Requirement for different Ca2+ pools in the activation of rabbit platelets: II. Phospholipase activity

The effects of extracellular Ca²⁺ concentration and the putative antagonist of intracellular Ca²⁺ movement, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) on platelet phospholipase activity and thromboxane B₂ synthesis were examined in rabbit platelets stimulated by platelet activating factor, thrombin and ionophore A23187. TMB-8 markedly inhibited the platelet activating factor-induced decrease in [¹⁴C]arachidonate content in platelet phsophatidylacholine and phosphatidylinositol, while showing minimal effects on thrombin-induced phospholipase activation. A23187 stimulation of these processes was inhibited to an intermediated degree by TMB-8. In contrast, extracellular Ca²⁺ removal inhibited phospholipase activity to a similar degree with all three stimuli. Moreover, the threshold concentration of extracelullar Ca²⁺ for phospholiphase activation, as measured by thromboxane B₂ synthesis, was similar for platelet activating factor- and thrombin-stimulated platelets. The data provide evidence that, while platelet activating factor and thrombin may, to some extent, have similar requirements for extracellular Ca²⁺, they utilize a TMB-8 sensitive step to different degrees during activation of platelet phospholipase.     

The Role of Phosphatidylinositol 4,5 Bisphosphate Breakdown in Cell-Surface Receptor Activation

Abstract

The activation of Ca²⁺-mobilising receptors on hepatocytes and many other cells leads to a prompt reduction in the cellular content of inositol phospholipids. The primary event which underlies these changes is, most probably, a phospholipase C-catalysed attack upon phosphatidylinositol 4,5 bisphosphate. The receptor-mediated breakdown of this lipid in stimulated cells is: (i) not mediated by an increase in cytosol [Ca²⁺] and (ii) closely coupled to receptor occupation. Phosphatidylinositol 4,5 bisphosphate degradation may be studied by measuring the appearance of the water-soluble product, inositol trisphosphate (and its metabolites: inositol bisphosphate and inositol monophosphate), in stimulated cells. Recent evidence indicates that inositol trisphosphate and the lipid soluble product of phosphatidylinositol 4,5 bisphosphate breakdown, 1,2 diacylglycerol, may act as ‘second messengers’ which mediate the effects of many extracellular signals in stimulated cells.     

Inhibition of polyphosphoinositide phosphodiesterase by aminoglycoside antibiotics

The calcium-activated phosphodiesteratic hydrolysis of³²P-labeled phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate in prelabeled nerve ending membranes is inhibited by the aminoglycosides neomycin and gentamicin, and to a lesser extent, by streptomycin. The inhibition is overcome by increasing concentrations of Ca²⁺, indicating that the aminoglycosides exert their effect by displacing Ca²⁺ from lipid.Dedicated to Professor Yasuzo Tsukada.     

Ca2+ Influx through Store-operated Calcium Channels Replenishes the Functional Phosphatidylinositol 4,5-Bisphosphate Pool Used by Cysteinyl Leukotriene Type I Receptors

Oscillations in cytoplasmic Ca²⁺ concentration are a universal mode of signaling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca²⁺ oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂), the source of the Ca²⁺-releasing second messenger inositol trisphosphate. Here we show that cytoplasmic Ca²⁺ oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pretreated with Li⁺, an inhibitor of inositol monophosphatases that prevents PIP₂ resynthesis. In Li⁺-treated cells, cytoplasmic Ca²⁺ signals evoked by an agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knockdown of the phosphatidylinositol 4-phosphate 5 (PIP5) kinases α and γ resulted in rapid loss of the intracellular Ca²⁺ oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca²⁺ oscillations, and these could not be rescued by inositol or PI4P. In Li⁺-treated cells, recovery of the cytoplasmic Ca²⁺ oscillations in the presence of inositol or PI4P was suppressed when Ca²⁺ influx through store-operated Ca²⁺ channels was inhibited. After rundown of the Ca²⁺ signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent inositol trisphosphate-dependent Ca²⁺ release. Therefore, leukotriene and P2Y receptors utilize distinct membrane PIP₂ pools. Our findings show that store-operated Ca²⁺ entry is needed to sustain cytoplasmic Ca²⁺ signaling following leukotriene receptor activation both by refilling the Ca²⁺ stores and by helping to replenish the PIP₂ pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.     

Maintenance of hormone-sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol 4-kinase IIIalpha

Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca²⁺ signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks (α or β) is responsible for supplying phosphoinositides during agonist-induced Ca²⁺ signaling. Using inhibitors that discriminate between the α- and β-isoforms of type III PI4Ks, PI4KIIIα was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂], and Ca²⁺ signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P₂ levels in ³²P-labeled cells, but only PI4KIIIα down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P₂ resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca²⁺ signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIIIα enzyme in maintaining plasma membrane phosphoinositides.     

Mechanism of desensitization of neutrophil response to N-formylmethionylleucylphenylalanine by slow rate of receptor occupancy. Studies on changes in Ca2+ concentration and phosphatidylinositol turnover

Previous studies on the regulation of responses of neutrophils to fMet-Leu-Phe have demonstrated the relevance of the role of the rate of occupation of the receptors by the stimulant. When this rate is decreased by presenting the peptide to neutrophils over a period of time by means of an infusion pump, the activation of the respiratory burst and of the secretion is greatly depressed or is absent. This paper deals with further investigations on the mechanisms of this desensitization, which previous results have shown to consist of an uncoupling between the ligand-receptor complexes and the target for cell responses, caused by the deceleration of the initial rate of occupation of the receptors. The data presented here demonstrate that this desensitization is not linked to the formation of a negative intermediate such as cAMP, but is associated with: (i) a depression of the rate and magnitude of the phosphatidylinositol response (activation of phospahtidylinositol turnover measured as modification of incorporation of [³²P]P_i and [³H]glycerol into phosphatidylinositol and phosphatidic acid); (ii) a deceleration of the rate of the release of bound Ca²⁺, without a decrease in the total quantity of Ca²⁺ liberated (measured as fluorescence changes of chlorotetracycline treated neutrophils); (iii) a slower rise of cytosolic free Ca²⁺ concentration [Ca²⁺]_i, without a decrease in the magnitude of the final increase of [Ca²⁺]_i (monitored with Quin 2). These findings, which are discussed in relation to the recent hypotheses on the transduction reactions of receptor-mediated stimuli for neutrophil responses, are consistent with a mechanism of desensitization involving decreased production of diacylglycerol by the hydrolysis of phosphatidylinositol and deficient activation of Ca²⁺-phospholipid-dependent protein kinase C.     

Changes in [3H]inositol-labelled phosphoinositides of pig platelets in response to thrombin

Pig platelet phosphoinositides have been labelled with [³H]inositol and then treated with thrombin in the absence of Ca²⁺. There was a loss of labelled phosphatidylinositol 4,5-bisphosphate between 30 and 60 s after the addition of thrombin but the general picture was of increased labelling over a 4-min period. Labelling of phosphatidylinositol 4-phosphate showed no period of loss but there was an early loss of phosphatidylinositol and no increased labelling during the 4-min incubation. The small amount of lysophosphatidyl[³H]inositol in the platelets was not affected by thrombin treatment. Thrombin caused loss of [¹⁴C]arachidonate-labelled phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol.     

CRAC channel is inhibited by neomycin in a Ptdlns(4,5)P2‐independent manner

Depletion of intracellular Ca²⁺ stores evokes store‐operated Ca²⁺ entry through the Ca²⁺ release‐activated Ca²⁺ (CRAC) channels. In this study, we found that the store‐operated Ca²⁺ entry was inhibited by neomycin, an aminoglycoside that strongly binds phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2). Patch clamp recordings revealed that neomycin blocked the CRAC currents reconstituted by co‐expression of Orai1 and Stim1 in HEK293 cells. Using a rapamycin‐inducible PtdIns(4,5)P2‐specific phosphatase (Inp54p) system to manipulate the PtdIns(4,5)P2 in the plasma membrane, we found that the CRAC current was not altered by PtdIns(4,5)P2 depletion. This result suggests that PtdIns(4,5)P2 is not required for CRAC channel activity, and thereby, neomycin inhibits CRAC channels in a manner that is independent of neomycin–PtdIns(4,5)P2 binding. Copyright © 2015 John Wiley & Sons, Ltd.     

Phosphatidylinositol hydrolysis and phosphatidylinositol 4′,5′-diphosphate hydrolysis are separable responses during secretagogue action in the rat pancreas

Rat pancreatic fragments and acinar preparations were incubated in vitro to characterize further the changes in phosphoinositide metabolism that occur during secretagogue action. Two distinct responses were discernible. The first response, most notably involving a decrease in phosphatidylinositol content, was (a) observed at lower carbachol concentrations in dose-response studies, (b) inhibited by incubation in Ca²⁺-free media containing 1 mM EGTA, (c) associated with increases in inositol monophosphate production, and (d) provoked by all tissue secretagogues (carbachol, cholecystokinin, secretin, insulin, dibutyryl cAMP and the ionophore A23187), regardless of whether their mechanism of action primarily involved Ca²⁺ mobilization or cAMP generation. This decrease in phosphatidylinositol content was at least partly due to phospholipase C (and/or D) activation, as evidenced by the increase in inositol monophosphate. The second response, most notably involving markedly increased incorporation of ³²PO₄ into phosphatidic acid and phosphatidylinositol, was (a) observed at higher carbachol concentrations, (b) not influenced by incubation in Ca²⁺-free media containing 1 mM EGTA, and (c) associated with increases in inositol triphosphate production. This ³²PO₄ turnover response was probably largely the result of phospholipase C-mediated hydrolysis of phosphatidylinositol 4′,5′-diphosphate, which, as shown previously, also occurs at higher carbachol concentrations and is insensitive to comparable EGTA-induced Ca²⁺ deficiency. This phosphatidylinositol 4′,5′-diphosphate hydrolysis response was only observed in the action of agents (carbachol and cholecystokinin) which mobilize Ca²⁺ via activation of cell surface receptors. The present results indicate that phosphatidylinositol and phosphatidylinositol 4′,5′-diphosphate hydrolysis are truly separable responses to secretagogues acting in the rat pancreas. Furthermore, phosphatidylinositol 4′,5′-diphosphate, rather than phosphatidylinositol hydrolysis is more likely to be associated with receptor activation and Ca²⁺ mobilization.     

Depression of voltage-activated Ca2+ release in skeletal muscle by activation of a voltage-sensing phosphatase

Phosphoinositides act as signaling molecules in numerous cellular transduction processes, and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) regulates the function of several types of plasma membrane ion channels. We investigated the potential role of PtdIns(4,5)P₂ in Ca²⁺ homeostasis and excitation–contraction (E-C) coupling of mouse muscle fibers using in vivo expression of the voltage-sensing phosphatases (VSPs) Ciona intestinalis VSP (Ci-VSP) or Danio rerio VSP (Dr-VSP). Confocal images of enhanced green fluorescent protein–tagged Dr-VSP revealed a banded pattern consistent with VSP localization within the transverse tubule membrane. Rhod-2 Ca²⁺ transients generated by 0.5-s-long voltage-clamp depolarizing pulses sufficient to elicit Ca²⁺ release from the sarcoplasmic reticulum (SR) but below the range at which VSPs are activated were unaffected by the presence of the VSPs. However, in Ci-VSP–expressing fibers challenged by 5-s-long depolarizing pulses, the Ca²⁺ level late in the pulse (3 s after initiation) was significantly lower at 120 mV than at 20 mV. Furthermore, Ci-VSP–expressing fibers showed a reversible depression of Ca²⁺ release during trains, with the peak Ca²⁺ transient being reduced by ∼30% after the application of 10 200-ms-long pulses to 100 mV. A similar depression was observed in Dr-VSP–expressing fibers. Cav1.1 Ca²⁺ channel–mediated current was unaffected by Ci-VSP activation. In fibers expressing Ci-VSP and a pleckstrin homology domain fused with monomeric red fluorescent protein (PLCδ₁PH-mRFP), depolarizing pulses elicited transient changes in mRFP fluorescence consistent with release of transverse tubule–bound PLCδ₁PH domain into the cytosol; the voltage sensitivity of these changes was consistent with that of Ci-VSP activation, and recovery occurred with a time constant in the 10-s range. Our results indicate that the PtdIns(4,5)P₂ level is tightly maintained in the transverse tubule membrane of the muscle fibers, and that VSP-induced depletion of PtdIns(4,5)P₂ impairs voltage-activated Ca²⁺ release from the SR. Because Ca²⁺ release is thought to be independent from InsP₃ signaling, the effect likely results from an interaction between PtdIns(4,5)P₂ and a protein partner of the E-C coupling machinery.     

Interplay between Calmodulin and Phosphatidylinositol 4,5-Bisphosphate in Ca2+-induced Inactivation of Transient Receptor Potential Vanilloid 6 Channels

The epithelial Ca²⁺ channel transient receptor potential vanilloid 6 (TRPV6) undergoes Ca²⁺-induced inactivation that protects the cell from toxic Ca²⁺ overload and may also limit intestinal Ca²⁺ transport. To dissect the roles of individual signaling pathways in this phenomenon, we studied the effects of Ca²⁺, calmodulin (CaM), and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) in excised inside-out patches. The activity of TRPV6 strictly depended on the presence of PI(4,5)P₂, and Ca²⁺-CaM inhibited the channel at physiologically relevant concentrations. Ca²⁺ alone also inhibited TRPV6 at high concentrations (IC₅₀ = ∼20 μm). A double mutation in the distal C-terminal CaM-binding site of TRPV6 (W695A/R699E) essentially eliminated inhibition by CaM in excised patches. In whole cell patch clamp experiments, this mutation reduced but did not eliminate Ca²⁺-induced inactivation. Providing excess PI(4,5)P₂ reduced the inhibition by CaM in excised patches and in planar lipid bilayers, but PI(4,5)P₂ did not inhibit binding of CaM to the C terminus of the channel. Overall, our data show a complex interplay between CaM and PI(4,5)P₂ and show that Ca²⁺, CaM, and the depletion of PI(4,5)P₂ all contribute to inactivation of TRPV6.     

Phospholipid turnover in isolated rat pancreatic acini. Consideration of the relative roles of phospholipase A2 and phospholipase C

The purpose of the present study was to explore the interaction of phosphatidylinositol breakdown and the turnover of arachidonic acid in isolated rat pancreatic acini by using receptor agonists and the calcium ionophore ionomycin. Acini prelabelled with myo-[³H]inositol in vivo responded to carbachol with a rapid breakdown of phosphatidylinositol. In the presence of [³²P]P_i, carbachol increased labelling of phosphatidic acid and phosphatidylinositol within 1 and 5 min respectively. Carbachol also rapidly stimulated the incorporation of [¹⁴C]arachidonic acid into phosphatidylinositol within 2 min, and the peptidergic secretagogue caerulein caused the loss of radioactivity from phospholipids prelabelled with arachidonic acid. Ca²⁺ deprivation partially impaired the stimulatory action of carbachol on arachidonic acid turnover. In contrast with its stimulatory effects on [³²P]P_i and [¹⁴C]arachidonate incorporation, carbachol inhibited the incorporation of the saturated fatty acid stearic acid into phosphatidylinositol. Whereas ionomycin stimulation of phosphatidylinositol breakdown and [³²P]P_i labelling of phospholipids was slower in onset and less effective than carbachol stimulation, the ionophore effectively promoted (arachidonyl) phosphatidylinositol turnover within 2 min. These results implicate two separate pathways for stimulated phosphatidylinositol degradation in the exocrine pancreas, involving phospholipases A₂ and C. Whereas mobilization of cellular Ca²⁺ appears sufficient to cause activation of phospholipase A₂ and amylase secretion, additional events triggered by receptor activation may be required to act in concert with Ca²⁺ to optimally stimulate phospholipase C. The nature of the interaction between phospholipases A₂ and C and their specific physiological roles in pancreatic secretion remain to be elucidated.     

Differences in Ca2+ mobilization induced by alpha-adrenergic agonist and phosphatidic acid in cultured hepatocytes

In an attempt to elucidate the relationship between phosphatidylinositol breakdown and alpha-adrenergic responses, effects of phosphatidic acid and phosphatidylinositol related metabolites on Ca²⁺ mobilization and glucose output in cultured hepatocytes were examined. Norepinephrine induced the net ⁴⁵Ca²⁺ efflux from preloaded cells and stimulated glucose output via alpha-adrenergic receptor stimulation, whereas phosphatidic acid caused ⁴⁵Ca²⁺ uptake to cells and did not stimulate glucose output. Myo-inositol-monophosphate, diglyceride and arachidonic acid, which are released by phosphatidylinositol breakdown, had no effect on ⁴⁵Ca²⁺ efflux and glucose output in cells. These results suggest that phosphatidic acid and phosphatidylinositol related metabolites can not mimic the alpha-adrenergic actions in cultured hepatocytes.     

Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

TransMEMbrane 16A (TMEM16A) is a Ca²⁺-activated Cl⁻ channel that plays critical roles in regulating diverse physiologic processes, including vascular tone, sensory signal transduction, and mucosal secretion. In addition to Ca²⁺, TMEM16A activation requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). However, the structural determinants mediating this interaction are not clear. Here, we interrogated the parts of the PI(4,5)P₂ head group that mediate its interaction with TMEM16A by using patch- and two-electrode voltage-clamp recordings on oocytes from the African clawed frog Xenopus laevis, which endogenously express TMEM16A channels. During continuous application of Ca²⁺ to excised inside–out patches, we found that TMEM16A-conducted currents decayed shortly after patch excision. Following this rundown, we show that the application of a synthetic PI(4,5)P₂ analog produced current recovery. Furthermore, inducible dephosphorylation of PI(4,5)P₂ reduces TMEM16A-conducted currents. Application of PIP₂ analogs with different phosphate orientations yielded distinct amounts of current recovery, and only lipids that include a phosphate at the 4′ position effectively recovered TMEM16A currents. Taken together, these findings improve our understanding of how PI(4,5)P₂ binds to and potentiates TMEM16A channels.     

Molecular cloning and functional expression of chromaffin cell scinderin indicates that it belongs to the family of Ca2+-dependent F-actin severing proteins

Scienderin is a Ca⁺-dependent actin filament severing protein present in chromaffin cells, platelets and a variety of secretory cells. It has been suggested that scinderin is involved in chromaffin cell F-actin dynamics and that this actin network controls the delivery of secretory vesicles to plasma membrane exocytotic sites. Moreover, scinderin redistribution and activity may be regulated by pH and Ca²⁺ in resting and stimulated cells. Here we describe the molecular cloning, the nucleotide sequence and the expression of bovine chromaffin cell scinderin cDNA. The fusion protein obtained cross-reacts with native scinderin antibodies and binds phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PIP₂) and actin in a Ca⁺-dependent manner. Antibodies raised against the fusion protein produced the same cellular staining patterns for scinderin as anti-native scinderin. Nucleotide and amino acid sequence analysis indicate that scinderin has six domains each containing three internal sequence motifs, two actin and two PIP₂ binding sites and has 63 and 53% homology with gelsolin and villin. These data indicate that scinderin is a novel member of the family of Ca²⁺-dependent F-actin severing proteins which includes gelsolin and villin.Abbreviations PIP₂ phosphatidylinositol 4,5 bisphosphate - PKC protein kinase C - Sc scinderin - PS phosphatidyl serine - F-Sc scinderin fusion protein - PCR polymerase chain reaction     

Evidence that cyclic AMP may regulate Ca2+-mobilization and phospholipases in thrombin-stimulated human platelets

The regulation of human platelet responses by cyclic AMP (cAMP) has been investigated by measuring thrombin-stimulated serotonin release, Ca²⁺ uptake and phospholipase activity. Thrombin-induced 1,2-diacylglycerol (DG) formation as a result of phospholipase C activation was inhibited by pretreatment with dibutyryl cAMP (dbcAMP) in a dose-dependent manner. Subsequent failure to produce phosphatidic acid (PA), which is converted from 1,2-DG by phosphorylation and would serve as intracellular Ca²⁺ ionophore, appeared to parallel the decrease in Ca²⁺ uptake activity. Phospholipase A₂ activity, monitored by the production of [³H]lysophosphatidylcholine and [³H]lysophosphatidylethanolamine, was also suppressed by dbcAMP. These data indicate that the intracellular cAMP level may be closely associated with Ca²⁺ uptake and phospholipases activation. In addition, it is suggested that alteration of intracellular cAMP regulates phospholipase activation and consequently platelet responses, perhaps by controlling available Ca²⁺ content.     

Evidence for coupling of phospatidic acid formation and calcium influx in thrombin-activated human platelets

The time-sequential relationship between Ca²⁺ flux, phospholipid metabolism and platelet activation have been examined. Thrombin-activation caused a marked enhancement in ⁴⁵Ca²⁺ influx and a decrease in extracellular Ca²⁺ concentration measured by murexide dye, which occurred in parallel with the conversion of 1,2-diacylglycerol (DG) to phosphatidic acid (PA). The incorporated ⁴⁵Ca²⁺ was located mainly in cytosolic fraction. The influx of Ca²⁺ was observed to commence prior to the onset of lysophospholipids formation and subsequent liberation of arachidonic acid. These data provide evidence which indicates a coupling between the rapid PI-turnover and the active Ca²⁺ influx, in which phosphatidic acid (PA) may serve as a Ca²⁺ ionophore.     

A key role for dense granule secretion in potentiation of the Ca2+ signal arising from store-operated calcium entry in human platelets

Recent work has demonstrated a role for Na⁺/Ca²⁺ exchange in potentiation of the Ca²⁺ entry elicited through the human platelet store-operated channel by controlling a Mn²⁺-impermeable Ca²⁺ entry pathway. Here we demonstrate that this involves control over the secretion of dense granules by a Na⁺/Ca²⁺ exchanger (NCX) and so autocrine signalling between platelets. NCX inhibition reduced dense granule secretion. The reduction in SOCE elicited by NCX inhibition could be reversed by the addition of uninhibited donor cells, their releasate alone, or exogenous ADP and 5-HT. The use of specific receptor antagonists indicated that ATP, ADP and 5-HT all played a role in NCX-dependent autocrine signalling between platelets following thapsigargin stimulation, by activating Mn²⁺-impermeable Ca²⁺ entry pathways. These data provide further insight into the mechanisms underlying the known interrelationship between platelet Ca²⁺ signalling and dense granule secretion, and suggest an important role for the NCX in potentiation of platelet activation via dense granule secretion and so autocrine signalling. Our results caution the interpretation of platelet Ca²⁺ signalling studies involving pharmacological or other manipulations that do not assess possible effects on NCX activity and dense granule secretion.     

Human platelet phospholipase A2 activity is responsive in vitro to pH and Ca2+ variations which parallel those occurring after platelet activation in vivo

Secretion of human platelet dense granule contents in response to epinephrine and other weak agonists requires the prior liberation of membrane-esterified arachidonic acid by a phospholipase A₂ enzyme species whose activity is regulated by Na⁺/H⁺ exchange (e.g., Sweatt et al. (1986) J. Biol. Chem. 261, 8660–8673 and Banga et al. (1986) Proc. Natl. Acad. Sci. USA 83, (197–9201). Based on our earlier findings in intact platelets, we postulated that the alkalinization of the platelet interior that accompanies accelerated activity of the Na⁺/H⁺ antiporter enables the phospholipase A₂ enzyme to function at ambient or low concentrations of intraplatelet Ca²⁺. To test the hypothesis that the Ca²⁺ dependence of platelet phospholipase A₂ activity is influenced by changes in intraplatelet pH that occur following platelet activation, we characterized the Ca²⁺ dependence of this enzyme as a function of changes in pH (from pH 6.8–8.0), since it is within this range that intraplatelet pH changes occur following platelet activation. Phospholipase A₂ enzymatic activity in platelet particulate preparations was detectable in the presence of micromolar concentrations of Ca²⁺ (EC₅₀ 1–2 μM) and plateaued above 10 μM Ca²⁺. Enzymatic activity measured at 4.8 μM Ca²⁺ was increased by raising the pH from 5.5 to 8.0 (EC₅₀ 7.4), was optimal at pH 8.0 and declined at more alkaline values. Furthermore, increases in pH from pH 6.8 to pH 8.0 not only increased maximal enzymatic activity but also enabled detection of enzymatic activity at lower Ca²⁺ concentrations. The interdependent regulation of phospholipase A₂ activity by changes in pH and Ca²⁺ suggests that phospholipase A₂ could serve to integrate changes in intracellular pH and available Ca²⁺ that occur subsequent to activation of human platelets by epinephrine and other weak agonists.     

Structural preferences of phosphatidylinositol and phosphatidylinositol-phosphatidylethanolamine model membranes influence of Ca2+ and Mg2+

The structural preferences of soya phosphatidylinositol in isolation and in mixtures with soya phosphatidylethanolamine, and the influence of Ca²⁺ and Mg²⁺ on these preferences, have been examined employing ³¹P-NMR and freeze-fracture techniques. It is shown that phosphatidylinositol assumes the bilayer organization on hydration both in the presence and absence of Ca²⁺ and Mg²⁺. In mixed systems with H_II phase) phosphatidylethanolamine, phosphatidylinositol induces lipidic particle structure at low (<10 mol%) concentrations and bilayer structure at higher levels. In systems containing 15 or 20 mol% phosphatidylinositol, Ca²⁺ (but not Mg²⁺) can induce H_II phase structure. The results indicate that phosphatidylinositol is a more effective agent than other acidic phospholipids for stabilizing bilayer structure, particularly when high levels of divalent cations are present. These findings are discussed in terms of functional roles of phosphatidylinositol and mechanisms whereby Ca²⁺ induces structural reorganization in mixed systems containing acidic phospholipids and phosphatidylethanolamine.