Effects of collagen, ionophore A23187 and prostaglandin E1 on the phosphorylation of specific proteins in blood platelets

Human platelets that had been preincubated with 5-hydroxy[(3)H]tryptamine and [(32)P]P(i) were stirred with various agents; the secretion of 5-hydroxy[(3)H]tryptamine from platelet granules and the radioactivity of platelet [(32)P]phosphopolypeptides separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis were then measured. Exposure of the platelets to collagen fibres or ionophore A23187 selectively increased the phosphorylation of polypeptides with apparent mol.wts. of 47000 (P47) and 20000 (P20) by approx. 3-fold, in association with the release of 5-hydroxy[(3)H]tryptamine. The 47000-mol.wt. phosphopolypeptide (P47) was clearly separated from platelet actin by the electrophoresis system used. Prostaglandin E(1), which inhibits platelet function by increasing platelet cyclic AMP, decreased the phosphorylation of polypeptides caused by collagen as well as the release of 5-hydroxy[(3)H]tryptamine. Prostaglandin E(1) also selectively increased the phosphorylation of distinct polypeptides with apparent mol.wts. of 24000 (P24) and 22000 (P22) by approx. 2-fold. As the phosphorylation reactions caused by collagen are probably mediated by an increase in Ca(2+) concentration in the platelet cytosol and may have a role in the release reaction [Haslam & Lynham (1977) Biochem. Biophys. Res. Commun.77, 714-722; (1978) Thromb. Res.12, 619-628], we suggest that a cyclic AMP-dependent phosphorylation of the 24000- and/or 22000-mol.wt. polypeptides caused by prostaglandin E(1) may initiate processes that decrease the Ca(2+) concentration in the cytosol, so inhibiting both the Ca(2+)-dependent phosphorylation reactions and the release reaction. Treatment of platelets with prostaglandin E(1) did not inhibit the increased phosphorylation of polypeptides with apparent mol.wts. of 47000 and 20000 (P47 and P20) caused by ionophore A23187, which may therefore short-circuit cyclic AMP-dependent mechanisms that decrease the Ca(2+) concentration in the platelet cytosol. As prostaglandin E(1) did inhibit the release of 5-hydroxy[(3)H]tryptamine by ionophore A23187, cyclic AMP may also inhibit the release reaction by additional mechanisms.     

The regulation of brain mitochondrial calcium-ion transport. The role of ATP in the discrimination between kinetic and membrane-potential-dependent calcium-ion efflux mechanisms.

Mitochondria from guinea-pig cerebral cortex incubated in the presence of Pi or acetate are unable to regulate the extramitochondrial free Ca2+ at a steady-state which is independent of the Ca2+ accumulated in the matrix. This is due to the superimposition on kinetically regulated Ca2+ cycling of a membrane-potential-dependent reversal of the Ca2+ uniporter. The latter efflux is a consequence of a low membrane potential, which correlates with a loss of adenine nucleotide loss from the matrix, enable the mitochondria to maintain a high membrane potential and allow the mitochondria to buffer the extramitochondrial free Ca2+ precisely when up to 200 nmol of Ca2+/mg of protein is accumulated in the matrix. The steady-state extramitochondrial free Ca2+ is maintained as low as 0.3 microM. The Na+-activated efflux pathway is functional in the presence of ATP and oligomycin and accounts precisely for the change in steady-state free Ca2+ induced by Na+ addition. The need to distinguish carefully between kinetic and membrane-potential-dependent efflux pathways is emphasized and the competence of brain mitochondria to regulate cytosolic free Ca2+ concentrations in vivo is discussed.     

Isolation of inner cell masses from mouse blastocysts by immunosurgery or exposure to the calcium ionophore A23187.

Two techniques have been evaluated for their use in routinely isolating inner cell masses from mouse blastocysts by destroying the trophectoderm. The most efficient method of immunosurgery was a 15-min incubation in a 1:50 dilution of rabbit anti-mouse spleen antiserum followed by a 30--60-min incubation in guinea pig complement (1:10). Alternatively, inner cell masses were isolated by incubating blastocysts in 10(-5) M calcium ionophore A23187 in medium devoid of calcium and magnesium ions. Inner cell masses re-exposed to immunosurgery or the ionophore were less susceptible to lysis than the trophectoderm had been. The presence of the zona pellucida reduced trophectoderm lysis by immunosurgery in antiserum dilutions greater than 1:100, but had no effect when in the presence of ionophore. Inner cell masses were consistently isolated from expanded blastocysts which had been collected 78 h after ovulation and cultured in vitro for 24 h before exposure to ionophore or immunosurgery, whereas blastocysts which had developed for the full 102 h in vivo were frequently unaffected.     

Identification of glycoprotein Ib beta as one of the major proteins phosphorylated during exposure of intact platelets to agents that activate cyclic AMP-dependent protein kinase

Platelet function is inhibited by prostaglandin E1, prostaglandin I2, or forskolin, agents that increase the intracellular concentration of cyclic AMP. The inhibition appears to result from cyclic AMP-stimulated phosphorylation of specific intracellular proteins. One of the major increases in phosphorylation occurs in a polypeptide of Mr = 24,000 (P24). In this study, an effort was made to identify P24. Platelets prelabeled with [32P]phosphate were incubated with prostaglandin E1, prostaglandin I2, or forskolin. Proteins that became phosphorylated were detected by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. Several lines of evidence indicated that P24 was the beta-subunit of the plasma membrane glycoprotein (GP) Ib, a glycoprotein that is essential for the adhesion of platelets to damaged subendothelium, for the rapid response of platelets to thrombin, and for the attachment of the membrane skeleton to the cytoplasmic face of the plasma membrane. P24 co-migrated with GP Ib beta on reduced gels (Mr = 24,000) and also on nonreduced gels (when GP Ib beta is disulfide-linked to GP Ib alpha and migrates with Mr = 170,000). Like GP Ib beta, P24 was associated with actin filaments in Triton X-100 lysates. Like GP Ib beta, it was selectively associated with filaments of the membrane skeleton and was released from filaments when the Ca2+-dependent protease was active. Antibodies against GP Ib immunoprecipitated P24 from platelet lysates. Finally, exposure of Bernard-Soulier platelets (which lack GP Ib) to prostaglandin E1 resulted in phosphorylation of other polypeptides, but not of P24. These studies show that P24, one of the major polypeptides phosphorylated when platelets are exposed to agents that inhibit platelet function by increasing the concentration of cyclic AMP, is the beta-subunit of GP Ib.     

Possible role of prostaglandin E1 in the affective disorders and in alcoholism.

Prostaglandin (PG)E1 may play an important part in the affective disorders, with an excess being present in mania and a deficiency in depression. Platelets from manic patients produce more PGE1 than normal while those from depressive patients produce less. Ethyl alcohol stimulates PGE1 production whereas lithium inhibits it. Alcoholics will tend to have raised PGE1 concentrations while drinking, but, because precursor supplies are limited, when alcohol concentrations fall PGE1 concentrations may fall sharply leading to depression. PGE1 biosynthesis may be affected by nutritional factors including essential fatty acids, pyridoxine, vitamin C, and zinc. Nutritional approaches may be of value in both depression and alcoholism.     

Macrophage membrane proteins: Possible role in the regulation of priming for enhanced respiratory burst activity

Brief exposure of macrophages to the proteolytic enzymes papain, elastase, or trypsin primed them for enhanced production of superoxide anion (O₂⁻) in response to stimulation by phorbol myristate acetate (PMA). Priming by trypsin was achieved at 0 °C, at which temperature trypsin functions as a protease but is not internalized, supporting the concept that protease priming depends on modification of the plasma membrane. Analysis of external membrane proteins after radioiodination of intact cells and separation by gel electrophoresis indicated that papain treatment of macrophages resulted in the cleavage of a membrane protein with a molecular weight of approximately 305K. Membranes from macrophages primed by elicitation with Corynebacterium parvum also demonstrated a reduced amount of the membrane protein at approximately 305 kDa, as well as a reduction of a protein at about 270 kDa. Lipopolysaccharideelicited macrophages showed a reduced amount of a protein at about 175 kDa. Continuous spectrophotometric assays of O₂⁻ release from adherent macrophages indicated that after exposure to a stimulus, protease-treated cells produced O₂⁻ more quickly than did control cells (reduced lag time). Inhibitors of protein synthesis augmented the priming effect of papain when added with the protease. These results suggest that protease-induced priming results from inactivation of a membrane protein (or proteins) that exerts a down-regulating effect on the respiratory burst.     

Calcium-dependent regulation of progesterone production by isolated rat granulosa cells: effects of the calcium ionophore A23187, prostaglandin E2, dl-isoproterenol and cholera toxin

The role of calcium in the regulation of ovarian steroidogenesis was investigated in granulosa cells from estradiol-treated immature rats. Incubation of granulosa cells with various calcium channel blockers (verapamil, cobalt or manganese) and a calcium chelator (EGTA) resulted in marked decreases in progesterone production in response to follicle-stimulating hormone (FSH), cholera toxin, prostaglandin E2, dl-isoproterenol and dibutyryl cyclic AMP (Bt2cAMP). Cyclic AMP production, however, was unaffected by treatment with EGTA and verapamil at concentrations which attenuated steroidogenesis (0.1-1.0 mM and 125 microM, respectively). Two inhibitors of the calcium-dependent regulatory protein, calmodulin [trifluoperazine, 40 microM and 1[bis-(p-chlorophenyl)methyl] 3-[2,4-dichloro-beta-(2,4- dichlorobenzyloxy )-phenethyl]imidazolium chloride, ( R24571 ) 20 microM] significantly inhibited both cyclic AMP and progesterone production elicited by these stimulatory agents. Over the concentration range of 62.5 ng/ml-1.0 micrograms/ml, the calcium ionophore A23187 increased basal progesterone production in a dose-dependent manner, with half-maximal stimulation at approximately 0.14 microgram/ml. Maximal steroidogenic response to the calcium ionophore (1 microgram/ml) however, was only 50% of that evoked by FSH (0.33 microgram/ml). A23187 (0.5 microgram/ml) significantly enhanced progesterone production stimulated by a low concentration of FSH (0.025 microgram/ml) but failed to potentiate the maximally stimulatory action of the gonadotropin (0.33 microgram/ml). These findings support our earlier suggestion that the calcium-calmodulin system plays a central role in the gonadotropic regulation of ovarian steroidogenesis and suggest that a transmembrane flux of extracellular calcium may be an important and common step in the mechanism of stimulation of granulosa cell progesterone production.     

Comparison of the effects of phorbol 12-myristate 13-acetate and prostaglandin E1 on calcium regulation in human platelets.

We compared the effects of phorbol 12-myristate 13-acetate (PMA) with those of prostaglandin E1 (PGE1) on the calcium transient in intact platelets and on 45Ca2+ uptake in saponin-treated platelets and microsomal fractions to determine the roles of protein kinase C and cyclic AMP in calcium sequestration. In intact platelets, PMA, like PGE1, stimulated the return of the calcium transient to resting values after a thrombin stimulus, but only the PGE1 effect was reversed by adrenaline. Both PMA and PGE1, when added before saponin, stimulated ATP-dependent 45Ca2+ uptake into the permeabilized platelets. Thrombin also stimulated 45Ca2+ uptake into saponin-treated platelets. Uptake of 45Ca2+ was increased in microsomal preparations from platelets pretreated with PMA or PGE1. PMA did not increase the cyclic AMP content of control or thrombin-treated platelets, and it induced a pattern of protein phosphorylation in 32P-labelled platelets different from that with PGE1. In correlation with the increased uptake of calcium in the saponin-treated preparation, we measured a rapid translocation of protein kinase C from supernatant to cell fraction after the addition of PMA. Our results suggest that activation of protein kinase C enhances calcium sequestration independently of an effect on cyclic AMP content in platelets. This activation could play a physiological role in the regulation of the calcium transient.     

Calcium-ion transport by intact synaptosomes. Intrasynaptosomal compartmentation and the role of the mitochondrial membrane potential.

1. The pathways and the fate of glutamate carbon and nitrogen were investigated in isolated guinea-pig kidney-cortex tubules. 2. At low glutamate concentration (1 mM), the glutamate carbon skeleton was either completely oxidized or converted into glutamine. At high glutamate concentration (5 mM), glucose, lactate and alanine were additional products of glutamate metabolism. 3. At neither concentration of glutamate was there accumulation of ammonia. 4. Nitrogen-balance calculations and the release of 14CO2 from L-[1-14C]glutamate (which gives an estimation of the flux of glutamate carbon skeleton through alpha-oxoglutarate dehydrogenase) clearly indicated that, despite the absence of ammonia accumulation, glutamate metabolism was initiated by the action of glutamate dehydrogenase and not by transamination reactions as suggested by Klahr, Schoolwerth & Bourgoignie [(1972) Am. J. Physiol. 222, 813-820] and Preuss [(1972) Am. J. Physiol. 222, 1395-1397]. Additional evidence for this was obtained by the use of (i) amino-oxyacetate, an inhibitor of transaminases, which did not decrease glutamate removal, or (ii) L-methionine DL-sulphoximine, an inhibitor of glutamine synthetase, which caused an accumulation of ammonia from glutamate. 5. Addition of NH4Cl plus glutamate caused an increase in both glutamate removal and glutamine synthesis, demonstrating that the supply of ammonia via glutamate dehydrogenase is the rate-limiting step in glutamine formation from glutamate. NH4Cl also inhibited the flux of glutamate through glutamate dehydrogenase and the formation of glucose, alanine and lactate. 6. The activities of enzymes possibly involved in the glutamate conversion into pyruvate were measured in guinea-pig renal cortex. 7. Renal arteriovenous-difference measurements revealed that in vivo the guinea-pig kidney adds glutamine and alanine to the circulating blood.     

Inhibition of cyclic nucleotide phosphodiesterase during exposure to WI-38 cells to prostaglandin E1.

Short term incubation of WI-38 cultures with 5.7 micron prostaglandin E1 (PGE1) caused cyclic AMP phosphodiesterase activity in fibroblast homogenates to fall by 25 to 35% as compared to controls. The PGE1-induced decline in phosphodiesterase activity coincided with a rapid increase in intracellular cyclic AMP levels in response to the hormone and was rapidly reversed by washing the cultures free of the prostaglandin before homogenizing the cells. The effect of PGE1 on WI-38 phosphodiesterase activity was localized to the enzyme form(s) present in 27,000 times g supernatant fractions of cell homogenates. These data suggest that the pattern of cyclic AMP accumulation in WI-38 fibroblasts exposed to PGE1 may be related, at least in part, to decreased phosphodiesterase activity during hormone stimulation.     

Selective modulation of the human platelet thromboxane A2/prostaglandin H2 receptor by eicosapentaenoic and docosahexaenoic acids in intact platelets and solubilized platelet membranes.

We previously demonstrated that nonesterified as well as esterified eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) inhibit U46619-induced platelet aggregation and [3H]U46619 specific binding to washed human platelets. It was also demonstrated that esterification of these fatty acids resulted in a decrease in the affinity of [3H]U46619 for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. In order to investigate the specificity of this inhibition, the effects of 20:5n-3 and 22:6n-3 on the function and binding of the platelet alpha 2-adrenergic receptor were studied. It was found that neither 20:5n-3 nor 22:6n-3 (nonesterified or esterified) altered epinephrine-induced aggregation or [3H]yohimbine specific binding. Moreover, Scatchard analysis revealed that esterification with either 20:5n-3 or 22:6n-3 did not alter the dissociation constant for [3H]yohimbine binding. Modulation of the TXA2/PGH2 receptor by 20:5n-3 and 22:6n-3 was next evaluated using CHAPS- and digitonin-solubilized platelet membranes. [3H]SQ29,548 dissociation constants of 26.5 nM and 20.8 nM were measured for CHAPS and digitonin-solubilized membranes, respectively. Competitive binding experiments in these solubilized preparations revealed that 20:5n-3 or 22:6n-3 blocked [3H] SQ29,548 binding with IC50 values in the range of 6-15 microM, while concentrations of these fatty acids of up to 100 microM showed no effect on [3H]yohimbine binding. On the other hand, the IC50 values for inhibition of [3H] SQ29,548 binding by linoleic acid (18:2n-6) and gamma-linolenic acid (18:3n-6) were in the range of 150 microM. Furthermore, 18:2n-6 and 18:3n-6 showed similar inhibitory effects on [3H]yohimbine binding. Finally, competition binding studies performed in a partially purified TXA2/PGH2 receptor preparation also demonstrated inhibition of [3H]SQ29,548 binding by 20:5n-3 and 22:6n-3. Collectively, these findings support the notion that 20:5n-3 and 22:6n-3 can selectively and directly modulate TXA2/PGH2 receptor function, and that this mechanism of action may contribute to the antiplatelet activity associated with diets rich in these fatty acids.     

The role of divalent cations in the regulation of microtubule assembly. In vivo studies on microtubules of the heliozoan axopodium using the ionophore A23187

Low concentrations of calcium and magnesium ions have been shown to influence microtubule assembly in vitro. To test whether these cations also have an effect on microtubules in vivo, specimens of Actinosphaerium eichhorni were exposed to different concentrations of Ca++ and Mg++ and the divalent cation ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Experimental degradation and reformation of axopodia were studied by light and electron microscopy. In the presence of Ca++ and the ionophore axopodia gradually shorten, the rate of shortening depending on the concentrations of Ca++ and the ionophore used. Retraction of axopodia was observed with a concentration of Ca++ as low as 0.01 mM. After transfer to a Ca++-free solution containing EGTA, axopodia re-extend; the initial length is reached after about 2 h. Likewise, reformation of axopodia of cold-treated organisms is observed only in solutions of EGTA or Mg++, whereas it is completely inhibited in a Ca++ solution. Electron microscope studies demonstrate degradation of the axonemal microtubular array in organisms treated with Ca++ and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. No alteration was observed in organisms treated with Mg++ or EGTA plus ionophore. The results suggest that, in the presence of the ionophore, formation of axonemal microtubules can be regulated by varying the Ca++ concentration in the medium. Since {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 tends to equilibrate the concentrations of divalent cations between external medium and cell interior, it is likely that microtubule formation invivo is influenced by micromolar concentrations of Ca++. These concentrations are low enough to be of physiological significance for a role in the regulation of microtubule assembly in vivo.     

Targeting of proteins to the outer envelope membrane uses a different pathway than transport into chloroplasts.

The chloroplastic envelope is composed of two membranes, inner and outer, each with a distinct set of polypeptides. Like proteins in other chloroplastic compartments, most envelope proteins are synthesized in the cytosol and post-translationally imported into chloroplasts. Considerable knowledge has been obtained concerning protein import proteins. We isolated a cDNA clone from pea that encodes a 14-kilodalton outer envelope membrane protein. The precursor form of this protein does not possess a cleavable transit peptide and its import into isolated chloroplasts does not require either ATP or a thermolysin-sensitive component on the chloroplastic surface. These findings, together with similar observations made with a spinach chloroplastic outer membrane protein, led us to propose that proteins destined for the outer membrane of the chloroplastic envelope follow an import pathway distinct from that followed by proteins destined for other chloroplastic compartments.     

Stimulation of prostaglandin E1 binding to human blood platelet membrane by insulin and the activation of adenylate cyclase

Incubation of human platelet-rich plasma with physiological amounts of insulin resulted in the increase of the binding of prostaglandin E1 by more than 2-fold when compared to the control platelets. Scatchard analysis of the binding of the prostaglandin to the hormone-treated platelets showed that the increased binding was due to the increase of receptor numbers rather than the increase of affinity of the binding sites. The membranes prepared from the insulin-treated platelets also showed similar enhanced binding of the prostaglandin. However, addition of insulin directly to the membranes isolated from the platelets which had not been previously incubated with the hormone failed to show any effect. This increased binding of the prostanoid to the membranes prepared from the insulin-treated platelets resulted in the stimulation of adenylate cyclase by more than 2-fold when compared with the control of membrane preparation by the prostaglandin alone. In contrast, treatment of platelets with the hormone which increased the prostanoid binding to these cells did not influence the cyclic AMP phosphodiesterase activity of either the membrane or cytosolic fraction. The increase in the cellular level of cyclic AMP by prostaglandin E1 was 2-fold greater in the hormone-treated cells than in the case of untreated platelets stimulated by the agonist alone. The incubation of platelet-rich plasma with insulin, as expected, decreased the amount of prostaglandin E1 needed to inhibit platelet aggregation by more than 50% when compared to the control platelets.     

The role of plasma membrane STIM1 and Ca2 +entry in platelet aggregation. STIM1 binds to novel proteins in human platelets

Ca^2 + elevation is essential to platelet activation. STIM1 senses Ca^2 + in the endoplasmic reticulum and activates Orai channels allowing store-operated Ca^2 + entry (SOCE). STIM1 has also been reported to be present in the plasma membrane (PM) with its N-terminal region exposed to the outside medium but its role is not fully understood. We have examined the effects of the antibody GOK/STIM1, which recognises the N-terminal region of STIM1, on SOCE, agonist-stimulated Ca^2 + entry, surface exposure, in vitro thrombus formation and aggregation in human platelets. We also determined novel binding partners of STIM1 using proteomics. The dialysed GOK/STIM1 antibody failed to reduced thapsigargin- and agonist-mediated Ca^2 + entry in Fura2-labelled cells. Using flow cytometry we detect a portion of STIM1 to be surface-exposed. The dialysed GOK/STIM1 antibody reduced thrombus formation by whole blood on collagen-coated capillaries under flow and platelet aggregation induced by collagen. In immunoprecipitation experiments followed by proteomic analysis, STIM1 was found to extract a number of proteins including myosin, DOCK10, thrombospondin-1 and actin. These studies suggest that PM STIM1 may facilitate platelet activation by collagen through novel interactions at the plasma membrane while the essential Ca^2 +-sensing role of STIM1 is served by the protein in the ER.     

Comparison of the effects of prostacyclin (PGI2), prostaglandin E1 and D2 on platelet aggregation in different species.

The activity of prostacyclin (PGI2), PGE1 or PGD2 as inhibitors of platelet aggregation in plasma from human, dog, rabbit, rat, sheep and horse was investigated. Prostacyclin was the most potent inhibitor in all species. PGD2 was a weak inhibitor in dog, rabbit and rat plasma whereas PGE1 and prostacyclin were highly active. Theophylline or dipyridamole potentiated the inhibition of human platelet aggregation by prostacyclin, PGE1 or PGD2. Compound N-0164 abolished the inhibition by PGD2 of human platelet aggregation but did not inhibit the effects of PGE1 or prostacyclin. The results suggest that prostacyclin and PGE1 act on similar sites on platelets which are distinct from those for PGD2.     

A possible role of carbohydrate moieties in prostaglandin D2 and prostaglandin E2 receptor proteins from the porcine temporal cortex.

The binding activities of prostaglandins (PGs) D2 and E2 were measured after deglycosylation of P2 membranes prepared from the porcine temporal cortex in order to investigate the role of carbohydrate moieties in the receptor binding. PGD2 and PGE2 binding activities were significantly decreased by pretreatment with various exoglycosidases, such as neuraminidase for PGE2 binding, alpha-mannosidase and beta-galactosidase for PGD2 binding, and beta-N-acetylhexosaminidase for both. Further, peptide N-glycohydrolase F and endo-alpha-N-acetylgalactosaminidase, which are specific for the cleavage of N-glycan and O-glycan linkages, respectively, in glycoproteins were used. Pretreatment with either of them also reduced both PGD2 and PGE2 binding activities. The reduction was dependent on the pretreatment time and enzyme concentration. The time courses of the reduction were typically characterized by a marked increase in the nonspecific bindings. Scatchard plot analysis revealed that the reduction was caused by a decrease in the affinity rather than one in the maximal binding capacity. The specificity of the binding sites thereby shifted to be more nonspecific without affecting the order of the relative affinities among PGs for the binding sites. These results suggest that the carbohydrate moieties on PG receptor proteins of the brain are essential for the expression of their binding activities.