N-ethylmaleimide inhibits Ca2+ influx induced by collagen or arachidonate on rabbit platelets

N-Ethylmaleimide dose dependently inhibited platelet aggregation induced by collagen or arachidonate but did not inhibit the aggregation by thrombin or ionophore A23187 within the concentrations tested. [3H]Arachidonate release from membrane phospholipids of the collagen-stimulated platelets was inhibited by N-ethylmaleimide in parallel with the inhibition of aggregation, but not in response to A23187. N-Ethylmaleimide prevented 45Ca2+ influx into platelet cells from outer medium induced by collagen, and also inhibited the increase in the concentration of cytoplasmic free Ca2+, which probably results from Ca2+ influx, as monitored by quin2 fluorescence, under stimulation with arachidonate. The concentration of N-ethylmaleimide giving a complete inhibition of Ca2+ influx was consistent with that required to inhibit collagen- or arachidonate-induced aggregation. Prostaglandin metabolism from arachidonate to thromboxane A2 was not disturbed by N-ethylmaleimide, while phosphatidate formation induced by arachidonate was slightly inhibited by it at concentrations at which aggregation was completely inhibited. These data suggest that N-ethylmaleimide preferentially suppresses increase in cytoplasmic free Ca2+ which is linked to thromboxane A2-receptor occupation in collagen- or arachidonate-stimulated platelets, probably due to blockage of Ca2+ influx through Ca2+-channel protein, thereby inhibiting aggregation induced by these agonists.     

An ATP-dependent and inositol trisphosphate-sensitive Ca2+ pool linked with microfilaments of the parietal cell

In digitonin-permeabilized parietal cells, myo-inositol 1,4,5-trisphosphate (Ins P3) or Ca2+ ionophore (A23187) increased the cytosolic Ca2+ concentration due to the intracellular Ca2+ release. Addition of ATP decreased the cytosolic Ca2+ concentration due to the rapid Ca2+ re-uptake into the same or similar pool which releases Ca2+ from a non-mitochondrial location (measured by quin2/AM and 45Ca2+). Cytochalasin B failed to increase the cytosolic Ca2+ concentration in response to Ins P3 or A23187 and even failed to decrease the cytosolic Ca2+ concentration in response to ATP. This implies that the ATP-dependent and Ins P3-sensitive Ca2+ pool is linked with the microfilaments of the parietal cell. In intact parietal cells, A23187 increased the amino[14C]pyrine accumulation (an index of acid secretion), that was independent of medium Ca2+. This increase of acid secretion was inhibited by the pretreatment with cytochalasin B. This suggests that medium Ca2+-independent acid secretion (by A23187) is regulated by the microfilaments. Therefore, there is a close relationship between the intracellular Ca2+ metabolism, microfilaments and acid secretion.     

Fluorescence labeling of the C-terminus of proteins with a puromycin analogue in cell-free translation systems

L-Arginine uptake and Ca(2+) changes in unstirred platelets activated by thrombin, collagen and Ca(2+) ionophore A23187 were evaluated. Thrombin did not affect L-arginine uptake at short incubation times (2-15 min), but at prolonged times slowed down the amino acid transport. Collagen was ineffective. A23187 decreased the L-arginine uptake in a dose-dependent manner, producing the maximal inhibition at 5 microM. In FURA 2-loaded platelets collagen did not modify Ca(2+) basal level, thrombin induced a late Ca(2+) rise and A23187 dose-dependently increased cytosolic Ca(2+), eliciting the highest increase at 5 microM. It is likely that L-arginine uptake is inversely modulated by Ca(2+) concentrations and is inhibited during platelet stimulation with agonists which induce cytosolic Ca(2+) elevation.     

Inhibition of platelet aggregation by unsaturated fatty acids through interference with a thromboxane-mediated process

cis- and trans-unsaturated fatty acids with 18 carbon atoms (oleic, linoleic, elaidic and linolelaidic acid) inhibited aggregation of washed rabbit platelets stimulated with collagen, arachidonic acid and U46619 when in the same concentration ranges. Thrombin-induced aggregation was not affected by any of them. Saturated fatty acid (stearic acid) had no effect on this response. The inhibition is independent of the induced change in membrane fluidity, since trans-isomers could not induce the change in fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Unsaturated fatty acids, except linoleic acid, did not interfere with the formation of thromboxane B2 from exogenously added arachidonic acid. All the unsaturated fatty acids only slightly inhibited the arachidonic acid liberation by phospholipase A2 in platelet lysate. This indicates that the unsaturated fatty acids may block a process after formation of thromboxane A2 in response to collagen and arachidonic acid. The increase in phosphatidic acid formation stimulated with U46619 was inhibited dose dependently by each of the unsaturated fatty acids but that stimulated with thrombin was not affected by any of them. Phospholipase C activity measured by diacylglycerol formation in unstimulated platelet lysate was not inhibited by the fatty acids. The elevation of cytosolic free Ca2+ induced by arachidonic acid or U46619 and Ca2+ influx by collagen were inhibited almost completely at the same concentration as that which inhibited their aggregation. These data suggest that the unsaturated fatty acids were intercalated into the membrane and inhibited collagen- and arachidonic acid-induced platelet aggregation by causing a significant suppression of the thromboxane A2-mediated increase in cytosolic free Ca2+, probably due to interference with the receptor-operated Ca2+ channel.     

Inhibition of amylase secretion from differentiated AR4-2J pancreatic acinar cells by an actin cytoskeleton controlled protein tyrosine phosphatase activity.

Disruption of the actin cytoskeleton in AR4-2J pancreatic acinar cells led to an increase in cytosolic protein tyrosine phosphatase activity, abolished bombesin-induced tyrosine phosphorylation and reduced bombesin-induced amylase secretion by about 45%. Furthermore, both tyrosine phosphorylation and amylase secretion induced by phorbol ester-induced activation of protein kinase C were abolished. An increase in the cytosolic free Ca2+ concentration by the Ca2+ ionophore A23187 had no effect on tyrosine phosphorylation but induced amylase release. Only when added together with phorbol ester, the same level of amylase secretion as with bombesin was reached. This amylase secretion was inhibited by about 40%, by actin cytoskeleton disruption similar to that induced by bombesin. We conclude that actin cytoskeleton-controlled protein tyrosine phosphatase activity downstream of protein kinase C activity regulates tyrosine phosphorylation which in part is involved in bombesin-stimulated amylase secretion.     

Dependence of cytoplasmic calcium transients on the membrane potential in isolated nerve endings of the guinea pig

The relation of changes in internal, free Ca2+, measured with arsenazo III, to the membrane potential, measured with the cyanine dye di-S-C2(5) or 86Rb+ distribution ratio, was studied in isolated guinea pig cortical nerve endings. Depolarization of the plasma membrane with veratridine or gramicidin as well as addition of ionophore A23187 led to an increase in cytosolic Ca2+. Only the response to veratridine was inhibited by tetrodotoxin. The dependence of the depolarization-induced increase in intraterminal, free Ca2+ on the membrane potential between about -50 to 0 mV was sigmoidal. A maximal increase in cytosolic Ca2+ was reached when the membrane potential was depolarized from the resting level, about -64 mV, to about -40 mV. These results show that in isolated nerve endings the activation of voltage-sensitive Ca2+ channels concomitantly leads to an increase in cytosolic, free Ca2+. Comparison of the results of the present study with the previous electrophysiological observations indicate that Ca2+ channels in synaptosomes, presynaptic nerve terminals of the squid giant synapse and cardiac cells have essentially similar voltage dependency.     

Phosphorylation-dependent and -independent pathways of platelet aggregation.

We have used the non-specific inhibitor of protein kinases, staurosporine, to investigate the role of protein phosphorylation during aggregation, the mobilization of intracellular Ca2+ (Ca2+)i and intracellular pH (pHi) in thrombin-stimulated platelets. The concentration of staurosporine chosen for these studies, 1 microM, was previously reported to inhibit protein phosphorylation completely but to have no effect on the activation of phospholipase C in thrombin-stimulated human platelets [Watson, McNally, Shipman & Godfrey (1988) Biochem. J. 249, 345-350]. Aggregation induced by phorbol dibutyrate is slow (several minutes) and is inhibited completely by staurosporine. In contrast, aggregation induced by thrombin, platelet-activating factor or ionophore A23187 is rapid (occurs within 60 s), and is slowed, but not inhibited, in the presence of staurosporine. On the other hand, staurosporine causes a small potentiation of the peak [Ca2+]i signal induced by thrombin and a marked increase in the half-life of decay of this signal, but has no effect on pHi. Under conditions designed to prevent an increase in [Ca2+]i (presence of Ni2+ to prevent Ca2+ entry, and depletion of the intracellular Ca2+ stores), aggregation induced by thrombin resembles that by phorbol dibutyrate and is now inhibited completely by staurosporine. Taken together, these results provide evidence for two signalling pathways for aggregation, a relatively rapid phosphorylation-independent route mediated by Ca2+ and a slower, phosphorylation-dependent, pathway mediated by protein kinase C. Since staurosporine slows aggregation induced by thrombin, it appears that under normal conditions these pathways interact synergistically.     

血小板激活时胞质游离钙浓度与两种颗粒数变化的相关性

用电镜形态计量法检测血小板α颗粒（αＧ）和致密颗粒（ｄＧ）的数密度,用钙荧光指示剂Ｆｕｒａ２检测血小板胞质游离Ｃａ＾２＋浓度（「Ｃａ＾２＋」ｉ）,观察到在钙离子导体Ａ２３１８７作用下,血小板「Ｃａ＾２＋」ｉ明显升高。凝血酶与ＡＤＰ也都分别引起「Ｃａ＾２＋」ｉ升高,且有浓度依赖性,选用三种激动剂的不同量以反映血小板不同程度激活时,测定「Ｃａ＾２＋」与颗粒数密度,分析两者间的相关性,发现αＧ和ｄＧ的数     

Inhibitory effects of phenol derivatives on bovine platelet aggregation and their effects on Ca2+ mobilization

The effects of phenol derivatives on aggregation of bovine platelets induced by ADP, thrombin, platelet activating factor, collagen and A23187 were investigated. The phenol derivatives inhibited all these induced aggregations except that by the calcium ionophore. The derivatives each inhibited the aggregations induced by ADP, thrombin, platelet activating factor and collagen, respectively, within a similar concentration range. A linear relation was found between the inhibitory potencies of the phenol derivatives and their partition coefficients between n-octanol and water (Poct values), suggesting that their interaction with hydrophobic regions of the cell was important for inhibition. Fluorescence analyses with fura-2-loaded platelets showed that in the concentration ranges in which the phenol derivatives inhibited aggregation, they also inhibited agonist-induced increases in Ca2+ both in the presence and absence of extracellular Ca2+. Moreover, a high correlation was found between the inhibitory effects of the derivatives on aggregation and their effects on Ca2+ mobilization. These results suggest that inhibition of platelet aggregation by phenol derivatives is mainly due to inhibition of the increase in cytoplasmic Ca2+ by inhibition of both intracellular Ca2+ mobilization and Ca2+ uptake.     

Inhibition by antioxidants of agonist evoked cytosolic Ca++ increase, ATP secretion and aggregation of aspirinated human platelets

The synthetic antioxidants butylated hydroxytoluene (BHT), nordihydroguaiaretic acid and the one-electron donor 1,1'-dimethylferrocene, inhibit cytosolic Ca++ increase, shape change, aggregation and ATP secretion in aspirinated washed human platelets stimulated by thrombin, vasopressin and platelet-activating factor. The antioxidants also inhibit cytosolic Ca++ increase originating from intracellular stores in the presence of EGTA. The effect of phorbol ester (TPA), which promotes platelet aggregation and secretion without raising the cytosolic Ca++, is also antioxidant-sensitive. Since agonist activation of aspirinated platelets does not involve cyclooxygenase or lipoxygenase metabolites, it is suggested that other yet unknown free radical-dependent pathways are involved in the mechanism of platelet activation, both in the protein kinase C-independent events leading to the cytosolic Ca++ increase, and in those, largely protein kinase C-dependent, leading to aggregation and ATP secretion.     

Forskolin and prostacyclin inhibit fluoride induced platelet activation and protein kinase C dependent responses

Platelet activation (cytosolic [Ca2+] increase, aggregation and ATP secretion) was induced with A1F-4. This agent presumably interacts with a G protein which appears to mediate the coupling of the receptors for Ca mobilizing hormones and phospholipase C. All the A1F-4 evoked responses were inhibited by treatment with forskolin or prostacyclin, agents known to increase cellular cAMP. Thus the G protein-phospholipase C system appears to be the site of cAMP inhibition. Unexpectedly forskolin and prostacyclin also inhibited secretion and aggregation induced by the activators of protein kinase C, diglyceride and phorbol ester, suggesting that cAMP can also inhibit directly the protein kinase C dependent responses.     

Intracellular calcium fluxes in human platelets

Fluorescence changes and secretory responses have been measured on addition of various excitatory agonists to platelets loaded with the cytosolic Ca2+ probe, Quin 2 or with chlortetracycline as a probe for membrane-associated Ca2+. When extracellular [Ca2+] is decreased to less than 0.1 microM by addition of EGTA a linear correlation is observed between the extent of increase in cytosolic [Ca2+] and the extent of mobilisation of membrane-associated Ca2+ on stimulation by maximal doses of five excitatory agonists. A similar linear correlation between the increase in cytosolic [Ca2+] and the extent of ATP secretion is observed over the thrombin dose/response curve. Similar EC50 values are observed for ATP secretion, the increase in cytosolic [Ca2+] and the decrease in chlortetracycline fluorescence induced by thrombin. However, the decrease in chlortetracycline fluorescence shows a sigmoidal relationship with the increase in cytosolic [Ca2+] and a hyperbolic relationship with ATP secretion over this dose/response curve. Addition of prostaglandin D2 prior to thrombin causes parallel inhibition of the increase in cytosolic [Ca2+] and the decrease in chlortetracycline fluorescence induced by this agonist. However, addition of prostaglandin D2 after thrombin reverses the increase in cytosolic [Ca2+] induced by this agonist but fails to cause a similar reversal of the decrease in chlortetracycline fluorescence. The data provide further evidence supporting the proposal that chlortatracycline can be used as a probe to monitor mobilisation of membrane-associated Ca2+ but suggest that, in platelets stimulated in the effective absence of extracellular Ca2+, both Ca2+ mobilisation and Ca2+ removal can under some conditions involve sites which are not monitored by this probe.     

Synergistic responses in human platelets. Comparison between aggregation, secretion and cytosolic Ca2+ concentration

Synergistic interaction between ADP, adrenaline, 5-hydroxytryptamine (5HT) and [8-arginine]vasopressin is not observed for the aggregatory response of aspirin-treated human platelets when this response is estimated directly from the decrease in the number of single platelets in the suspension. This finding is in marked contrast with prior reports of synergistic interaction between these agonists when the rate and extent of the aggregometer response is estimated from the increase in the light transmittance of the suspension, using a platelet aggregometer. We propose that the apparent synergistic response detected using the aggregometer results from the inability of this instrument to respond during the initial phase of aggregation. Significant synergistic interaction is observed for the increase in cytosolic [Ca2+] induced by addition of the ADP/5HT and, to a lesser extent, of the ADP/vasopressin agonist pairs as compared with that caused by addition of the individual agonists. This effect is not, however, typical of the system since increases in cytosolic [Ca2+] induced by addition of the ADP/thrombin or 5HT/vasopressin agonist pairs are no greater than the sum of the responses to these agonists added separately. Addition of collagen prior to ADP or 11,9-epoxymethanoprostaglandin H2 (U46619) fails to enhance the increase in cytosolic [Ca2+] induced by these latter agonists. Adrenaline, when added prior to non-saturating concentrations of U46619, thrombin, vasopressin or ADP, significantly enhances the increase in cytosolic [Ca2+] induced by these agonists in platelets suspended in media containing less than 0.1 microM or 1 mM Ca2+. However, adrenaline fails to enhance the increase in cytosolic [Ca2+] induced by the divalent cation ionophore, ionomycin. Enhancement by adrenaline of Ca2+ influx induced by U46619, thrombin and ADP has been shown by using Mn2+ as probe. Adrenaline also enhances the extent of [3H]5HT secretion induced by U46619, thrombin and vasopressin but fails to increase that induced by ADP in this aspirin-treated preparation. These results are in part consistent with the postulate that adrenaline, acting via an alpha 2-adrenoceptor, modulates receptor--phospholipase-C coupling. However, such modulation does not appear to involve inhibition of adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)     

The actions of Ca2+ ionophores on rat basophilic (2H3) cells are dependent on cellular ATP and hydrolysis of inositol phospholipids. A comparison with antigen stimulation

Calcium-specific ionophores are used widely to stimulate Ca2+-dependent secretion from cells on the assumption that permeabilization of the cell membranes to Ca2+ ions leads to a rise in concentration of cytosolic Ca2+ ([Ca2+]i), which in turn serves as a signal for secretion. In this way, events that precede mobilization of Ca2+ ions via receptor stimulation are bypassed. One such event is thought to be the rapid hydrolysis of membrane inositol phospholipids to form inositol phosphates and diacylglycerol. Accordingly, rat leukemic basophil (2H3) cells can be stimulated to secrete histamine either with the ionophores or by aggregation of receptors for IgE in the plasma membrane. We find, however, that ionophore A23187 stimulates secretion of histamine only at concentrations (200-1000 nM) that stimulate hydrolysis of membrane inositol phospholipids. The extent of hydrolysis of inositol phospholipids was dependent on the concentration of ionophore and the presence of external Ca2+ ions and correlated with the magnitude of the secretory response. A similar correlation between secretion and hydrolysis of inositol phospholipids was observed in response to the Ca2+-specific ionophore, ionomycin. Although this hydrolysis (possibly a consequence of elevated [Ca2+]i) was less extensive than that induced by aggregation of receptors, it may govern the secretory response to A23187. The studies revealed one paradox. The rise in [Ca2+]i depended on intracellular ATP levels, when either an ionophore or antigen was used as a stimulant irrespective of whether hydrolysis of inositol phospholipids was stimulated or not. The concept of how the ionophores act, therefore, requires critical reevaluation.     

Comparative studies on the energetics of platelet responses induced by different agonists.

The correlation between energy consumption and platelet responses induced by collagen, A23187 and ADP was investigated and compared with the energetics of thrombin-stimulated platelets established in earlier work. Aggregation, measured as single-platelet disappearance, and secretion correlated quantitatively with the increment but not with the total consumption of energy, suggesting that the former reflects the energy cost of these responses. The cost of complete aggregation was 2-3 mumol of ATP equivalents/10(11) platelets with collagen, ADP and thrombin as the stimulus. The cost of complete dense-granule secretion was 0.5-0.8 mumol of ATP equivalents/10(11) platelets with all agonists tested. The cost of combined secretion of alpha-granule and acid hydrolase granule contents was 5-7 mumol of ATP equivalents/10(11) platelets with thrombin and collagen. However, in the presence of A23187 much more energy was consumed during aggregation and secretion. Also ADP triggered more energy consumption during secretion than was seen with the other inducers. The effect of inhibitors of aggregation and secretion was investigated in thrombin-stimulated platelets. Raising the cellular cyclic AMP content sharply decreased the increment in energy consumption as well as aggregation and secretion. The cytoskeleton-disrupting agents cytochalasin B and colchicine left the increment in energy consumption intact, but decreased the basal consumption seen in unstimulated platelets. This was accompanied by normal (cytochalasin B) or diminished (colchicine) aggregation and secretion. Apart from the latter exception, all inhibitors decreased secretion and incremental energy consumption in parallel, thereby preserving the energy-versus-secretion relationship established in earlier work. In contrast, aggregation and energy consumption varied independently, suggesting that the coupling with energy consumption is much weaker for this response.     

Antiplatelet effect of butylidenephthalide

Butylidenephthalide inhibited, in a dose-dependent manner, the aggregation and release reaction of washed rabbit platelets induced by collagen and arachidonic acid. Butylidenephthalide also inhibited slightly the platelet aggregation induced by PAF and ADP, but not that by thrombin or ionophore A23187. Thromboxane B2 formation caused by collagen, arachidonic acid, thrombin and ionophore A23187 was in each case markedly inhibited by butylidenephthalide. Butylidenephthalide inhibited the aggregation of ADP-refractory platelets, thrombin-degranulated platelets, chymotrypsin-treated platelets and platelets in the presence of creatine phosphate/creatine phosphokinase. Its inhibition of collagen-induced aggregation was more marked at lower Ca2+ concentrations in the medium. The aggregability of platelets inhibited by butylidenephthalide could be recovered after the washing of platelets. In human platelet-rich plasma, butylidenephthalide and indomethacin prevented the secondary aggregation and blocked ATP release from platelets induced by epinephrine. Prostaglandin E2 formed by the incubation of guinea-pig lung homogenate with arachidonic acid could be inhibited by butylidenephthalide, indomethacin and aspirin. It is concluded that the antiplatelet effect of butylidenephthalide is mainly due to an inhibitory effect on cyclo-oxygenase and may be due partly to interference with calcium mobilization.     

Role of Ca2+ in H+ transport by rabbit gastric glands studied with A23187 and BAPTA, an incorporated Ca2+ chelator

The role of Ca2+ in stimulation of H+ gastric secretion by cAMP-dependent and -independent secretagogues was studied in isolated rabbit glands using Ca2+ ionophore, A23187, and an intracellular Ca2+ chelator (BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) incorporated as its acetoxymethyl ester (BAPTA-AM). Acetylcholine (ACh), tetragastrin (TG), histamine and forskolin induced a transitory increase of intracellular Ca2+ concentration, [Ca2+]i, measured in gastric glands loaded with Ca2+-sensitive dye fura-2, and provoked an acid secretory response evaluated with aminopyrine accumulation ratio (AP ratio). The Ca2+-ionophore A23187 also induced an increase in [Ca2+]i and in AP ratio. cAMP-dependent secretagogues were more potent stimulants of acid secretion than cAMP-independent secretagogues. cAMP analogue, 8-bromo-adenosine 3',5'-cyclic monophosphate (8-BR-cAMP) induced an increase in AP ratio without modifying [Ca2+]i. BAPTA-AM (5-25 microM) induced a transient decrease of resting [Ca2+]i which returned to basal level due to extracellular Ca2+ entry. Increases in [Ca2+]i produced by ACh and TG were abolished by BAPTA and those produced by Ca2+ ionophore A23187 were partially buffered. BAPTA inhibited in a dose-dependent manner H+ secretion induced by cholinergic and gastrinergic stimulants in the presence of cimetidine. A23187 increased the AP ratio to values similar to those obtained with ACh or TG and was not inhibited by BAPTA. BAPTA partially inhibited (40%) the increase in AP ratio induced by forskolin and histamine inspite of the complete inhibition of the Ca2+ response. BAPTA did not inhibit the response to 8-BR-cAMP. BAPTA inhibition of forskolin stimulation was reversed by A23187 and the response was potentiated. These results indicate that ACh and TG response are completely dependent on an increase of [Ca2+]i. The response to cAMP-dependent agonists histamine and forskolin depend both on Ca2+ and cAMP. For forskolin stimulation the response may be the result of a potentiation between Ca2+ and cAMP.     

Factors influencing the response of human blood platelets to analogues of ADP which may act as partial agonists at the ADP receptor.

1. The prior addition of non-aggregating concentrations of the divalent cation ionophore, A-23187, causes human platelets to aggregate in response to a subsequent addition of the 2',3'-dialdehyde and 2',3'-dialcohol derivatives of ADP (oADP and or ADP). Previous studies [Pearce et al. (1978) Eur. J. Biochem. 88, 543--555] have shown that these derivatives act as partial agonists at the platelet ADP receptor inducing only the transition from discoid to globular morphology ('shape change'). A secretion response is also observed on addition of a low concentration of ionophore A-23187 prior to orADP. These responses are not observed if ionophore A-23187 is added prior to the 2',3'-dialdehyde and 2',3'-dialcohol derivatives of ATP (oATP and or ATP) and are markedly inhibited by prior addition of the ADP antagonist, adenosine 5'-[beta, gamma-methylene]triphosphate. 2. The aggregation response to oADP in the presence of ionophore A-23187 is reduced but not eliminated by addition of 3 mM EGTA when studies are performed in heparinised platelet-rich plasma. Additions of 3 mM EGTA in citrated platelet-rich plasma, or of 4 mM EDTA in either system completely inhibits this response. Inhibitors which are reported to elevate the intracellular concentration of adenosine 3':5'-monophosphate (cyclic AMP) or to prevent Ca2+ movement also inhibit the aggregation response to oADP which is observed in the presence of ionophore A-23187. 3. Prior addition of inhibitors of adenylate cyclase fails to cause an aggregation response to subsequent addition of oADP or orADP. Certain of these inhibitors enhance and prolong the shape change response to oADP or orADP but only at concentrations an order of magnitude in excess of those required to antagonise inhibition by agents such as prostaglandin E1, which act by increasing the concentration of cyclic AMP. 4. The concentration of prostaglandin E1, adenosine or papaverine required to inhibit shape change induced by oADP is one to two orders of magnitude lower than that required to inhibit shape change induced by ADP. 5. Prior addition of oADP decreases the lag phase in the response of human platelets to arachidonate while also increasing the concentration required to observe half-maximal response, and causing a decrease in the extent of the response. Prior addition of oATP also diminishes the extent of this response and increases the concentration of arachidonate required but has no effect on the lag phase. 6. The data suggest that oADP and orADP are capable only of acting as partial agonists at the ADP receptor because of a defective ability to increase cytosolic Ca2+ concentration. The defect is rectified by the presence of low concentrations of ionophore A-23187, which promotes mobilisation of Ca2+ from an intracellular store. The results do not appear consistent with the thesis that a decrease in platelet cyclic AMP is an initiating event in aggregation induced by ADP, but do support a model which implicates cyclic AMP in depletion of cytosolic Ca2+.     

Cytosolic and stored calcium antagonistically control tyrosine phosphorylation of specific platelet proteins.

Depletion of intracellular calcium stores appears to increase plasma membrane permeability for calcium by an as yet obscure mechanism. We found that the Ca2+ ionophore, A23187, and thrombin elevate cytosolic calcium ([Ca2+]i) equally and cause tyrosine phosphorylation of a 130-kDa protein and to a lesser extent 80- and 60-kDa proteins. Chelation of [Ca2+]i by 1,2-bis(2-aminophenoxyethane)-N,N,N',N'-tetraacetic acid/acetomethoxy ester decreased thrombin-induced tyrosine phosphorylation responses. These results suggested that [Ca2+]i elevation promotes tyrosine phosphorylation. Tyrosine phosphorylation persisted in the presence or absence of extracellular calcium after thrombin stimulation but subsided rapidly after A23187 addition if extracellular calcium was present. When Ca2+/ATPase activity, which is apparently required to maintain calcium stores, is inhibited by low temperature, tyrosine phosphorylation of the 130-kDa protein occurs. Rewarming platelets reverses tyrosine phosphorylation only if extracellular calcium is present. Thapsigargin, a calcium ATPase inhibitor, also induces tyrosine phosphorylation of the 130-kDa protein and prevents dephosphorylation of this protein when added prior to rewarming. These observations suggest that homeostatic levels of calcium in storage compartments favor tyrosine dephosphorylation of specific proteins. Thus the levels of [Ca2+]i and stored calcium appear to control tyrosine phosphorylation antagonistically. Tyrosine phosphorylation may play a role in regulating calcium channel function.     

Characteristics of intracellular calcium changes required for augmentation of phorbol ester-stimulated pancreatic enzyme secretion

Previous studies demonstrated that Ca2+ ionophores augment the pancreatic enzyme secretion caused by phorbol esters. The present study was performed to determine the nature of the cellular Ca2+ effects responsible for the augmentation. Relatively low concentrations (0.3-1.0 microM) of the nonfluorescent Ca2+ ionophore, 4-bromo-A23187 (Br-A23187), did not measurably increase free cytosolic Ca2+ ([Ca2+]i) and caused little or no enzyme release from guinea pig pancreatic acini. However, these concentrations of Br-A23187 augmented the amylase release caused by the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA). This augmentation occurred in the absence of extracellular Ca2+ as long as the intracellular agonist-sensitive pool contained Ca2+. Greater concentrations of Br-A23187 (3-10 microM) alone caused transient increases in [Ca2+]i and transient increases in amylase release. Although not resulting in an increase in [Ca2+]i, the low concentrations of Br-A23187 caused release of Ca2+ from the intracellular agonist-sensitive pool. These results suggest that Ca2+ mediates enzyme release by two distinct mechanisms in the pancreatic acinar cell. First, an increase in [Ca2+]i alone mediates enzyme release. Second, Ca2+ release from the agonist-sensitive pool not resulting in a measurable increase in [Ca2+]i augments enzyme release stimulated by a phorbol ester. The second effect of Ca2+ may be due to a small localized change in cell Ca2+ or an induction of cytosolic Ca2+ oscillations.