Stimulus-response (S-R) coupling in platelets requires an intermediary other than an elevation in cytosolic free calcium ([Ca2+]i). While an increase in [Ca2+]i is essential in S-R coupling, effecting phosphorylation of myosin of relative molecular mass (Mr) 20,000 (20 K), platelet activation is also associated with phosphorylation of a 40K protein, which can occur in the absence of changes in [Ca2+]i. The 40K protein is the substrate for protein kinase C (PKC). Mounting evidence suggests that activation of PKC by diacylglycerol is the other signal involved in S-R coupling. Although phosphorylation of the 40K protein is associated with certain platelet functional responses, no precise role has been accredited to it. Recently, we and others have described several proteins (collectively known as lipocortin) which inhibit phospholipase A2 (PLA2). One of the most conspicuous proteins of this group is a 40K peptide whose inhibitory activity can be suppressed by prior phosphorylation. We hypothesized that the 40K protein described in platelets may possess anti-PLA2 activity and that phosphorylation by PKC, suppressing its inhibitory activity, may represent the mechanism underlying mobilization of arachidonic acid, the precursor of prostaglandins. The results of the present study strongly support this hypothesis.

1 The presence of b 3 -adrenoceptors and the low a nity state of the b 1 -adrenoceptor (formerlỳ putative b 4 -adrenoceptor') was investigated in ring preparations of rat isolated aorta preconstricted with phenylephrine or prostaglandin F 2a (PGF 2a ). Relaxant responses to isoprenaline, selective b 3 -adrenoceptor agonists (BRL 37344, SR 58611A, CL 316243) and non-conventional partial agonists (CGP 12177A, cyanopindolol, pindolol) were obtained. 2 In phenylephrine-constricted, but not PGF 2a -constricted rings, relaxations to isoprenaline showed a propranolol-resistant component. 3 In phenylephrine-constricted rings, relaxations to BRL 37344 (pEC 50 , 4.64) and SR 58611A (pEC 50 , 4.94) were not antagonized by the selective b 3 -adrenoceptor antagonist SR 59230A (41 mM). CL 316243 (4100 mM) failed to produce relaxation. In PGF 2a -constricted rings only SR 58611A produced relaxation, which was not a ected by SR 59230A (43 mM). 4 Non-conventional partial agonists produced relaxation in phenylephrine-constricted but not PGF 2a -constricted rings. The relaxation to CGP 12177A was una ected by SR 59230A (41 mM) or by CGP 20712A (10 mM), reported to block the low a nity state of the b 1 -adrenoceptor. Abbreviations: DMSO, dimethyl sulphoxide; pEC 50 , negative logarithm of the concentration (M) of relaxant that produces 50% of its maximum response; PG, prostaglandin; R max , %, maximum % relaxation

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1 b-adrenoceptor-mediated relaxation was investigated in ring preparations of rat isolated thoracic aorta. Rings were pre-constricted with a sub-maximal concentration of noradrenaline (1 mM) and relaxant responses to cumulative concentrations of b-adrenoceptor agonists obtained. 2 The concentration-response curve (CRC) to isoprenaline was shifted to the right by propranolol (0.3 mM) with a steepening of the slope. Estimation of the magnitude of the shift from EC 50 values gave a pA 2 of 7.6. Selective b 1 -and b 2 -adrenoceptor antagonists, CGP 20712A (0.1 mM) and ICI 118551 (0.1 mM), respectively, produced 4 and 14 fold shifts of the isoprenaline CRC.