Barrie Ashby - US grants

Platelet aggregation and secretion can be inhibited by increase in the level of cyclic AMP caused by stimulation of adenylate cyclase. Endogenous agents generally act through specific receptors to stimulate the enzyme. Forskolin, from the medicinal plant Coleus forskholii, also stimulates adenylate cyclase and offers great potential for control of platelet activation. In contrast to receptor-mediated activation, forskolin acts directly on the catalytic unit or a closely associated subunit. Similarly, the inhibitor, dideoxyadenosine, interacts directly with the enzyme rather than receptors. The proposal will contribute to the long-term objective of understanding the role of adenylate cyclase in platelet responsiveness by studies on the catalytic unit. The enzyme will be characterized in terms of interaction with its substrate; with activators, such as Mn++ and forskolin; and with inhibitors such as adenosine and purine-site analogs. The possibility that the enzyme is influenced by Ca++ ions, acting through calmodulin or a Ca++ binding component of the catalytic unit, will be examined. Platelet adenylate cyclase will be solubilized using detergents and activity stabilized by ATP or forskolin. The catalytic unit will be resolved from GTP-regulatory components by gel-filtration. Attempts will be made to reconstitute the catalytic and regulatory components in terms of the ability of the regulatory component to restore responsiveness of the catalytic component to Gpp(NH)p and fluoride. The catalytic component will be further purified by a combination of dodecyl-agarose, ATP-agarose and forskolin-agarose columns, as well as appropriate conventional techniques. At each stage in the purification the responsiveness of the enzyme to activators or inhibitors will be determined. The molecular weight and subunit structure of the enzyme will also be determined at each stage, by hydrodynamic methods and by sodium dodecyl sulfate gel electrophoresis. Attempts will be made to correlate the structure with responsiveness to effectors. In particular, efforts will be made to identify and isolate a putative forskolin binding subunit and reconstitute it with the isolated catalytic unit.

The proposal will employ molecular cloning methods to characterize prostaglandin receptor subtypes in human erythroleukemia (HEL) cells and vascular smooth muscle cells and correlate the subtypes with patterns of cyclic AMP metabolism in both cell types. Prostaglandins are potent stimulators of cyclic AMP formation in platelets and vascular smooth muscle cells, leading to inhibition of platelet aggregation and relaxation of vascular smooth muscle so that therapeutic use of prostaglandins as antiplatelet agents may be offset by unwanted vasodilation. Platelets and smooth muscle cells may possess different prostaglandin stimulatory receptors that may be targeted selectively by different drugs. Platelet adenylate cyclase appears to be controlled by both stimulatory and Inhibitory prostaglandin receptors that act together to maintain platelet homeostasis while allowing the cells to respond rapidly to appropriate activators. Similar two-receptor control of cAMP formation occurs in HEL cells, a megaryocytic cell line with many properties of platelets. Prostaglandin receptors coupled to inhibition (EP3 subtype) and activation (EP2 subtype) of adenylate cyclase have been successfully cloned and expressed in this laboratory from HEL cells. Low stringency hybridization will be used to clone the stimulatory IP prostaglandin receptor. Cloned receptors will be used to probe receptor expression in HEL cells and in reconstitution studies to determine the validity of the two receptor model of prostaglandin regulation: stimulatory and inhibitory receptors will be transfected into CHO cells alone and together to examine corresponding patterns of cAMP formation. The activity of splice variants of the EP3 receptor subtype will be examined. Prostaglandin subtypes on vascular smooth muscle cells will be compared with HEL cell receptors by cloning techniques including Northern blot analysis, high fidelity PCR screening of vascular smooth muscle total RNA, and if necessary by cloning the corresponding vascular smooth muscle receptors. Further studies will correlate radioligand prostaglandin binding with receptor subtypes expressed on the cells and with patterns of cyclic AMP metabolism that can be analyzed by computer modeling.