP in electropermeabilized neutrophils incubated with 100 nM free Ca2+. This response, however, was strongly enhanced in the presence of 1 microM Ca2+. Our results indicate that changes in [Ca2+]i are essential for the activation of PLD by fMLP, but probably do not constitute the sole activation signal. In addition, our data provide evidence that PLD activation is important, but not necessary, for activation of the neutrophil respiratory burst.     

Prostaglandins inhibit proliferation of the murine P815 mastocytoma by decreasing cytoplasmic free calcium levels [( Ca+2]i)

Prostaglandins inhibit the proliferation of the murine P815 mastocytoma. The mechanism of this antitumour activity remains undefined. In several cell systems, the action of PGs is inhibited at the cell surface receptor by pertussis toxin likely through regulatory G proteins involved in the inhibition of adenyl cyclase or activation of phospholipase C. We therefore determined the effect of prostaglandins on the biochemical consequences of activation of these pathways; i.e. concentrations of cyclic AMP (cAMP) and cytosolic free Ca+2 concentrations [( Ca/2]i) respectively. PGD2 (6 ug/mL), PGE1 (10 ug/mL) and PGB1 (50 ug/mL) maximally inhibited (3H)-thymidine incorporation to DNA. PGF2 alpha did not affect DNA synthesis. PGE1 (10 ug/mL) induced a three fold increase in cAMP concentrations. In contrast, the other prostaglandins did not alter cAMP concentrations. Maximal growth inhibitory doses of PGD2, PGE1 and PGB1 decrease [Ca+2]i, as measured by the fluorescence of Indo-1, from 320 +/- 5 nM to 172 +/- 20 nM, 161 +/- 12 nM, and 151 +/- 18 nM respectively. PGF2 alpha did not alter [Ca+2]i. Therefore, in contrast to the effects on cAMP, the decrease in [Ca+2]i was concordant with the inhibition of DNA synthesis. This suggests that PGs may inhibit proliferation through decreasing [Ca+2]i in the P815 mastocytoma.     

Possible Existence of Two Subsets of Platelet-Activating Factor Receptor to Mediate Polyphosphoinositide Breakdown and Calcium Influx in Neuroblastoma × Glioma Hybrid NG 108–15 Cells

Platelet-activating factor (PAF) initiated polyphosphoinositide (polyPI) breakdown and a rise of intracellular calcium concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG 108-15 cells. The accumulation of [3H]inositol trisphosphate and [3H]inositol bisphosphate was evident within 15 s after PAF stimulation, peaked at 1 min, and then gradually decayed. The increase in [3H]inositol monophosphate level was observed at 30 s, plateaued in 5 min, and was sustained up to 10 min in the presence of 10 mM LiCl. On the other hand, the rise of [Ca2+]i evoked by PAF reached a peak within 8-12 s and returned to basal levels within 1 min as measured in fura 2-loaded cells. When cells were suspended in Ca(2+)-depleted medium, the PAF-induced [Ca2+]i rise was reduced by 80%, indicating that the increase of [Ca2+]i was predominantly due to the Ca2+ influx from an extracellular source. Both PAF-induced accumulation of 3H-labeled inositol phosphates and [Ca2+]i elevation were concentration dependent with EC50 values of approximately 1 x 10(-10) and 5 x 10(-8) M, respectively. The PAF analogs 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine and 1-O-hexadecyl-2-O-methyl-rac-glycerol-3-phosphocholine were much poorer agonists at eliciting the same responses in these cells. Pretreatment of cells with pertussis toxin caused a substantial inhibition of PAF-induced accumulation of 3H-inositol phosphates. In contrast, the rise in [Ca2+]i was not significantly affected by toxin treatment at the same concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipase C-beta and ovarian sex steroids in pig granulosa cells.

We compared the membrane effects of estradiol, progesterone, and androstenedione in a single experimental model, the ovarian granulosa cells collected from immature Large White sows. We measured changes in cytosolic free calcium concentration ([Ca2+]i) in confluent Fura-2 loaded cells. We used pharmacological tools and polyclonal phospholipase C-beta (PLC-beta) antibodies. Each steroid (0.1 pM to 1 nM) transiently increased intracellular calcium concentration ([Ca2+]i) within 5 sec. They mobilized Ca2+ from the endoplasmic reticulum as shown by using two phospholipase C inhibitors, neomycin and U-73122. Ca2+ mobilization involved PLC-beta1 for progesterone, PLC-beta2 for estradiol and PLC-beta4 for androstenedione. A pertussis toxin-insensitive G protein was involved in the effects of progesterone on Ca2+ mobilization whereas estradiol and androstenedione effects were mediated via a pertussis toxin-sensitive G-protein. Ca2+ influx from the extracellular milieu was involved in the increase in [Ca2+]i induced by progesterone and estradiol, but not by androstenedione. Influx of Ca2+ was independent of Ca2+ mobilization from calcium stores, and it was suggested that L-type Ca2+ channels for estradiol and T-type Ca2+ channels for progesterone were involved. The three steroids had no effect on cAMP. Rapid effects of progesterone, estradiol, and androstenedione involved a direct action on cell membrane elements such as PLC-beta, G-proteins, and calcium channels, and these mechanisms were hormone-specific.     

ATP-Stimulated Ca2+ Influx and Phospholipase D Activities of a Rat Brain-Derived Type-2 Astrocyte Cell Line, RBA-2, Are Mediated Through P2X7 Receptors

This study characterizes and examines the P2 receptor-mediated signal transduction pathway of a rat brain-derived type 2 astrocyte cell line, RBA-2. ATP induced Ca2+ influx and activated phospholipase D (PLD). The ATP-stimulated Ca2+ influx was inhibited by pretreating cells with P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), in a concentration-dependent manner. The agonist 2'- and 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) stimulated the largest increases in intracellular Ca2+ concentrations ([Ca2+]i); ATP, 2-methylthioadenosine triphosphate tetrasodium, and ATPgammaS were much less effective, whereas UTP, ADP, alpha,beta-methylene-ATP, and beta,gamma-methylene-ATP were ineffective. Furthermore, removal of extracellular Mg2+ enhanced the ATP- and BzATP-stimulated increases in [Ca2+]i. BzATP stimulated PLD in a concentration- and time-dependent manner that could be abolished by removal of extracellular Ca2+ and was inhibited by suramin, PPADS, and oxidized ATP. In addition, PLD activities were activated by the Ca2+ mobilization agent, ionomycin, in an extracellular Ca2+ concentration-dependent manner. Both staurosporine and prolonged phorbol ester treatment inhibited BzATP-stimulated PLD activity. Taken together, these data indicate that activation of the P2X7 receptors induces Ca2+ influx and stimulates a Ca2+-dependent PLD in RBA-2 astrocytes. Furthermore, protein kinase C regulates this PLD.     

Manipulation of Intracellular Calcium in NCB-20 Cells

A number of lines of evidence indicate that the Ca2+ and cyclic AMP signalling systems interact in NCB-20 cells. However, to date, the regulation of [Ca2+]i homeostasis has not been studied in this cell line. The present study aimed to clarify our understanding of [Ca2+]i homeostasis in these cells and to evaluate tools that manipulate [Ca2+]i, independently of protein kinase C effects. Bradykinin, by a B2-receptor, elevated [Ca2+]i by a pertussis-toxin-insensitive mechanism. The BK-stimulated [Ca2+]i rise originated from intracellular sources, without a contribution from Ca2+ entry mechanisms. The effect of BK was precluded by pretreatment with thapsigargin and ionomycin--compounds that elevated [Ca2+]i independent of phospholipase C activation. Both compounds, however, exerted effects in addition to stimulating release of Ca2+ from BK-sensitive stores; the BK-sensitive Ca2+ pool was a subset of the thapsigargin-sensitive pool; ionomycin strongly stimulates Ca2+ entry. Activation of protein kinases A and C attenuated the duration of the BK-induced rise in [Ca2+]i, without affecting the peak [Ca2+]i, suggesting interference with the BK response at a step downstream of the activation of phospholipase C. Application of these approaches should enhance the delineation of the consequences of Ca2+ mobilization on cyclic AMP accumulation.     

Extracellular ATP causes Ca2(+)-dependent and -independent insulin secretion in RINm5F cells. Phospholipase C mediates Ca2+ mobilization but not Ca2+ influx and membrane depolarization

The mechanism by which extracellular ATP stimulates insulin secretion was investigated in RINm5F cells. ATP depolarized the cells as demonstrated both by using the patch-clamp technique and a fluorescent probe. The depolarization is due to closure of ATP-sensitive K+ channels as shown directly in outside-out membrane patches. ATP also raised cytosolic Ca2+ [( Ca2+]i). At the single cell level the latency of the [Ca2+]i response was inversely related to ATP concentration. The [Ca2+]i rise is due both to inositol trisphosphate mediated Ca2+ mobilization and to Ca2+ influx. The former component, as well as inositol trisphosphate generation, were inhibited by phorbol myristate acetate which uncouples agonist receptors from phospholipase C. This manoeuvre did not block Ca2+ influx or membrane depolarization. Diazoxide, which opens ATP-sensitive K+ channels, attenuated membrane depolarization and part of the Ca2+ influx stimulated by ATP. However, the main Ca2+ influx component was unaffected by L-type channel blockers, suggesting the activation of other Ca2+ conductance pathways. ATP increased the rate of insulin secretion by more than 12-fold but the effect was transient. Prolonged exposure to EGTA dissociated the [Ca2+]i rise from ATP-induced insulin secretion, since the former was abolished and the latter only decreased by about 60%. In contrast, vasopressin-evoked insulin secretion was more sensitive to Ca2+ removal than the accompanying [Ca2+]i rise. Inhibition of phospholipase C stimulation by phorbol myristate acetate abrogated vasopressin but only reduced ATP-induced insulin secretion by 34%. These results suggest that ATP stimulates insulin release by both phospholipase C dependent and distinct mechanisms. The Ca2+)-independent component of insulin secretion points to a direct triggering of exocytosis by ATP.     

Signal transduction by neutrophil immunoglobulin G Fc receptors. Dissociation of intracytoplasmic calcium concentration rise from inositol 1,4,5-trisphosphate.

The signal transduction mechanisms involved in the regulation of phagocytosis are largely unknown. We have recently shown that in neutrophils, when IgG-mediated phagocytosis is stimulated by formyl-methionyl-leucyl-phenyl-alanine (fMLP), the enhanced ingestion is dependent on the increase in [Ca2+]i which results from ligation of Fc receptors by the IgG-coated target (Rosales, C., and Brown, E. (1991) J. Immunol. 146, 3937-3944). Now, we have studied the mechanism by which this rise in [Ca2+]i occurs. Aggregated IgG, the monoclonal antibody 3G8 (which recognizes Fc receptor type III), and insoluble immune complexes caused an increase in [Ca2+]i. The rise in [Ca2+]i induced by Fc receptor ligation was resistant to pertussis toxin. In contrast, fMLP induced a rise in [Ca2+]i which was inhibited by pertussis toxin. fMLP-induced [Ca2+]i was accompanied by an accumulation of inositol 1,4,5-trisphosphate (IP3) which peaked by 15 s, and which was also abolished by pertussis toxin. IP3 accumulation after aggregated IgG, 3G8, or insoluble immune complexes was much less than after fMLP. Unlike [Ca2+]i rise induced by Fc receptor ligation, this small increase in IP3 was inhibited by pertussis toxin. These data demonstrated that the [Ca2+]i increase induced by Fc receptor ligation is not mediated by IP3. Immediate pretreatment of human polymorphonuclear neutrophils with optimal doses of fMLP also reduced subsequent increase in [Ca2+]i rise from thapsigargin, a sesquiterpene lactone tumor promoter that releases intracellular Ca2+ from IP3-sensitive stores without IP3 turnover. Similarly, to its effects on thapsigargin, fMLP inhibited the [Ca2+]i rise upon subsequent immune complex binding. Pretreatment of cells with immune complexes also prevented subsequent [Ca2+]i rise from thapsigargin and fMLP. These data demonstrate that IgG Fc receptor ligation and fMLP activation of human polymorphonuclear neutrophils use distinct signal transduction mechanisms to release Ca2+ from the same thapsigargin-sensitive intracellular pool. In contrast to fMLP, signal transduction for increased [Ca2+]i after Fc receptor stimulation does not involve a pertussis toxin-sensitive G protein, and is independent of IP3.     

Critical intracellular Ca2+ concentration for all-or-none Ca2+ spiking in single smooth muscle cells.

Neurotransmitters induce contractions of smooth muscle cells initially by mobilizing Ca2+ from intracellular Ca2+ stores through inositol 1,4,5-trisphosphate (InsP3) receptors. Here we studied roles of the molecules involved in Ca2+ mobilization in single smooth muscle cells. A slow rise in cytoplasmic Ca2+ ([Ca2+]i) in agonist-stimulated smooth muscle cells was followed by a wave of rapid regenerative Ca2+ release as the local [Ca2+]i reached a critical concentration of approximately 160 nM. Neither feedback regulation of phospholipase C nor caffeine-sensitive Ca(2+)-induced Ca2+ release was found to be required in the regenerative Ca2+ release. These results indicate that Ca(2+)-dependent feedback control of InsP3-induced Ca2+ release plays a dominant role in the generation of the regenerative Ca2+ release. The resulting Ca2+ release in a whole cell was an all-or-none event, i.e. constant peak [Ca2+]i was attained with agonist concentrations above the threshold value. This finding suggests a possible digital mode involved in the neural control of smooth muscle contraction.     

Characteristics of collagen-induced Ca2+ mobilization in bovine platelets

We characterized the collagen-induced increase in cytosolic Ca2+ ([Ca2+]i) of bovine platelets loaded with the Ca2+ indicator Fura-PE3/AM. Collagen (10 micrograms/ml)-induced increase in [Ca2+]i was only partially inhibited by aspirin, a cyclooxygenase inhibitor, or adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS, a P2Y1 receptor antagonist), while in human platelets it was almost completely suppressed by aspirin. Collagen-induced increase in [Ca2+]i of bovine platelets was inhibited by U73122 (0.3-5 microM), a phospholipase C inhibitor. Collagen (10 micrograms/ml) increased production of inositol 1,4,5-trisphosphate, which was prevented by pretreatment with U73122 (5 microM). Collagen (10 micrograms/ml) accelerated Mn2+ entry, since the rate of Fura-PE3 quenching by Mn2+ was enhanced by 13-fold following stimulation with collagen. U73122 inhibited the acceleration of Mn2+ entry induced by collagen. PGE1 (2.5 microM) partially inhibited the collagen (50 micrograms/ml)-induced increase in [Ca2+]i in bovine platelets but not in human platelets. The data suggest that collagen-induced Ca2+ mobilization in bovine platelets is mediated by phospholipase C. The Ca2+ mobilization in bovine platelets is different from that in human ones as to the dependency on arachidonic acid metabolites and sensitivity to PGE1.