Lack of inhibition of thrombin‐induced rise in intracellular Ca<sup>2+</sup> levels and 5‐hydroxytryptamine secretion by 1‐oleoyl‐2‐acetylglycerol in human platelets

Krishnamurthi, Sushila; Joseph, Sunil; Kakkar, Vijay V.

doi:10.1016/0014-5793(86)80281-2

Cited by 13 publications

(4 citation statements)

References 21 publications

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“…1, control). Earlier reports, including our own [6], have demonstrated an inhibition of thrombin-induced elevation of [Ca2+Ii by PMA, but subsequent to this, we have been able to demonstrate that OleAcGro (62-125 pM) has no inhibitory effect on thrombin-induced [Ca2+Ii elevations [l 11. Fig.…”

Section: Resultssupporting

confidence: 65%

1,2‐Dioctanoylglycerol but not 1‐oleoyl‐2‐acetylglycerol inhibits agonist‐induced platelet responses

Krishnamurthi

Joseph

Kakkar

1987

European Journal of Biochemistry

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1. The effect of the membrane-permeable diacylglycerol analogues, 1,2-dioctanoylglycero1 (OcozGro) and 1 -oleoyl-2-acetyl-glycerol (OleAcGro) on agonist-induced platelet activation processes were compared with those of the phorbol ester, phorbol12-myristate 13-acetate (PMA), using appropriately labelled washed human platelets.2. Pre-treatment (10 -300 s) with OcozGro (1 5 -60 pM) or PMA (16 nM) before addition of thrombin (0.2 U/ ml) or, addition of these agents 10-20 s after thrombin, resulted in a significant reduction (20-80%) in the extent of thrombin-induced intracellular CaZ ' ([Ca2'Ii) mobilisation and arachidonate/thromboxane B2 release.OleAcGro (62-125 pM) had no effect on thrombin-induced [Ca2+Ii elevations but had a slight (15%) inhibitory effect on thrombin-induced arachidonate release with a 5-min pre-incubation. Addition of OcozGro, PMA orOleAcGro on their own caused no rise in [Ca2 'Ii levels or arachidonate release.3. Collagen (20 pg/ml) induced substantial arachidonate release without a detectable rise in [Ca2+Ii. Pretreatment (10-300 s) with OcozGro (15-60 pM), PMA (16 nM) or OleAcGro (62 pM) before collagen addition or addition of these agents 30 -60 s after collagen addition resulted in a significant potentiation of arachidonate release (1.2 -2-fold over control), even though thromboxane B2 formation in response to collagen was inhibited in the presence of OcozGro or PMA.4. Both Oco2Gro and PMA had dual effects on 5-hydroxytryptamine secretion induced by thrombin or collagen. Short pre-incubations ( < 2 min) with these agents caused a potentiation of sub-maximal agonist-induced secretion, while not affecting secretion induced by maximal agonist concentrations. With longer pre-incubation times (5 -15 min) however, a significant reduction in the level of agonist-induced secretion in the presence of Oco2Gro or PMA was observed. Inhibition of secretion was also observed in platelets treated with indomethacin (10 pM), suggesting that inhibition of thromboxane B2 formation alone does not account for inhibition of 5-hydroxytryptamine secretion. OleAcGro had no inhibitory effects on agonist-induced secretion even though it potentiated it (with < 2-min incubations) at sub-maximal agonist concentrations.5. Time courses of phosphorylation of a 45-kDa protein, a marker of protein kinase C activation, in 32P-labelled platelets showed that while OcozGro (60 pM) and PMA (16 nM) caused a 4-5-fold increase in 32P-labelling of this protein over a 5-min incubation period, OleAcGro (62-125 pM) caused a 1.5-fold increase in labelling which was only maintained for a 10-30-s period. The inability of OleAcGro to exert any significant inhibitory effects on agonist-induced platelet responses may therefore be due to insufficient activation of protein kinase C and/or phosphorylation of the 45-kDa protein. Hence, OcozGro may be a better tool as a diacylglycerol analogue. However, the potentiatory effects of OleAcGro with short pre-incubations (agonist-induced 5-hydroxytryptamine secretion and collagen-induced arachidonat...

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Section: Resultssupporting

confidence: 65%

1,2‐Dioctanoylglycerol but not 1‐oleoyl‐2‐acetylglycerol inhibits agonist‐induced platelet responses

Krishnamurthi

Joseph

Kakkar

1987

European Journal of Biochemistry

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“…Previous workers [10,[14][15][16][17] have postulated a relationship between protein kinase C activation and the inhibitory effects of PMA, but it is interesting that our recent studies [31] with 1-oleoyl-2-acetylglycerol have revealed that the DAG mimic has no inhibitory effect on the thrombin-induced rise in [Ca2+], levels or 5HT secretion. Although I-oleoyl-2-acetylglycerol may have inhibitory effects on agonist-induced Ptdlns(4,5)P2 breakdown [16,32] even in our system, it seems that these effects may be too weak to inhibit platelet responses such as secretion.…”

Section: Resultsmentioning

confidence: 98%

“…On the other hand, it may be significant that both PMA and 1-oleoyl-2-acetylglycerol [31] were able to potentiate agonist-induced 5HT secretion in the absence of a significant rise in [Ca2+]i and endogenous thromboxane formation. Although the synergistic effects of agonist and PMA on 5HT secretion in this study are only apparent with the short preincubations with PMA (10 s-2 min), the existence of synergism even with the 5 min preincubations is implied by the finding that a 5 min preincubation with PMA totally suppressed U46619 (3 ,tM)-induced rise in [Ca2+], levels, but had no noticeable effect on 5HT secretion.…”

Section: Resultsmentioning

confidence: 99%

Synergistic potentiation of 5-hydroxytryptamine secretion by platelet agonists and phorbol myristate acetate despite inhibition of agonist-induced arachidonate/thromboxane and β-thromboglobulin release and Ca2+ mobilization by phorbol myristate acetate

Krishnamurthi¹,

Joseph²,

Kakkar³

1986

Biochemical Journal

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Previous studies have demonstrated an inhibition of agonist-induced inositol phospholipid breakdown and intracellular Ca2+ ([Ca2+]i) mobilization by phorbol esters in platelets. In this study, we have examined the effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced granule secretion and correlated it with agonist-induced [Ca2+]i mobilization, arachidonate and thromboxane (Tx) release in human platelets. With increasing times of incubation with PMA (10 s-5 min), the rise in [Ca2+]i induced by thrombin and the TxA2 mimetic, U46619, was increasingly inhibited (90-100% with 5 min incubation) and, correlating with this, thrombin-induced [3H]arachidonate, TxB2 and beta-thromboglobulin (beta TG) release were also inhibited. In addition, the conversion of exogenously added arachidonate to TxB2 was inhibited (50-80%) by a 10 s-5 min pretreatment with PMA. However, secretion of 5-hydroxy[14C]tryptamine (5HT) induced by thrombin or U46619 was not inhibited by 10 s-2 min incubations with PMA and, on the contrary, with low agonist concentrations, was potentiated by PMA in the absence of a significant rise in [Ca2+]i or endogenous Tx formation, to levels significantly greater than or equal to the sum of that obtained when agonist and PMA were added separately. With longer times of incubation with PMA (5 min), these synergistic effects became less pronounced as inhibitory effects of PMA on agonist-induced [14C]5HT secretion became apparent. The results indicate that, while PMA may cause an inhibition of agonist-induced [Ca2+]i mobilization resulting in an inhibition of agonist-induced arachidonate, TxB2 and beta TG release, its effects on agonist-induced 5HT secretion may be complicated by [Ca2+]i-independent synergistic effects of agonist and PMA.

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“…Phorbol esters, the tumour promotors that activate protein kinase C, were also shown to cause phosphorylation and desensitization (Sibley et al, 1984b), and a1-receptors, which stimulate phosphoinositide hydrolysis, are phosphorylated and desensitized by incubation with phorbol esters (Leeb-Lundberg et al, 1985). It has been reported that incubation of platelets with phorbol esters or oleoylacetylglycerol can inhibit their responses to thrombin (MacIntyre et al, 1985;Rittenhouse & Sasson, 1985;Zavoico et al, 1985; but see Krishnamurthi et al, 1986), but this is not specific for thrombin (Maclntyre et al, 1985;. Although there is no evidence of phosphorylation of platelet receptors by protein kinase C, Katada et al (1985) demonstrated protein kinase C-catalysed phosphorylation of the 40 kDa cc-subunit of the inhibitory GTP-binding protein of the adenylate cyclase system in platelet membranes.…”

Section: Discussionmentioning

confidence: 99%

Thrombin-induced phosphoinositide hydrolysis in platelets. Receptor occupancy and desensitization

Huang¹,

Detwiler²

1987

Biochemical Journal

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The relationship between occupancy of thrombin receptors on platelets and enhanced phosphoinositide hydrolysis was analysed by examination of the dose-response relationship, the effects of thrombin inhibitors and the contribution of secondary effects. Washed human platelets were labelled with [3H]inositol, and agonist-induced accumulation of labelled inositol phosphates was measured. The dose-response curves and the time courses for alpha-thrombin- or gamma-thrombin-induced accumulation of inositol phosphates were similar to those for dense-granule secretion. Addition of the thrombin inhibitor hirudin to thrombin-activated platelets revealed that the continuous presence of active thrombin was required to maintain the accumulation of labelled inositol phosphates; the total production of inositol phosphates increased with longer periods of exposure to thrombin, reaching a maximum between 5 and 10 min. After activation with thrombin, the ability of a second, greater, addition of thrombin to induce additional phosphoinositide hydrolysis decreased with time; it was absent within 10 min after the first addition. The failure to sustain accumulation of labelled inositol phosphates or to respond to a second addition of thrombin beyond 10 min was not due to depletion of the pool of labelled precursors, because the platelets retained their ability to respond to collagen. Addition of ADP-consuming enzymes decreased sensitivity to thrombin, but inhibition of cyclo-oxygenase with indomethacin did not impair the thrombin-induced hydrolysis of phosphoinositides. It was concluded that thrombin-induced hydrolysis of phosphoinositides has characteristics consistent with mediation by a receptor that is similar to that that triggers dense-granule secretion, requires continuous presence of active thrombin to be maintained, is mediated by a receptor that displays thrombin-induced desensitization, and is only partially enhanced by secondary agents.

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Lack of inhibition of thrombin‐induced rise in intracellular Ca²⁺ levels and 5‐hydroxytryptamine secretion by 1‐oleoyl‐2‐acetylglycerol in human platelets

Abstract: The effect of I-oleoyl-2-acetylgIy~ro1 (OAG) on the thrombin-endued rise in intracellular Ca2+ levels (

Cited by 13 publications

References 21 publications

1,2‐Dioctanoylglycerol but not 1‐oleoyl‐2‐acetylglycerol inhibits agonist‐induced platelet responses

1,2‐Dioctanoylglycerol but not 1‐oleoyl‐2‐acetylglycerol inhibits agonist‐induced platelet responses

Synergistic potentiation of 5-hydroxytryptamine secretion by platelet agonists and phorbol myristate acetate despite inhibition of agonist-induced arachidonate/thromboxane and β-thromboglobulin release and Ca2+ mobilization by phorbol myristate acetate

Thrombin-induced phosphoinositide hydrolysis in platelets. Receptor occupancy and desensitization

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Lack of inhibition of thrombin‐induced rise in intracellular Ca2+ levels and 5‐hydroxytryptamine secretion by 1‐oleoyl‐2‐acetylglycerol in human platelets

Abstract: The effect of I-oleoyl-2-acetylgIy~ro1 (OAG) on the thrombin-endued rise in intracellular Ca2+ levels (

Cited by 13 publications

References 21 publications

1,2‐Dioctanoylglycerol but not 1‐oleoyl‐2‐acetylglycerol inhibits agonist‐induced platelet responses

1,2‐Dioctanoylglycerol but not 1‐oleoyl‐2‐acetylglycerol inhibits agonist‐induced platelet responses

Synergistic potentiation of 5-hydroxytryptamine secretion by platelet agonists and phorbol myristate acetate despite inhibition of agonist-induced arachidonate/thromboxane and β-thromboglobulin release and Ca2+ mobilization by phorbol myristate acetate

Thrombin-induced phosphoinositide hydrolysis in platelets. Receptor occupancy and desensitization

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Lack of inhibition of thrombin‐induced rise in intracellular Ca²⁺ levels and 5‐hydroxytryptamine secretion by 1‐oleoyl‐2‐acetylglycerol in human platelets