Ca2+.Calmodulin-dependent release of arachidonic acid for renal medullary prostaglandin synthesis. Evidence for involvement of phospholipases A2 and C

The present study examined (a) the source of arachidonic acid for Ca2+-stimulated renal inner medullary prostaglandin synthesis, (b) the Ca2+-dependence of enzymes of the phospholipase A2 and C pathways, and (c) the role of calmodulin in these Ca2+ actions. Ca2+ plus the ionophore A23187 stimulated (2-4-fold) release of labeled arachidonate, diglyceride, prostaglandin E2 or F2 alpha from inner medullary slices with a concomitant fall in labeled phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride (W-7) (10-100 microM) abolished or suppressed Ca++-stimulated immunoreactive prostaglandin E, labeled arachidonate and prostaglandin release, and the fall in labeled phospholipids but did not suppress labeled diglyceride or inositol accumulation. Studies in subcellular fractions demonstrated a particulate phospholipase A2 activity and a phosphatidylinositol-specific phospholipase C activity which was predominantly soluble (80%). W-7 or trifluoperazine (25 microM) abolished Ca2+-stimulated phospholipase A2 activity and particulate phospholipase C activity but were without effect on soluble phospholipase C. W-7 (100 microM) was without effect on Ca2+-stimulated diglyceride lipase and phosphatidic acid-specific phospholipase A2 activities. Hypertonic urea at concentrations that pertain in the inner medulla of hydropenic rats in vivo inhibited Ca2+-induced increases in labeled arachidonate release and immunoreactive prostaglandin E in slice incubates and Ca2+-responsive phospholipase C and A2. The results are consistent with the involvement of phospholipase A2, C, or both in the Ca2+ (+A23187)-stimulated release of free arachidonate for prostaglandin synthesis and support a role for calmodulin in Ca2+ activation of phospholipase A2 and particulate phospholipase C.     

Effects of bivalent cations on prostaglandin biosynthesis and phospholipase A2 activation in rabbit kidney medulla slices.

The bivalent cations Ca2+, Mg2+, Co2+, Mn2+, Sr2+ and Ba2+ were compared for their stimulatory or inhibitory effect on prostaglandin formation in rabbit kidney medulla slices. Ca2+, Mn2+ and Sr2+ ions stimulated prostaglandin generation up to 3--5-fold in a time- and dose-dependent manner (Ca2+ greater than Mn2+ congruent to Sr2+). The stimulation by Mn2+ (but not by Sr2+) was also observed in incubations of medulla slices in the presence of Ca2+. Mg2+ and Co2+ ions were without significant effects on either basal or Ca2+-stimulated prostaglandin synthesis. The stimulatory effects of Ca2+, Mn2+ and Sr2+ on medullary generation of prostaglandin E2 were found to correlate with their stimulatory effects on the release of arachidonic acid and linoleic acid from tissue lipids. The release of other fatty acids was unaffected, except for a small increase in oleic acid release. As both arachidonic acid and linoleic acid are predominantly found in the 2-position of the glycerol moiety of phospholipids, the stimulation by these cations of prostaglandin E2 formation appears to be mediated via stimulation of phospholipase A2 activity.     

Synergistic effect of magnesium and calcium ions in the activation of phospholipase A2 of liver macrophages

In cell-free extracts of rat liver macrophages (Kupffer cells) phospholipase A2 was found to be strongly activated at free Ca2+ concentrations from 100 nM to 1 microM in the presence of 4 mM free Mg2+. This is within the range of intracellular free Ca2+ reported for basal and various stimulated conditions, respectively. Ca2+ alone increased phospholipase A2 activity at high Ca2+ concentrations (1 mM) whereas Mg2+ alone had only little stimulatory effect. Calmodulin does not seem to participate in the regulation of phospholipase A2 although it relieved the inhibition of phospholipase A2 activity by calmodulin antagonists.     

Modulation of Na, K-ATPase activity by prostaglandin E1 and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin

Adenylyl cyclase is activated by prostaglandin E and inhibited by mu-opioids. Since cAMP-related events influence the activity of the Na Pump and its biochemical correlate Na,K-ATPase in many systems, we tested the hypothesis that prostaglandin E1 and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), a mu-opioid agonist, have opposing actions on Na,K-ATPase activity. Studies were conducted with alamethicin-permeabilized SH-SY5Y human neuroblastoma cells. Prostaglandin E1 (1 microM) transiently inhibited Na,K-ATPase activity for 10-15 min. A direct activator of protein kinase A, 8-Br-cAMP (150 and 500 microM), also inhibited, but more rapidly and for a shorter duration. Both DAMGO (1 microM) and Rp-adenosine 3',5'-cyclic monophosphorothioate (500 microM), a protein kinase A-inhibitor, reversed the inhibitory effect of prostaglandin E1. DAMGO alone (1 microM) stimulated Na,K-ATPase activity up to nearly three-fold control activity. The stimulatory action of DAMGO was blocked by cyclosporine A (2 microM), an inhibitor of calcineurin, and was dependent on Ca2+ entry through nifedipine-sensitive Ca2+ channels. In the presence of 1 mM EGTA, DAMGO inhibited Na,K-ATPase activity. DAMGO-induced inhibition was blocked by the inositol 1,4,5-trisphosphate receptor antagonist xestospongin C (1 microM). Na,K-ATPase is poised to modulate neuronal excitability through its roles in maintaining the membrane potential and transmembrane ion gradients. The differential effects of prostaglandin E1 and opioids on Na,K-ATPase activity may be related to their actions in hyperalgesia.     

Calcium dependence of the stimulatory action of hypertonicity on renal medullary prostaglandin synthesis

Previous studies have shown that hypertonic mannitol or NaCl increases the release of [3H]arachidonate and immunoreactive prostaglandin E in inner medullary slices incubated in Ca2+-free media containing EGTA. By contrast, the stimulation of these parameters by ionophore A23187 and by arginine-vasopressin are abolished in Ca2+-free media plus EGTA. In the present study, the effects of Ca2+ deprivation and the intracellular Ca2+ antagonist TMB-8 [8-N,N-diethylamino)octyl-3,4,5 -trimethoxybenzoate-HCl) were further examined to assess the Ca2+ dependence of the actions of different stimuli of prostaglandin E synthesis in rat renal inner medulla. Ca2+-free media without EGTA abolished increases in [3H]arachidonate and immunoreactive prostaglandin E release induced by ionophore A23187, but not those induced by arginine-vasopressin, suggesting that different pools of Ca2+ subserve expression of the actions of these two stimuli. At low concentrations, TMB-8 (10-25 microM) inhibited increases in [3H]arachidonate and immunoreactive prostaglandin E release induced by arginine-vasopressin, but did not influence effects of Ca2+ plus ionophore A23187 or hypertonicity on these parameters. At higher concentrations (100-500 microM), TMB-8 suppressed effects of ionophore A23187, hyperosmolar NaCl and mannitol on immunoreactive prostaglandin E and [3H]arachidonate release from slices. The effects of a sub-optimal inhibitory concentration of TMB-8 on ionophore A23187 actions were overcome by increasing Ca2+ in the media from 1.5 to 5 mM. Ca2+ deprivation, or concentrations of EGTA or TMB-8, that were effective in suppressing increases in immunoreactive prostaglandin E induced by ionophore A23187, arginine-vasopressin or hypertonicity, did not modify increases in immunoreactive prostaglandin E induced by exogenous arachidonate. Moreover, in microsomal fractions of inner medulla, TMB-8 suppressed Ca2+-dependent increases in phospholipase A2 and C activities, an effect which was competitive with Ca2+. Thus, Ca2+ deprivation and TMB-8 act at a step in the immunoreactive prostaglandin E synthetic pathway proximal to cyclooxygenase activity, and probably at the level of Ca2+-dependent acyl hydrolase activity. The results with TMB-8 indicate that an intracellular pool of Ca2+ is involved in expression of the actions of hypertonicity to increase [3H]arachidonate release and immunoreactive prostaglandin E in inner medulla.     

Detergent Effects on the Phosphatidylinositol-Specific Phospholipase C in Rat Brain Synaptosomes

In the presence of Ca2+ (2.5 mM) and using [14C]arachidonoyl phosphatidylinositol (PI) membrane as substrate, phosphatidylinositol-specific phospholipase C (PI-PLC) (EC 3.1.4.10) in rat brain synaptosomes was activated by deoxycholate but not taurocholate. Calcium stimulated enzymic hydrolysis by both detergents, but the stimulatory effect of taurocholate was less than that of deoxycholate. Peak stimulation for deoxycholate was observed at 1 mg/ml, whereas that for taurocholate was 4 mg/ml. When 1 mM EDTA was added to the taurocholate (4 mg/ml) and Ca2+ (3.5 mM) system, synaptosomal PI-PLC activity was greatly stimulated, to almost the same level as the deoxycholate + Ca2+ system. This system required the presence of all three factors, and EGTA could not effectively replace EDTA in the stimulatory action. The detergent-induced hydrolysis of synaptosomal PI by the deoxycholate + Ca2+ and the taurocholate + Ca2+ + EDTA systems was strongly inhibited by divalent metal ions such as Zn2+, Cu2+, Pb2+, and Fe2+, whereas Mg2+ and Ca2+ were ineffective. Nevertheless, only the deoxycholate + Ca2+ system was responsive to enzyme inhibition by membrane-perturbing agents such as lysophospholipids and free fatty acids. The specific requirement for EDTA in the taurocholate system may be due to the release of a pool of inhibitory divalent metal ions from the membranes.     

Effect of diarachidonin on prostaglandin E2 synthesis in rabbit kidney medulla slices.

The effect of diarachidonin on the synthesis of prostaglandin E2 in rabbit kidney medulla slices was examined. The addition of diarachidonin stimulated prostaglandin E2 production in a dose-dependent manner. At three concentrations (10, 50 and 100 microM), increases in prostaglandin E2 formation induced by exogenous diarachidonin were 2-fold greater than those induced by exogenous arachidonic acid. Diacylglycerol or phosphatidic acid from egg lecithin had little or no effect on prostaglandin E2 production. Moreover, EGTA failed to inhibit diarachidonin-stimulated prostaglandin E2 formation, indicating that the stimulatory effect of diarachidonin is not mediated through the activation of endogenous phospholipase A2 (including phosphatidic acid-specific phospholipase A2). These results are discussed in the light of our former hypothesis that arachidonic acid release from kidney medulla phospholipids might occur through the sequential action of a phospholipase C coupled to diacylglycerol and monoacylglycerol lipases [Fujimoto, Akamatsu, Hattori & Fujita (1984) Biochem. J. 218, 69-74].     

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E1 or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. p-Bromophenacyl bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: arachidonic acid greater than collagen greater than thrombin greater than ionophore A23187. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca2+ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca2+ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.     

Properties of particulate, membrane-associated and soluble guanylate cyclase from cardiac muscle, skeletal muscle, cerebral cortex and liver.

1. Guanylate cyclase of washed particles and plasma membranes showed S-shaped progress curves when titrated with either GTP or Mn2+ ions; similar results were obtained with Triton X-100-solubilized enzyme preparation from washed particles. Hill plots of these data revealed multiple metal-nucleotide and free-metal binding sites. 2. Guanylate cyclase of supernatant fractions displayed typical Michaelis-Menten properties when enzyme required excess of (free) Mn2+ (over GTP) for maximal activities; Ka (free Mn2+) was about 0.15-0.25 mM at subsaturating concentrations of GTP. 4 MnATP, MnADP, and MnGDP were found to increase the activities of both particulate and superantant enzyme, when MnGTP concentration was below saturation and free Mn2+ ion concentration was low (less than 100 muM); MnATP (50muM-1 mM) inhibited both these activities at high free Mn2+ concentration (1.5 mM) and inhibition of the particulate enzyme was greater than that of supernatant enzyme. 5. Ca2+ ions stimulated supernatant-enzyme activity; the stimulatory concentration of Ca2+ ions depended on the concentration of Mn2+ and GTP. 6. A modest stimulation of particulate guanylate cyclase by pyrophosphate (0.02-1 mM) was observed; the pyrophosphate effect appeared to be competitive with respect to GTP. At a higher concentration (2 mM), pyrophosphate produced a marked inhibition of particulate enzyme; the nature of inhibitory effect appeared complex. 7. Inorganic salts (e.g. NaCl, KCl, LiBr, NaF) produced inhibition of particulate enzyme; the degree of inhibition of Triton X-100-stimulated activity was less than that of unstimulated activity. 9. Treatment of sarcolemmal or microsomal membranes with either phospholipase C or trypsin decreased, whereas phospholipase A increased, the activity of guanylate cyclase.     

Aluminum enhances the stimulatory effect of NaF on prostaglandin E2 synthesis in a clonal osteoblast-like cell line, MOB 3-4, in vitro

The effect of NaF on prostaglandin E2 (PGE2) synthesis in a clonal osteoblast-like cell line, MOB 3-4, was examined in the presence of Al3+. The MOB 3-4 cell line, which was derived from neonatal mouse calvaria, displays many osteoblastic characteristics, including the biosynthesis of PGE2. In the absence of Al3+, 1 mM NaF increased PGE2 synthesis (per well) to about 340% of the control level, whereas NaF at lower concentrations (below 0.1 mM) did not show such a significant effect. In the presence of 10 microM Al3+, NaF concentrations ranging from 0.01 to 1 mM increased PGE2 synthesis in a dose-dependent manner, though 10 microM Al3+ had no effect by itself. Similar effects were observed on alkaline phosphatase (ALP) activity per well, but a stimulatory effect of NaF on protein synthesis was observed only in the presence of 10 microM Al3+. These data demonstrated that PGE2 synthesis per protein was increased by NaF alone, and this effect was markedly enhanced by the addition of AlCl3. ALP activity per protein was, however, significantly increased by NaF in the absence of AlCl3. Taken together with our previous finding that Al3+ enhances the NaF-induced Ca2+ mobilization in MOB 3-4 cells, these results suggest that F- combined with Al3+ (i.e., AlF4-) is a more potent stimulator of PGE2 synthesis in cells than F- alone, and that the AlF4- -enhanced PGE2 synthesis may be caused by an increase in cytosolic free Ca2+ concentration during activation of the G protein by AlF4-.     

Role of Ca2+ and cyclic nucleotides in the control of prostaglandin E production in the rat anterior pituitary gland.

In an attempt to relate changes in the intracellular concentration of prostaglandin E to the secretion process, two agents known to increase cyclic nucleotide concentrations and hormone release were added to dispersed rat anterior pituitary cells. They caused increases in teh intracellular prostaglandin E concentrations. Increasing the K+ concentration in the medium (which stimulates hormone release) caused a rapid rise in prostaglandin E concentrations. The addition of the Ca2'onophore A23187 had a similar effect. The effects of changes in the K+ and Ca2+concentrations and the addition of EDTA were measured on the redistribution of radioactivity in pituitary glands prelabelled with [3H]arachidonic acid. Elevated K+ concentrations stimulated the transfer of label to prostaglandins and free arachidonic acid, suggesting an increased phospholipase A activity. On the other hand, the absence of extracellular CaCl2 and the addition of EDTA had the opposite effect, which could be cancelled by the addition of sufficient amounts. of CaCl2. It is concluded that the addition of agents that increase membrane permeability to bivalent cations probably results in an influx of Ca2+ and this appears to result in increased phospholipase A activity, which in turn leads to an increase in prostaglandin production.     

The role of intramembrane Ca2+ in the hydrolysis of the phospholipids of Escherichia coli by Ca2+-dependent phospholipases

Ca2+-dependent phospholipases A require Ca2+ concentrations in the millimolar range for optimal activity toward artificial substrates. Because Ca2+-dependent phospholipases A2 degrade the phospholipids of Escherichia coli, treated with the membrane-active antibiotic polymixin B equally well with and without added Ca2+ (Weiss, J., Beckerdite-Quagliata, S., and Elsbach, P. (1979) J. Biol. Chem. 254, 11010-11014), we have examined the possibility that intramembrane Ca2+ can provide the Ca2+ needed for phospholipase action. We studied the effect of Ca2+ depletion on the hydrolysis of the phospholipids of polymixin B-killed E. coli by 1) added pig pancreas phospholipase A2 in E. coli S17 (a phospholipase A-lacking mutant) and 2) endogenous Ca2+-dependent phospholipase A1 in the parent strain E. coli S15. Transfer of E. coli from nutrient broth (Ca2+ concentration approximately 3 X 10(-5) M) to Ca2+-depleted medium (Ca2+ concentration less than 10(-6)M) reduced polymixin B-induced hydrolysis by 50-75%, in parallel with a reduction of bacterial Ca2+ from 19.6 +/- 2.8 to 3.9 +/- 0.6 nmol (mean +/- standard error) per 3 X 10(10) bacteria. The bacterial Ca2+ content was repleted and the sensitivity of the bacterial phospholipids to hydrolysis by both exogenous phospholipase A2 (E. coli S17) and endogenous phospholipase A (E. coli S15) was restored by adding Ca2+ back to the suspensions. Complete restoration occurred at low Ca2+ levels in the reaction mixture (3 X 10(-5) - 10(-4) M) and required time, suggesting that hydrolysis was restored because bacterial Ca2+ stores were gradually replenished and not because extracellular Ca2+ concentrations were raised to levels that were still at least 10X lower than needed for optimal phospholipase A activity. This conclusion is supported by the finding that Ca2+ depletion or addition caused respectively decreased and increased release of lipopolysaccharides by EGTA (ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid), suggesting that the bacterial Ca2+ pool bound to lipopolysaccharides in the outer membrane shrinks or expands depending on extracellular Ca2+ levels. Thus, the cationic membrane-disruptive polymixin B, thought to compete with Mg2+ and Ca2+ for the same anionic sites on lipopolysaccharides, may liberate the Ca2+ near where the phospholipids are exposed to phospholipase.     

Calcium-requirement and its pH dependency of lysophosphoinositide-specific phospholipase C in porcine platelet membranes

The lysophosphoinositide-specific phospholipase C (lysoPI-PLase C) in porcine platelet membranes had an optimal pH of 9.2 and the activity at a physiological pH of 7.3 was 20% of the maximum in the absence of added divalent metals (Murase, S. et al. (1985) J. Biol. Chem. 260, 262). The activity was completely inhibited by 1 mM EGTA in the assay mixture but was restored by addition of excess Ca2+ or Mn2+, indicating that this is a metalloenzyme. However, membranes pretreated with 1 mM EGTA and washed with buffer retained full activity at a free Ca2+ concentration of 5 nM and no stimulation was observed by added Ca2+ at pH 9.2. In contrast to the results obtained at pH 9.2, addition of Ca2+ stimulated lysoPI-PLase C activity severalfold at pH 7.3, apparently by shifting down the optimal pH and broadening the pH profile. The effect of Ca2+ at pH 7.3 was to enhance Vmax with no significant change in Km value. The stimulatory effect of Ca2+ at pH 7.3 alone did not appear to be of physiological significance since millimolar concentrations of Ca2+ were necessary to reach the maximum activity. However, a shift in pH had a profound effect on the Ca2+-dependency of the activity. A rise in 2 pH units increased the apparent affinity for Ca2+ 10,000-fold. These results indicate that the alkalinization and the rise in free Ca2+ concentration known to occur in stimulated platelets could synergistically provide conditions under which the lysoPI-PLase C exerts its activity when the substrate lysoPI is generated by phospholipase A.     

Cardiac glycosides stimulate phospholipase C activity in rat pinealocytes

Ouabain and related cardiac glycosides stimulate phospholipase C activity 5-fold in rat pinealocytes. The combined treatment of ouabain and norepinephrine, which also stimulates phospholipase C, produces an additive effect. The effects of either ouabain or norepinephrine are blocked by EGTA. However, there are notable differences. The stimulatory effect of ouabain is lost when extracellular Na+ is reduced to 20 mM and is not blocked by prazosin. In contrast, the stimulatory effect of norepinephrine is not blocked when extracellular Na+ is reduced to 20 mM but is blocked by prazosin. Ouabain appears to increase phospholipase C activity through a mechanism involving inhibition of Na+,K+-ATPase, and an accumulation of intracellular Na+ and Ca2+, not involving alpha 1-adrenoceptors. These findings raise the possibility that activation of phospholipase C might be a more general effect of cardiac glycosides.     

Quinacrine antagonizes the effects of Na,K-ATPase inhibitors on renal prostaglandin E2 release but not their effects on renin secretion

Previous results have demonstrated that two inhibitors of Na-and-K-activated adenosine triphosphatase (ouabain, vanadate) lead to stimulated prostaglandin E2 release and to inhibited renin secretion in the rat renal cortical slice preparation. It was speculated that stimulation of phospholipase A2 activity accounted for the effect on prostaglandin E2 release. We used the same preparation in the present experiments, and showed that another inhibitor of Na-and-K-activated adenosine triphosphatase (K-free incubation medium) stimulates prostaglandin E2 release and inhibits renin secretion. Quinacrine antagonized the stimulatory effects of ouabain, vanadate, and K-free medium on prostaglandin E2 release (consistent with phospholipase A2 involvement), but did not antagonize their inhibitory effects on renin secretion. Collectively, these observations lend further weight to the argument against a mediatory role of prostaglandin synthesis in the renin secretory process.     

Inhibitory and stimulatory effects of fluoride on the calcium pump of cardiac sarcoplasmic reticulum.

While studying the effects of membrane phosphorylation on active Ca2+ transport in cardiac sarcoplasmic reticulum (SR) we used NaF (a conventional phosphatase inhibitor) in the Ca2+ transport assay medium to suppress protein dephosphorylation by endogenous phosphatases. Unexpectedly, depending on the experimental conditions employed, NaF was found to cause a strong inhibitory or stimulatory effect on ATP-dependent, oxalate-facilitated Ca2+ uptake (Ca2+ pump) activity of SR. Investigation of this phenomenon using canine cardiac SR revealed the following. Exposure of SR to NaF in the absence of Ca2+ or ATP in the Ca2+ transport assay medium (prior to initiating Ca2+ transport by the addition of Ca2+ or ATP) promoted a striking concentration-dependent inhibitory effect of NaF (50% and 90% inhibition with approx. 4 and 10 mM NaF, respectively) on Ca2+ uptake by SR; the magnitude of inhibition did not differ appreciably with varying oxalate concentrations. In contrast, exposure of SR to NaF in the presence of both Ca2+ and ATP resulted in a concentration-dependent stimulatory effect of NaF (half-maximal stimulation at approx. 2.5 mM NaF with 2.5 mM oxalate in assay) on Ca2+ uptake; the magnitude of stimulation decreased with increasing oxalate concentration (greater than 2-fold at 1 mM oxalate, 10% at 5 mM oxalate). The inhibitory effect prevailed when SR was exposed to NaF in the presence of Ca2+ alone (without ATP) or ATP alone (without Ca2+). Both the inhibitory and stimulatory effects of NaF were specific to fluoride ion, as NaCl (1-10 mM) showed no effect on Ca2+ uptake by SR under identical assay conditions. A persistently less active state of the Ca2+ pump (evidenced by decreased Ca2+ transport rates) resulted upon pretreatment of SR with NaF in the absence of Ca2+ or ATP; presence of Ca2+ and ATP during pretreatment prevented this transition. The inhibitory action of NaF on the Ca2+ pump was accompanied by a two-fold increase in K0.5 for Ca2+ and decrements in Hill coefficient (nH) and Ca(2+)-stimulated ATP hydrolysis, as well as steady-state level of Ca(2+)-induced phosphoenzyme. The stimulatory effect of NaF, on the other hand, was associated with an increase in the ratio of Ca2+ transported/ATP hydrolysed with only minor changes, if any, in the above parameters. These findings imply that the divergent effects of fluoride are dependent on specific conformational states of the Ca(2+)-ATPase which evolve during the catalytic and ion transport cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of regulation of adenylate cyclase activity in human polymorphonuclear leukocytes by calcium, guanosyl nucleotides, and positive effectors.

This study presents the results of a kinetic investigation of adenylate cyclase in human polymorphonuclear leukocytes. In the presence of a saturating concentration of substrate (1 mM), the basal activity was increased severalfold by increasing Mg2+ from 1 to 25 mM. A Hill coefficient of 1.9 was obtained for Mg2+ or ATP. The data suggest cooperative interactions between the substrate binding sites in the neutrophil adenylate cyclase complex. It has been observed that guanyl-5'-yl imidodiphosphate (Gpp(NH)p) (S0.5 = 10 MUM) significantly increased and Ca2+ (S0.5 = 0.5 MM) significantly decreased only the Vmax without affecting the Hill coefficient or S0.5 for ATP. The Hill coefficients for Ca2+ or Gpp(NH)p were 0.9 and 0.8, respectively. The Hill coefficient for Ca2+ was not changed by the increased Gpp(NH)p concentrations. It appears that neutrophil adenylate cyclase has distinct binding sites for Gpp(NH)p and Ca2+, one for each compond. The binding of ligands is not changed by the other effectors and the action is directed only toward the Vmax of the enzyme. The stimulatory action of positive effectors (prostaglandin E1, isoproterenol, histamine) was enhanced by Gpp(NH)p and depressed by Ca2+. No preferential stimulation by Gpp(NH)p nor inhibition by Ca2+ of the action of the positive effectors has been found. The data suggests that only one type of catalytic subunit responds to the action of several positive effectors. Extracellular Gpp(NH)p or Ca2+ do not affect the cyclic adenosine 3':5'-monophosphate (cAMP) level in whole neutrophils and the effect of positive effectors on cAMP production is also not significantly changed by 5 mM Ca2+ or 0.1 mM Gpp(NH)p. Ionophore A23187 in the presence of 5 mM Ca2+ enhances Ca2+ entry into cells and decreases the basal cAMP formation. It appears that Gpp(NH)p or Ca2+ act only at the intracellular site of the adenylate cyclase complex.     

Selective inhibition of human platelet phospholipase A2 by buffering cytoplasmic calcium with the fluorescent indicator quin 2. Evidence for different calcium sensitivities of phospholipases A2 and C

Human platelets labelled with either [14C]arachidonic acid or [32P]orthophosphate were loaded or not with the Ca2+ fluorescent indicator quin 2. They were then incubated in the presence or in the absence of human thrombin (1 U/ml) in a medium where Ca2+ concentration was adjusted near zero or to 1 mM. Under these conditions, phospholipase A2 activity, as detected by the release of [14C]arachidonate and of its metabolites, or by the hydrolysis of [14C]phosphatidylcholine, was severely impaired in quin 2-loaded platelets upon removal of external Ca2+. However, Ca2+ was not required in non-loaded platelets, where a maximal phospholipase A2 activity was detected in the absence of external Ca2+. In contrast, phospholipase C action, as determined from the amounts of [14C]diacylglycerol, [14C]- or [32P]phosphatidic acid formed, appeared to be much less sensitive to the effects of quin 2 loading and of Ca2+ omission. By using various concentrations of quin 2, it was found that the inhibitory effect exerted against phospholipase A2 could be overcome by external Ca2+ only when the intracellular concentration of the calcium chelator did not exceed 2 mM. At higher concentrations averaging 3.5 mM of quin 2, phospholipase A2 activity was fully suppressed even in the presence of external Ca2+, whereas phospholipase C was still active, although partly inhibited. It is concluded that platelet phospholipase A2 requires higher Ca2+ concentrations than phospholipase C to display a maximal activity. By comparing platelet phospholipase A2 activity under various conditions with the values of cytoplasmic free Ca2+ as detected by quin 2 fluorescence, it is proposed that cytoplasmic free Ca2+ in control platelets stimulated with thrombin can attain concentrations above 1 microM, probably close to 5-10 microM, as recently determined with the photoprotein aequorin (Johnson, P.C., Ware, J.A., Cliveden, P.B., Smith, M., Dvorak, A.M. and Salzman, E.W. (1985) J. Biol. Chem. 260, 2069-2076).     

Solubilization and properties of Ca2+-dependent human platelet phospholipase A2

Using a sonicated dispersion of radiolabeled 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine as substrate, we found that phospholipase A2 activity of human platelets was enhanced 2.4-fold by albumin (1 mg/ml). The enzyme was recovered predominantly in the cytosolic fraction of platelets with less than a third of its activity being associated with the membrane fraction. In the presence of 24 mM n-octyl-beta-D-glucopyranoside (octylglucoside) phospholipase A2 was effectively (more than 90%) extracted from platelet lysates without solubilization of platelet membranes. Ion exchange chromatography of the soluble enzyme yielded a phospholipase A2 of unchanged total activity and great stability. This phospholipase A2 was active only in the presence of divalent cations (Ca2+ greater than Sr2+ greater than Mg2+ = 0), required albumin for optimal activity and exhibited exclusive positional specificity for the acyl ester bond at the 2-position of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. Indomethacin (500 microM), mepacrine (500 microM) and N-ethylmaleimide (4 mM) inhibited the phospholipase A2 by 69, 62 and 19%, respectively. The results are discussed in the light of previous findings on human platelet phospholipase A2.     

Modulation of phospholipase A2 activity associated with human sperm membranes by divalent cations and calcium antagonists

Phospholipase A2 activity in sonicates and acid extracts of ejaculated, washed human sperm was measured using [1-14C] oleate-labeled autoclaved E. coli and 1-[1-14C] stearoyl-2-acyl-3-sn- glycerophosphorylethanolamine as substrates. Phospholipase A was optimally active at pH 7.5, was calcium-dependent, and exclusively catalyzed the release of fatty acid from the 2-position of phospholipids. The activity was membrane-associated, and was solubilized by extraction with 0.18 N H2SO4. Acid extracts of human sperm had the highest specific activity (1709 nmols /h per mg), followed by mouse, rabbit and bull, which were 105, 36 and 1.7 nmols /h per mg, respectively. para-bromophenacyl bromide inhibited human sperm phospholipase A2 activity, but mepacrine was without effect. In the presence of 1.0 mM added CaCl2, phospholipase A2 activity was inhibited by Zn2+ and Mn2+; whereas Cu2+, Cd2+, Mg2+, or Sr2+ had no effect. Zn2+ stimulated activity at low concentrations (10(-6) to 10(-8) M), and inhibited activity in a dose-dependent manner at concentrations of 10(-5) M. The extent of stimulation by low concentrations of Zn2+ was dependent on Ca2+ concentration; at 10(-7) M, Zn2+ activity was stimulated 160% with 0.5 mM CaCl2, and only 120% with 1.0 mM CaCl2. At low concentrations (10(-5) to 10(-7) M), methoxyverapamil (D600) and trifluoperazine stimulated human sperm phospholipase A2 activity, and trifluoperazine but not D600 produced almost complete inhibition between 10(-5) and 10(-4) M of the drug. The significance of human sperm phospholipase A2 activity and its modulation by Ca2+, Zn2+ and Mn2+ in the sperm acrosome reaction is discussed.