Mark Bushfield

Regulation of platelet cytosolic free calcium by cyclic nucleotides and protein kinase C.

PAF elicits a rapid, concentration‐dependent elevation of platelet cytosolic free calcium ([Caf]), measured by quin2. Elevation of [Caf] is transient, and the rate of reversal increases with agonist concentration. Adenylate cyclase stimulants (PGI2, PGD2) and 8‐bromo cAMP; a guanylate cyclase stimulant (sodium nitroprusside) and 8‐bromo cGMP; and a protein kinase C stimulant (phorbol myristate acetate) block the elevation of [Caf] induced by PAF, and accelerate its reversal. These results suggest that cAMP, cGMP and 1,2‐diacylglycerol (DAG) could act as second messengers to regulate [Caf] in platelets. As PAF is known to stimulate platelet phosphoinositide hydrolysis (ergo DAG formation) but fails to elevate platelet cAMP or cGMP, it is proposed that DAG, via activation of protein kinase C, may act as an endogenous modulator of platelet [Caf]: an action that contributes to the role of DAG as a bi‐directional regulator of platelet reactivity.

Agonist-induced inositol phospholipid metabolism and Ca++ flux in human platelet activation.

Human platelet responsiveness may be modulated by a variety of agonists that combine with specific receptors on the platelet plasma membrane. Receptors for Thrombin, ADP, Thromboxane (Tx) A2, Platelet activating factor (PAF), Vasopressin (VP). Adrenaline, Prostaglandin (PG) I2, PGD2 and Adenosine, inter alia, have been identified by a variety of pharmacological techniques including radioligand binding analyses, homologous desensitisation and the use of specific antagonists1,2. Platelets are electrically non-exciteable3. Thus in order for platelets to respond to externally applied (exogenous) agonists, there must exist some information transfer system whereby events at the cell surface influence the rates of the key biochemical reactions that actually mediate the cellular response. These key biochemical reactions are controlled by stimulus-induced changes in the intracellular concentrations of second messenger molecules including cAMP, cytosolic free Ca++ (Caf) and 1,2-diacylglycerol (DAG)4,5. These second messengers activate specific protein kinases, respectively cAMP-dependent protein kinase. Ca++-calmodulin-dependent protein kinase and protein kinase C6,7 that mediate the phosphorylation and altered reactivity of specific target proteins.

Effects of protein kinase C activation on human platelet cyclic AMP metabolism

Treatment of intact human platelets with the tumour‐promoting phorbol ester, phorbol 12‐myristate 13‐acetate (PMA), specifically inhibited PGD2‐induced cyclic AMP formation without affecting the regulation of cyclic AMP metabolism by PGI2, PGE1, 6‐keto‐PGE1, adenosine or adrenaline. This action of PMA was: (i) concentration‐dependent; (ii) not mediated by evoked formation or release of endogenous regulators of adenylate cyclase activity (thromboxane A2 or ADP); (iii) mimicked by 1,2‐dioctanoylglycerol (DiC8) but not by 4α‐phorbol 12,13‐didecanoate (which does not activate protein kinase C); (iv) attenuated by Staurosporine. These results indicate that activation of protein kinase C in platelets may provide a regulatory mechanism to abrogate the effects of the endogenous adenylate cyclase stimulant PGD2 without compromising the effects of exogenous stimulants of adenylate cyclase (PGI2, 6‐keto‐PGE1, adenosine).

Glucagon activates two distinct signal transduction systems in hepatocytes, which leads to the desensitization of G-protein-regulated adenylate cyclase, the phosphorylation and inactivation of Gi-2 and the phosphorylation and stimulation of a specific cyclic AMP phosphodiesterase

A transient increase in the intracellular concentrations of cyclic AMP occurs as a result of the challenge of hepatocytes with glucagon. This event is determined by the initial rapid activation of adenylate cyclase, which is responsible for the production of cyclic AMP within the cell. Following on from this we observe the desensitization of adenylate cyclase; the A-kinase-mediated activation of the ‘dense-vesicle’, high affinity cyclic AMP phosphodiesterase; the phosphorylation and functional inactivation of the inhibitory G-protein Gi-2 and the establishment of a ‘selective’ insulin-resistant state. These events identify ‘interplay’ or ‘cross-talk’ occurring between distinct cellular signalling systems.