Priming of human polymorphonuclear leukocytes with granulocyte-macrophage colony-stimulating factor involves protein kinase C rather than enhanced calcium mobilisation.

Pretreatment of human polymorphonuclear leukocytes with the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) enhances leukotriene biosynthesis in response to a receptor agonist (e.g. N-formyl-methionyl-leucyl-phenylalanine, fMLP) or a Ca(2+)-ionophore (e.g. ionomycin). This priming effect could be traced back to an elevated release of arachidonic acid from the phospholipid pools and hence an increased leukotriene biosynthesis by 5-lipoxygenase. Preincubation of polymorphonuclear leukocytes with GM-CSF did not influence the basal intracellular Ca2+ level and does not enhance cytosolic free calcium after stimulation with fMLP or ionomycin. Only a small increase in the second Ca2+ phase after receptor agonist stimulation was found. However, the Ca(2+)-threshold level necessary for the liberation of arachidonic acid by phospholipase A2 was decreased from 350-400 nM calcium in untreated cells to about 250 nM calcium in primed cells. This allows phospholipase A2 to be activated by a release of calcium from intracellular stores and by ionomycin concentrations which are ineffective in untreated cells. Protein biosynthesis inhibitors like actinomycin D (10 micrograms/ml) and cycloheximide (50 micrograms/ml) had no effect on the enhanced leukotriene biosynthesis in primed cells after stimulation with ionomycin. However, staurosporine (200 nM), an inhibitor of protein kinase C totally abolished the priming effect of GM-CSF after stimulation with ionomycin. The priming effect of GM-CSF could be mimicked by phorbol myristate acetate (PMA; 1 nM) and no additive or synergistic effect was found on leukotriene biosynthesis by simultaneous pretreatment with PMA and GM-CSF and stimulation with either fMLP or ionomycin. These results provide evidence that the enhanced arachidonic acid release in GM-CSF-primed polymorphonuclear leukocytes after stimulation with either fMLP or ionomycin involves activation of protein kinase C which, by a still unknown mechanism, reduces the Ca2+ requirement of phospholipase A2.     

Purification and properties of protein kinase C from bovine polymorphonuclear leucocytes

A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from bovine polymorphonuclear leucocytes. The purified enzyme had a specific activity of 10 000 U/mg protein and on SDS gelelectrophoresis the Mr was 88 000. The enzyme activity was almost totally dependent upon phosphatidylserine and could be strongly activated by Ca2+ concentrations in the micromolar range. At lower concentrations of calcium (less than 1 X 10(-7) M) the enzyme was only activated by the simultaneous presence of phosphatidylserine and diolein. Phorbol 12-myristate 13-acetate mimicked the effect of diolein and partially activated the enzyme. Protein kinase C activity and the phorbolester binding protein co-purified throughout all the purification steps.     

Differential effects of phorbol ester on phenylephrine and vasopressin-induced Ca2+ mobilization in isolated hepatocytes

Receptor-mediated breakdown of PtdIns(4,5)P2 produces two cellular signals, Ins(1,4,5)P3, which can release intracellular Ca2+, and diacylglycerol, which activates a Ca2+- and phospholipid-dependent protein kinase (protein kinase C). This study assesses the significance of protein kinase C in relation to phenylephrine- and vasopressin-induced Ca2+ mobilization in hepatocytes. Phorbol ester (4 beta-phorbol-12-myristate-13-acetate), which can directly activate protein kinase C, had no effect either on Ca2+ efflux from the cell (measured with arsenazo III) or on Ca2+ influx (measured with Quin-2), processes which are inhibited and stimulated, respectively, by both phenylephrine and vasopressin. No evidence of synergism between phorbol ester pretreatment of hepatocytes and the Ca2+ ionophore (ionomycin)-mediated effects on the increase of cytosolic free Ca2+ and phosphorylase activation could be obtained. These findings suggest that protein kinase C is not obligatorily involved in the regulation of hepatocyte Ca2+ fluxes. Pretreatment of hepatocytes with phorbol ester (PMA) or 1-oleoyl-2-acetylglycerol totally inhibited the effects of phenylephrine in elevating the cytosolic free Ca2+; half-maximal inhibitory effects occurred at PMA and 1-oleoyl-2-acetylglycerol concentrations of 1 ng/ml and 12 micrograms/ml, respectively. In contrast, pretreatment with PMA had a much smaller effect on Ca2+ mobilization induced by vasopressin. These observations suggest that protein kinase C may be involved in "down-regulation" of the alpha 1-receptor in hepatocytes and may thus exert a negative influence on the Ca2+-signalling pathway.     

Mechanism of hepatic glycogen synthase inactivation induced by Ca2+-mobilizing hormones. Studies using phospholipase C and phorbol myristate acetate.

Incubation of hepatocytes with the protein kinase C activator and tumour promoter 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) produced a time- and concentration-dependent inactivation of glycogen synthase, but no change in phosphorylase. The same rate and extent of inactivation occurred in hepatocytes depleted of Ca2+ by treatment with the Ca2+ chelator EGTA. When hepatocytes were treated with the Ca2+-mobilizing hormone vasopressin (10 nM), the rate of glycogen synthase inactivation was similar to that observed with PMA (1 microM). Depletion of intracellular Ca2+ stores with EGTA abolished the ability of vasopressin to mobilize Ca2+ and activate phosphorylase without abolishing its ability to inactivate glycogen synthase and increase 1,2-diacylglycerol (DAG), the endogenous activator of protein kinase C. Protein kinase C, either in membranes or after partial purification, was shown to be activated in vitro by PMA in the presence of very low concentrations of Ca2+. Exogenous phospholipase C from Clostridium perfringens, at low concentrations, inactivated glycogen synthase and increased DAG without affecting cell Ca2+ or phosphorylase. It is proposed that the inactivation of glycogen synthase elicited by the Ca2+-mobilizing hormones is due, at least in part, to generation of DAG and activation of protein kinase C.     

Protein kinase C phosphorylation of protamine is Ca2+ independent, but the addition of DNA renders it Ca2+ dependent

Protamine is a unique substrate of protein kinase C for its Ca2+-independent phosphorylation. The interaction between protein kinase C and protamine and the effect of DNA on the interaction was studied. Protein kinase C was retained in a protamine-immobilized Sepharose 4B column, even in the absence of Ca2+ and was eluted with ammonium sulfate or L-arginine. The eluted enzyme was fully activated by phosphatidylserine alone, when protamine was used as substrate. When DNA was included in the assay system, the activity elicited by phosphatidylserine alone was inhibited. The DNA effect on the activity in the presence of both Ca2+ and phosphatidylserine was much lower than on the activity elicited by phosphatidylserine alone, thereby demonstrating the Ca2+ sensitivity of protamine phosphorylation.     

Effect of thapsigargin on cytosolic Ca2+ level and amylase release in rat parotid acinar cells.

At concentrations greater than 0.01 microM, thapsigargin (ThG) dose-dependently caused an increase in cytosolic free Ca2+ concentration ([Ca2+]i) in rat parotid acinar cells, as measured by the fluorescent Ca(2+)-indicator fura-2. In the absence of extracellular Ca2+, a transient increase in [Ca2+]i by ThG was observed, and subsequent addition of carbachol (CCh) did not produce a further [Ca2+]i response, suggesting that ThG released Ca2+ from the CCh-sensitive intracellular Ca2+ pool. Since ThG did not stimulate formation of inositol phosphates, the ThG-induced Ca2+ mobilization is independent of phosphoinositide breakdown. High concentrations (greater than 0.1 microM) of ThG induced amylase release from rat parotide acini, but the effect was very poor as compared with that of CCh or the protein kinase C activator, PMA (phorbol 12-myristate 13-acetate). Combined addition of ThG and PMA modestly potentiated amylase release induced by PMA alone. These results support the view that amylase release by muscarinic stimulation is mediated mainly by activation of protein kinase C rather than a rise in [Ca2+]i, although Ca2+ may modulate the secretory response.     

Local Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels stimulates production of an intracellular messenger and an intercellular pro-inflammatory signal

Ca2+ entry through store-operated Ca2+ channels drives the production of the pro-inflammatory molecule leukotriene C4 (LTC4) from mast cells through a pathway involving Ca2+-dependent protein kinase C, mitogen-activated protein kinases ERK1/2, phospholipase A2, and 5-lipoxygenase. Here we examine whether local Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane stimulates this signaling pathway. Manipulating the amplitude and spatial extent of Ca2+ entry by altering chemical and electrical gradients for Ca2+ influx or changing the Ca2+ buffering of the cytoplasm all impacted on protein kinase C and ERK activation, generation of arachidonic acid and LTC4 secretion, with little change in the bulk cytoplasmic Ca2+ rise. Similar bulk cytoplasmic Ca2+ concentrations were achieved when CRAC channels were activated in 0.25 mm external Ca2+ versus 2 mm Ca2+ and 100 nm La3+, an inhibitor of CRAC channels. However, despite similar bulk cytoplasmic Ca2+, protein kinase C activation and LTC4 secretion were larger in 2 mm Ca2+ and La3+ than in 0.25 mm Ca2+, consistent with the central involvement of a subplasmalemmal Ca2+ rise. The nonreceptor tyrosine kinase Syk coupled CRAC channel opening to protein kinase C and ERK activation. Recombinant TRPC3 channels also activated protein kinase C, suggesting that subplasmalemmal Ca2+ rather than a microdomain exclusive to CRAC channels is the trigger. Hence a subplasmalemmal Ca2+ increase in mast cells is highly versatile in that it triggers cytoplasmic responses through generation of intracellular messengers as well as long distance changes through increased secretion of paracrine signals.     

Involvement of intermediary metabolites in the pathway of extracellular Ca2+-induced mitogen-activated protein kinase activation in human fibroblasts.

Human fibroblasts in culture will grow in serum-free medium containing serum replacement factors, but without protein growth factors, as long as the Ca2+ level is 1.0-2.0 mM. When the Ca2+ is reduced to 0.1 mM, the cells stop cycling, but they can be reinduced to cycle by raising the Ca2+ level to 1.0 mM Ca2+ or to higher concentrations that result in activation of mitogen-activated protein kinase (MAPK). We now report that exposure of human fibroblasts to extracellular Ca2+ increased the level of inositol (1,4,5)-trisphosphate in the cytoplasm and caused a transient rise in the concentration of intracellular free Ca2+. Ca2+-induced MAPK activation was partly abolished by treatment of the cells with pertussis toxin. It was also decreased by treatment of cells with thapsigargin, which depletes intracellular Ca2+ stores; with phorbol 12-myristyl 13-acetate (PMA), which down-regulates protein kinase C (PKC); with the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide HCl (W-7), and calmidazolium (24571); as well as with lanthanum, a Ca2+ channel inhibitor. Ca2+ stimulation did not result in phosphorylation of the c-raf-1 protein. Our results suggest that extracellular Ca2+ stimulates MAPK activation through a pathway(s) involving a pertussis toxin-sensitive G protein, phospholipase C, intracellular free Ca2+, calmodulin, and PKC.     

Mechanism of polymorphonuclear leukocyte activation by myristate. Involvement of calcium ion and protein kinase C

The stimulative effects of myristate on the superoxide generation and depolarization of membrane potential of polymorphonuclear leukocytes (PMN) are particularly strong, yet myristate does not affect the intracellular free Ca2+ level ([Ca2+]i) in the presence of 1 microM free calcium in calcium-EGTA buffer. The half maximum concentration of myristate was 10 microM. Myristate inhibited the transitory changes in [Ca2+]i induced by formylmethionyl-leucyl-phenylalanine (FMLP), but stimulated further the FMLP-induced superoxide generation; these effects are similar to those of phorbol myristate acetate (PMA). The myristate-induced superoxide generation was partially inhibited by H-7, a specific inhibitor of protein kinase C. Myristate stimulated the activity of Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in a concentration-dependent manner in the presence of 10(-6) M Ca2+. The Ka was 100 microM. These results suggested that there is no relation between the superoxide generation and the [Ca2+]i change in PMNs and that the effects of myristate are similar to those of PMA against PMN.     

Regulation of protein kinase C activity by cooperative interaction of Zn2+ and Ca2+

cis-Fatty acids such as oleic acid or linoleic acid have been previously shown to induce full activation of protein kinase C in the absence of Ca2+ and phospholipids (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193; Murakami, K., Chan, S.Y., and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429). In this study, we have investigated the effects of various metal ions on protein kinase C activity without the interference of Ca2+ since cis-fatty acid requires no Ca2+ for protein kinase C activation. Here we report a specific interaction of Zn2+ with protein kinase C in either a positive or negative cooperative fashion in concert with Ca2+. At low concentrations (approximately 5 microM) of Ca2+, Zn2+ enhances protein kinase C activity induced by both oleic acid and phosphatidylserine/diolein. In contrast, Zn2+ inhibits the activity at higher concentrations (over 50 microM) of Ca2+. In the absence of Ca2+, Zn2+ shows no effect on protein kinase C activity. Our results suggest that Zn2+ does not recognize or interact with protein kinase C in the absence of Ca2+, that protein kinase C possesses high and low affinity Ca2+-binding sites, and that at least one Zn2+-binding site exists which is distinct from Ca2+-binding sites.     

Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids

Protein kinase C has been shown to be a phospholipid/Ca2+-dependent enzyme activated by diacylglycerol (Nishizuka, Y. (1984) Nature 308, 693-697; Nishizuka, Y. (1984) Science 225, 1365-1370). We have reported that unsaturated fatty acids (oleic acid and arachidonic acid) can activate protein kinase C independently of Ca2+ and phospholipid (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193). This study shows that other cis-fatty acids such as linoleic acid also fully activate protein kinase C in the same manner. None of the saturated fatty acids (C:4 to C:18) nor the detergents (sodium dodecyl sulfate and Triton X-100) tested here were as effective as oleic acid. Unlike oleic acid, these detergents strongly inhibited protein kinase C activity induced by Ca2+/phosphatidylserine (PS) and diacylglycerol. Lowering the critical micelle concentration of oleic acid by increasing ionic strength also strongly inhibited oleic acid activation of protein kinase C activity. Dioleoylphosphatidylserine activated protein kinase C effectively (Ka = 7.2 microM). On the other hand, dimyristoylphosphatidylserine, which contains saturated fatty acids at both acyl positions, failed to activate protein kinase C even in the presence of Ca2+. These observations suggest that: protein kinase C activation by free fatty acid is specific to the cis-form and is not due to their detergent-like action, cis-fatty acid activation is due to the direct interaction of protein kinase C with the monomeric form of cis-fatty acids and not with the micelles of fatty acids, and cis-fatty acids at acyl positions in PS are also important for Ca2+/PS activation of protein kinase C.     

Activation of protein kinase C by myristate and its requirements of Ca2+ and phospholipid

Myristate (C14:0) was found to significantly activate partially purified rat brain Ca(2+)- and phospholipid-dependent protein kinase (PKC). The Ka value, the concentration needed for half maximum activation, for C14:0 in the presence of 1 microM Ca2+ and 20 microM phosphatidylserine (PS) was 20 microM. This activation required Ca2+ and acidic phospholipid and was associated with a decreased Ka for Ca2+ of the enzyme to 10 microM in an analogous fashion as dioleoylglycerol (DO) or phorbol myristate acetate (PMA). The phospholipid requirement for the activation was concentration dependent and was inhibited by 1-(5-isoquinolinesulfonyl)-methylpiperazine dihydrochloride (H-7), a inhibitor of this enzyme. The concentration of H-7 required for half inhibition of the enzyme was about 15 microM and maximum inhibition was about 75%. The concentration profile of cytoplasmic proteins phosphorylated by C14:0-activated PKC was similar to that by PMA-activated PKC. The 47 kDa protein of guinea pig neutrophil was also phosphorylated by the C14:0-activated PKC. It is further discussed whether PKC can function as signal transduction for stimulus-mediated generation of superoxide in neutrophils.     

Ca2+-independent synergistic augmentation of O2- production by FMLP and PMA in HL-60 cells

N-Formyl-Met-Leu-Phe (FMLP) and phorbol 12-myristate 13-acetate (PMA) caused a synergistic augmentation of superoxide anion (O2-) production in neutrophil-like HL-60 cells differentiated with dibutyryl cAMP. The present study was undertaken to investigate the mechanism of the synergistic augmentation of O2- production. FMLP increased intracellular free Ca2+ concentration ([Ca2+]i), which was slightly suppressed by PMA and completely inhibited by an intracellular Ca2+ chelating agent, O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM). Although FMLP-induced O2- production was inhibited by BAPTA-AM, a major part of the synergistic augmentation of O2- production by FMLP and PMA remained after BAPTA-AM treatment, suggesting that a Ca2+-independent mechanism might be involved in the augmentation. FMLP and PMA caused an activation of phospholipase D (PLD) almost additively in a Ca2+-sensitive manner. The synergistic activation of mitogen-activated protein kinase (MAPK) was evoked by combined addition of PMA and FMLP in a BAPTA-AM resistant manner. However, PD98059, a MAPK kinase inhibitor, did not affect the synergistic augmentation of O2- production, although it potently inhibited the synergistic augmentation of tyrosine phosphorylation of MAPK. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, inhibited FMLP-induced O2- production, but it did not inhibit the synergistic augmentation of O2- production by PMA and FMLP. In contrast, staurosporine and GF109203X, protein kinase C inhibitors, reduced the synergistic augmentation induced by PMA and FMLP. In addition, pertussis toxin (PT) abolished the synergistic augmentation of O2- production. It is concluded that the synergistic augmentation of O2- production induced by PMA and FMLP is mediated through a PT-sensitive G protein and a protein kinase C in a Ca2+-independent manner.     

Intracellular Ca2+ mobilization and not calcium influx promotes phorbol ester-stimulated thromboxane A2 synthesis in human platelets.

Phorbol esters, potent activators of protein kinase C (PKC), greatly enhance the release of arachidonic acid and its metabolites (TXA2, HETES, HHT) by Ca2+ ionophores in human platelets. In this paper, we report the relationship between intracellular Ca2+ mobilization and external calcium influx into platelets and the ability of PMA plus A23187 to promote thromboxane A2 (TXA2) synthesis. The enhanced levels of TXA2 due to the synergistic stimulation of the platelets with A23187 and phorbol esters are not affected significantly by the presence of external Ca2+ or the calcium-chelator EGTA. PKC inhibitors, staurosporine and sphingosine, abolished phorbol myristate acetate (PMA) potentiation of TXA2 production which strongly supports the role of PKC in the synergism. Platelet aggregation is more sensitive to PMA and external calcium than TXA2 formation. PMA increased TXA2 production as much as 4-fold at low ionophore concentrations. The A23187-induced rise in [Ca2+]i was reduced by pretreatment of human platelets with phorbol esters, both in the presence and absence of EGTA, and staurosporine reversed this inhibitory effect. These results indicate that the synergistic stimulation of TXA2 production by A23187 and phorbol esters is promoted by intracellular Ca2+ mobilization and not by external calcium influx. Our data also suggest that PKC is involved in the regulation of Ca2+ mobilization from some specific intracellular stores and that PKC may also stimulate the Ca(2+)-dependent phospholipase A2 at suboptimal Ca2+i concentrations.     

Influence of ryanodine and inositol trisphosphate receptors inhibitions on Ca2+ exit from intracellular stores of porcine oocytes stimulated by prolactin and GTP

The influence of ryanodine and inositol triphosphate receptors inhibitors on Ca2+ exit from intracellular stores of porcine oocytes stimulated by prolactin and GTP was investigated using fluorescent dye chlortetracycline. Porcine oocytes were isolated from ovaries with yellow body. Ca2+ exit from intracellular stores of porcine oocytes activated by prolactin (5 and 50 ng/ml) in calcium free medium was decreased after treatment of oocytes by heparin (inhibitor of inositol triphosphate receptors) and was not changed after treatment of oocytes by ruthenium red (inhibitor of ryanodine receptors). Inhibition of protein kinase C did not affect on the Ca2+ exit stimulated by prolactin. GTP did not stimulate Ca2+ exit from intracellular stores of pig oocytes, and inhibitors of both calcium channels and proteinkinase C had no influence on this process. The joint action of prolactin and GTP did not result in additional Ca2+ exit from intracellular stores of oocytes after both pretreatment and untreatment by the inhibitor of protein kinase C. The data obtained testify to activation of IP3-sensitive receptors under effect of prolactin and in the absence of GTP influence on these receptors.     

Different requirements for protein kinase C activation and Ca2+-independent insulin secretion in response to guanine nucleotides. Endogenously generated diacylglycerol requires elevated Ca2+ for kinase C insertion into membranes

Electrically permeabilized RINm5F cells were used to assess the factors required for activation of protein kinase C (PKC) and insulin secretion. PKC was activated either by phorbol 12-myristate 13-acetate (PMA) or by the generation of endogenous diacylglycerol in response to the nonhydrolyzable guanine nucleotide analog guanosine 5'-O-(thiotriphosphate) (GTP gamma S). As shown previously, both PMA and GTP gamma S elicit Ca2+-independent insulin secretion. This effect was mimicked by guanyl-5'-yl imidodiphosphate (Gpp(NH)p) but not by guanosine 5'-O-(3-fluorotriphosphate) and guanosine 5'-O-(3-phenyltriphosphate) possessing only one negative charge in the gamma-phosphate group. The action of PMA was mediated by PKC, since the agent caused both phosphorylation of specific protein substrates and association of the enzyme with cellular membranes. This translocation was independent of the Ca2+ concentration employed. In contrast, GTP gamma S only promoted association of PKC with membranes at 10(-6) and 10(-5) M Ca2+ and failed to alter significantly protein phosphorylation in the absence of Ca2+. Neither Gpp(NH)p, which stimulates insulin release, nor the other two GTP analogs, increased the proportion of PKC associated with membranes. To verify that the Ca2+-dependent effect of GTP gamma S on PKC is due to activation of phospholipase C, we measured the generation of diacylglycerol. GTP gamma S indeed stimulated diacylglycerol production in the leaky cells by about 50% at Ca2+ concentrations between 10(-7) and 10(-5) M, an effect which was almost abolished in the absence of Ca2+. Thus, at 10(-7) M Ca2+, the concentration found in resting intact cells, the generated diacylglycerol was not sufficient to cause PKC insertion into the membrane, demonstrating that both elevated Ca2+ and diacylglycerol are necessary for translocation to occur. It is concluded that while PKC activation by PMA elicits Ca2+-independent insulin secretion, the kinase seems not to mediate the stimulatory action of GTP analogs in the absence of Ca2+.     

Activation of protein kinase C with cardiolipin-containing liposomes in relation to membrane-protein interaction

Many cytoplasmic proteins, including Ca2+- and phospholipid-dependent protein kinase (protein kinase C) of polymorphonuclear leukocytes (PMNs) associate in Ca2+-dependent manner with phospholipid liposomes containing cardiolipin (CL), as in the case of phosphatidylserine (PS)-containing liposomes. A crude protein kinase C fraction was purified by association of the enzyme with CL-containing liposomes (flotation method). The partially purified protein kinase C from rat brain or guinea pig PMN was activated by the CL-containing liposomes in the presence of dioleoylglycerol (DG) and Ca2+. This activation was analogous to that of PS. The half maximum activity was obtained with 20 microM CL in the presence of 1 microM Ca2+ and 5 microM DG. Many of the cytoplasmic proteins which associate with CL-containing liposomes were preferentially phosphorylated by membrane-associated protein kinase C in the presence of DG and Ca2+. These results suggest that the association of cytoplasmic protein kinase C with the membrane has an important role in regulation of protein kinase C activity in relation to the association of other cytoplasmic proteins to the membrane.     

Ca2+-phospholipid-dependent phosphorylation of vesicular preparations of myocardial sarcolemma

A Ca2+-phospholipid-dependent protein kinase C was isolated from the soluble fraction of bovine brain, using hydrophobic chromatography on phenyl-Sepharose CL-4B and high performance liquid chromatography on a Mono Q column. The enzyme had a specific activity of 822 nmol 32P/mg protein/min with histone H1 as a substrate. Phosphorylation of pig myocardium sarcolemma protein substrates was stimulated by Ca2+ and phosphatidylserine; the optimal concentrations of these compounds were 10(-4) M and 200 micrograms/ml, respectively. The value of Km(app) for Ca2+ was 3.10(-6) M. An addition of exogenous dioleine increased the enzyme affinity for Ca2+ which led to a decrease of Ca2+ concentration necessary for the maximal activation to occur. The optimal concentration of ATP needed for sarcolemmal preparation phosphorylation was 0.3-0.4 mM, which seems to be due to the high activity of sarcolemmal ATPases. The proteins phosphorylated in sarcolemmal preparations were identified, using SDS polyacrylamide gel electrophoresis with subsequent autoradiography. The 250, 140, 67, 58, 25 and 11 kD proteins appeared to be phosphorylated in the greatest degree. Since in myocardial sarcolemma protein kinase C predominantly phosphorylates the same proteins as does the cAMP-dependent protein kinase, it was assumed that protein kinase C can also play a role in the regulation of Ca2+-transporting systems of sarcolemma.     

Ca2+ sensitivity of phospholipid scrambling in human red cell ghosts.

The phospholipids in plasma membranes of erythrocytes, as well as platelets, lymphocytes and other cells are asymmetrically distributed, with sphingomyelin and phosphatidylcholine residing predominantly in the outer leaflet of the bilayer, and phosphatidylserine and phosphatidylethanolamine in the inner leaflet. It is known that Ca2+ can disrupt the phospholipid asymmetry by activation of a protein known as phospholipid scramblase, which affects bidirectional phospholipid movement in a largely non-selective manner. As Ca2+ also inhibits aminophospholipid translocase, whose Mg(2+)-ATPase activity is responsible for active translocation of aminophospholipids from the outer to the inner leaflet, it is important to accurately determine the sensitivity of scramblase to intracellular free Ca2+. In the present study we have utilized the favourable Kd of Mag-fura-2 for calcium in the high micromolar range to determine free Ca2+ levels associated with lipid scrambling in resealed human red cell ghosts. The Ca2+ sensitivity was measured in parallel to the translocation of a fluorescent-labelled lipid incorporated into the ghost bilayer. The phospholipid scrambling was found to be half-maximally activated at 63-88 microM free intracellular Ca2+. The wider applicability of the method and the physiological implications of the calcium sensitivity determined is discussed.     

Analysis of calcium homeostasis in activated human polymorphonuclear leukocytes. Evidence for two distinct mechanisms for lowering cytosolic calcium

The stimulation of polymorphonuclear leukocytes (PMNs) by chemoattractants triggers a rapid rise in cytosolic free calcium concentration(s) ([Ca2+]i), which quickly returns to base line, suggesting a role for calcium removal in the homeostasis of activated PMNs. To investigate cytosolic calcium homeostasis, PMNs were treated with a fluoroprobe and ionomycin to induce a sustained elevation of [Ca2+]i. The cells were then stimulated, and attenuation of the fluorescence signal was measured as an indication of calcium loss from the cytosol. The formyl peptide chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA), and 1,2-dioctanoyl-sn-glycerol, but not the inactive phorbol ester 4 alpha-phorbol didecanoate, induced a dose-dependent decrease in [Ca2+]i in ionomycin-pretreated cells. However, the decline in [Ca2+]i caused by PMA was sustained and occurred following a lag time, whereas the response to fMLP was immediate, lasted approximately 2 min, and then was followed by a return of [Ca2+]i to its initial level. The restoration of [Ca2+]i required extracellular calcium. Varying the ionomycin concentration allowed studies at different initial [Ca2+]i, which in untreated PMNs was approximately 135 nM. In contrast to fMLP, PMA did not lower calcium at concentrations below 200 nM. The decline in [Ca2+]i induced by fMLP, but not PMA, was blocked by pertussis toxin. In contrast, the decrease in [Ca2+]i caused by PMA and 1,2-dioctanoyl-sn-glycerol, but not fMLP, was inhibited by the protein kinase C antagonists staurosporine, H-7, and sphingosine. These results suggest that formyl peptide chemoattractants transiently stimulate an activity which lowers [Ca2+]i to normal intracellular levels. Activation of this process appears to be independent of protein kinase C. An additional cytosolic calcium lowering activity, dependent on protein kinase C, operates at [Ca2+]i above 200 nM. Thus, activated PMNs can use at least two processes for attentuation of elevated cytosolic calcium levels.