scholarly journals 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

1986 ◽  
Vol 238 (1) ◽  
pp. 193-199 ◽  
Author(s):  
S Krishnamurthi ◽  
S Joseph ◽  
V V Kakkar
Keyword(s):  
Phorbol Myristate Acetate ◽  
Phorbol Esters ◽  
Synergistic Effects ◽  
Human Platelets ◽  
Significant Rise ◽  
Inhibitory Effects ◽  
Myristate Acetate ◽  
Inositol Phospholipid ◽  
Granule Secretion ◽  
Inositol Phospholipid Breakdown

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.

scholarly journals Abolishment of inhibitory effects of 3'-deazaadenosine on Superoxide generation of guinea pig phagocytes by pre-exposure to phorbol myristate acetate

FEBS Letters ◽  
1986 ◽  
Vol 201 (2) ◽  
pp. 287-290 ◽  
Author(s):  
Katsuro Yagawa ◽  
Masayuki Nakanishi ◽  
Shinichiro Hayashi ◽  
Mariko Kaku ◽  
Yukito Ichinose ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Phorbol Myristate Acetate ◽  
Inhibitory Effects ◽  
Myristate Acetate ◽  
Superoxide Generation

R59022, A DIACYLGLYCEROL (DG) KINASE INHIBITOR POTENTIATES THROMBIN-INDUCED PLATELET AGGREGATION AND GRANULE RELEASE

1987 ◽  
Author(s):  
S K Joseph ◽  
S Krishnamurthi ◽  
V V Kakkar
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Kinase Inhibitor ◽  
Human Platelets ◽  
Maximal Concentration ◽  
Inhibitory Effects ◽  
Kinase C ◽  
Granule Secretion ◽  
Granule Release

R59022 is a recently described inhibitor of the enzyme DG kinase [1], which converts DG to phosphatidic acid. While R59002 inhibits DG conversion in platelets resulting in enhanced protein kinase C (PrkC) activation [1], little is known on its effect on other platelet responses. In this study, we have examined the effect of R59022 on agonist-induced platelet aggregation and [14C]-5-hydroxytryptamine (5HT) release using washed human platelets. With a sub-maximal concentration of thrombin (T, 0.05U/ml) R59022 (10-30μM) significantly potentiated T-induced platelet aggregation and [14C]-5HT release eg % [14C]-5HT release:- 0.05U/ml T-52±5,30μM R59022+T-76±8. Removal of external Ca2+ (ImM) using EGTA (5mM) reduced T-induced 5HT release but not the potentiation of it by R59022 eg EGTA+ 0.05U/ml T-36±6%, EGTA+R59022+T- 72±5%. These results show that in the presence of EGTA and R59022 the increased DG levels can compensate for the diminished rise in T-induced Ca/2+ mobilisation thus re-emphasizing the importance of DG in promoting granule secretion. In addition to inhibiting DG phosphorylation, R59022 also inhibits the phosphorylation of the DG analogue 1-oleoyl 2-acetylglycerol (OAG) [1]. OAG (63μM) with pre-incubation times of 10-60 sec, significantly potentiated threshold T (0.03U/ml)-induced [l4C]-5HT release, though with longer incubation times, this potentiatory effect was gradually lost eg 0.03U/ml T-l±0.3%, OAG+T (10 sec)- 33±4%, OAG+T (1 min)-11±3%, 0AG+veh.-0%. However, in the presence of R59022 (30μM), OAG retained its potentiatory effect for longer periods eg R59022+0AG+T (1 min)-45+10%, R59022+T-2±l%. With incubation times > 5 min the potentiatory effects of OAG were lost even in the presence of R59022. This is possibly due to the metabolism of OAG by DG lipase. Our results demonstrate that R59022, which has been reported to inhibit DG kinase leading to enhanced PrkC activation, also enhances agonist-induced platelet aggregation and 5HT release. It may therefore be a useful compound in elucidating further the role of DG in terms of both stimulatory and inhibitory effects on platelet activation.[1]. de Chaffoy de Coucelles, D. et al (1985) J Biol Chem 260, 15762.

EFFECT OF PROTEIN KINASE C (PrkC) ACTIVATORS ON AGONIST-INDUCED ARACHIDONATE RELEASE IN HUMAN PLATELETS

1987 ◽  
Author(s):  
S Krishnamurthi ◽  
S K Joseph ◽  
V V Kakkar
Keyword(s):  
Human Platelets ◽  
Significant Rise ◽  
The Other ◽  
Cyclooxygenase Inhibitor ◽  
Inhibitory Effects ◽  
Response Curves ◽  
Kinase C ◽  
Significant Release ◽  
Collagen Versus ◽  
Arachidonate Release

We have examined the effect of 1,2 dioctanoylglycerol (diCg) and phorbol 12-myristate 13-acetate (PMA), both activators of PrkC, on arachidonic acid (AA) release induced by thrombin, collagen and the Ca2+ ionophore, ionomycin, and correlated this with the levels of [Ca2+]i in quin 2-loaded platelets. Experiments were carried out using phenidone (200μM, a combined lipoxygenase and cyclooxygenase inhibitor)-treated washed, human platelets pre-labelled with [3H]-AA or quin 2 and, sub-maximal concentrations of thrombin, collagen and ionomycin. Incubation of platelets with diCg (30 or60μM) or PMA (16nM) over a 15 min period, resulted in no significant release of AA or rise in [Ca2+]i levels compared to that in resting platelets. However, thrombin (0.2U/ml) induced a 6-8% release of [3H]-AA as well as a rise in [Ca2+]i equalling 1μM and, addition of diCg (30-60μM) or PMA (16nM) 10 sec-5 min before or 10-20 sec after thrombin, resulted in a significant reduction (20-40%) of both responses. On the other hand, [3H]-AA release in response to collagen (20μg/ml) and ionomycin (6μM) were significantly potentiated by 1.2-1.9 fold and 1.5-3.0 fold over control respectively, by pre-or post-agonist additions of diCg or PMA.As collagen induced no significant rise in [Ca2+]i levels, the differential effects of diCg and PMA on collagen versus thrombin-induced AA release may be related to inhibitory effects on [Ca2+]i mobilisation induced by the latter. Dose-response curves of ionomycin-induced [Ca2+]i mobilisation and AA release revealed that potentiation of the latter by 10 sec pre-incubations of diCg or PMA, only occurred at [Ca2+]i levels> 1μM, with diCg and PMA not affecting the ionomycin-induced rise in [Ca2+]i. The results suggest that the effect of PrkC activators, on agonist-induced AA release, may depend firstly, on whether AA release is dependent on agonist-induced [Ca2+]i mobilisation and secondly, on whether the latter is inhibited by PrkC activation. Thus, the hypothesis regarding a stimulatory role for PrkC in AA release via lipocortin (Touqui et al, Nature 321, pl77, 1986) needs to be re-assessed in the light of these agonist-related mechanistic differences.

Regulation of platelet phospholipase C

1988 ◽  
Vol 320 (1199) ◽  
pp. 299-311 ◽  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Adenylyl Cyclase ◽  
Phospholipase C ◽  
Phorbol Esters ◽  
Human Platelets ◽  
Direct Effects ◽  
Inhibitory Effects ◽  
Factors Affecting ◽  
Kinase C

We have investigated factors affecting the activation of phospholipase C in human platelets. Prior exposure of platelets to phorbol esters that stimulate protein kinase C inhibits the activation of phospholipase C in response to a variety of receptor-directed agonists, including x- and y-thrombin and thromboxane A 2 analogues. Such activation has been assayed by measurements of accumulated InsP 3 (including Ins(l,4,5)P 3 and Ins(l,3,4)P 3 ) and PtdOH. Inhibition is not overcome by Ca 2+ ionophores, and substances that block or mimic Na+-H + exchange neither block nor mimic these inhibitory effects. Cyclic AMP and cyclic GMP, other agents known to inhibit phospholipase C activation, do not accumulate in platelets exposed to phorbol esters. Although a portion of the effects of phorbol ester on InsP 3 accumulation may be explained by 5-phosphomonoesterase activity, it is likely that more direct effects on phospholipase C are being exerted as well, and contribute the major inhibitory route. We have examined the susceptibility of adenylyl cyclase-associated G 1 and ‘G p ’- activated phospholipase C to inhibitory ADP-ribosylation by pertussis toxin-derived enzyme (S 1 protomer) administered to saponin-permeabilized platelets. The effects of a-thrombin on adenylyl cyclase can be inhibited by up to 50% by S 1 at which point inhibition of phospholipase C is barely detectable. Thromboxane A 2 analogues, which do not affect adenylyl cyclase (G 1 ), stimulate phospholipase C; this effect is not impaired by Sr We therefore propose that the inhibitory effects of phorbol esters on the activation of phospholipase C are not mediated primarily by effects on G 1 .

Acute effects of phorbol esters on receptor-mediated IP3, cAMP, and progesterone levels in rat granulosa cells

1989 ◽  
Vol 256 (3) ◽  
pp. E368-E374
Author(s):  
J. S. Davis ◽  
L. L. Weakland ◽  
R. G. Coffey ◽  
L. A. West
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Granulosa Cells ◽  
Phorbol Esters ◽  
Inositol Phosphate ◽  
Inhibitory Effect ◽  
Inhibitory Effects ◽  
Kinase C ◽  
Inositol Phospholipid

Luteinizing hormone (LH) stimulates the formation of adenosine 3',5'-cyclic monophosphate (cAMP) and inositol trisphosphate (IP3) in rat granulosa cells. This report describes the effects of protein kinase C activators on second messenger generation in isolated rat granulosa cells. The protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) completely inhibited LH-stimulated inositol phosphate accumulation. The inhibitory effects of TPA were rapid (5-15 min) and concentration dependent with 50 nM TPA producing maximally inhibitory effects. However 30-min incubations with 10-100 nM TPA had no effect on LH-stimulated cAMP or progesterone levels. The inhibitory effect of TPA could not be overcome by high concentrations of LH. TPA also inhibited gonadotropin-releasing hormone-stimulated phospholipase C activity, although to a much lesser extent. Increased inositol phosphate degradation and reduced inositol phospholipid synthesis were unlikely explanations for the effects of TPA. The results indicate that phorbol esters modulate the inositol phospholipid-phospholipase C transmembrane signaling system in rat granulosa cells. The results suggest that phorbol esters may alter the coupling of the hormone receptor complex to phospholipase C.

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