Krishnamurthi S, Joseph S, Kakkar V V
Biochim Biophys Acta. 1987 Mar 11;927(3):429-36. doi: 10.1016/0167-4889(87)90109-1.
We have previously demonstrated synergistic potentiation of secretion by phorbol 12-myristate 13-acetate (PMA) and platelet agonists such as thrombin and the thromboxane mimetic, U46619, with short (less than 2 min) pre-incubations of PMA, despite inhibition of agonist-induced [Ca2+]i mobilization and arachidonate/thromboxane release. In this study, the effect of PMA on 5-hydroxytryptamine secretion in relation to arachidonate/thromboxane B2 release induced by collagen as well as the 'weak agonists', ADP, adrenaline and platelet-activating factor (PAF), was investigated using human platelet-rich plasma. Short incubations (10-30 s) with PMA (400 nM) before agonist addition caused an inhibition (60-100%) of 5-hydroxy[14C]tryptamine secretion and thromboxane B2 formation in response to maximally effective doses of ADP (10 microM), adrenaline (10 microM) and PAF (0.5 microM) but potentiated collagen-induced 5-hydroxy[14C]tryptamine secretion and [3H]arachidonate/thromboxane release. However, a longer pre-incubation with PMA (5 min) caused a significant reduction (20-50%) in the extent of collagen-induced 5-hydroxy[14C]tryptamine secretion and thromboxane B2 formation as seen earlier with thrombin, although collagen-induced [3]arachidonate release was still unaffected. Pretreatment of platelets with the cyclo-oxygenase inhibitor, indomethacin (10 microM), abolished 5-hydroxy[14C]tryptamine secretion in response to the weak agonists and reduced collagen (2.5-10 micrograms/ml) -induced secretion by 50-90%, depending on the collagen concentration. Addition of PMA (400 nM) 10 s before these agonists in indomethacin-treated platelets resulted in synergistic interactions between agonist and PMA leading to enhanced 5-hydroxy[14C]tryptamine secretion, although this was notably less than the synergism observed previously between thrombin and PMA or U46619 and PMA. The results suggest that the effect of short incubations with PMA on 5-hydroxytryptamine secretion induced by 'thromboxane-dependent' agonists, such as those examined in this study, is determined by the effect on agonist-induced thromboxane synthesis. However, when endogenous thromboxane synthesis is blocked, weak agonists as well as collagen can synergize with PMA at potentiating 5-hydroxytryptamine secretion, albeit to a weaker extent than thrombin or U46619. The results also suggest that PMA has differential effects on arachidonate release induced by collagen and thrombin.
我们之前已经证明，用佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）与血小板激动剂（如凝血酶和血栓素类似物U46619）进行短时间（少于2分钟）预孵育，可协同增强分泌，尽管激动剂诱导的[Ca2+]i动员以及花生四烯酸/血栓素释放受到抑制。在本研究中，使用富含人血小板的血浆，研究了PMA对5 - 羟色胺分泌的影响，该影响与胶原以及“弱激动剂”ADP、肾上腺素和血小板活化因子（PAF）诱导的花生四烯酸/血栓素B2释放相关。在添加激动剂之前，用PMA（400 nM）进行短时间孵育（10 - 30秒），可抑制（60 - 100%）对最大有效剂量的ADP（10 microM）、肾上腺素（10 microM）和PAF（0.5 microM）的反应中5 - 羟[14C]色胺分泌和血栓素B2形成，但增强胶原诱导的5 - 羟[14C]色胺分泌和[3H]花生四烯酸/血栓素释放。然而，与PMA进行更长时间的预孵育（5分钟）会导致胶原诱导的5 - 羟[14C]色胺分泌和血栓素B2形成的程度显著降低（20 - 50%），这与之前观察到的凝血酶的情况类似，尽管胶原诱导的[3]花生四烯酸释放仍未受影响。用环氧化酶抑制剂吲哚美辛（10 microM）预处理血小板，可消除对弱激动剂的反应中5 - 羟[14C]色胺分泌，并根据胶原浓度将胶原（2.5 - 10微克/毫升）诱导的分泌减少50 - 90%。在吲哚美辛处理的血小板中，在这些激动剂加入前10秒添加PMA（400 nM），会导致激动剂与PMA之间的协同相互作用，从而增强5 - 羟[14C]色胺分泌，尽管这明显小于之前观察到的凝血酶与PMA或U46619与PMA之间的协同作用。结果表明，与PMA短时间孵育对“血栓素依赖性”激动剂（如本研究中所检测的那些）诱导的5 - 羟色胺分泌的影响，取决于对激动剂诱导的血栓素合成的影响。然而，当内源性血栓素合成被阻断时，弱激动剂以及胶原在增强5 - 羟色胺分泌方面可与PMA协同作用，尽管程度比凝血酶或U46619弱。结果还表明，PMA对胶原和凝血酶诱导的花生四烯酸释放有不同影响。
