REGULATION OF CALCIUM CHANNELS IN VASCULAR SMOOTH MUSCLE

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DESCRIPTION: Calcium ion channels in the membrane of vascular smooth muscle (VSM) cells play an important role in excitation-contraction coupling and in setting vasomotor tone. Ca2+ channels may be regulated either directly or indirectly by neurotransmitters, vasoactive hormones and therapeutic agents; regulatory processes may include the formation or release of intracellular messengers such as Ca2+, cAMP, cGMP, IP3/IP4, and diacylglycerol. The objective of the proposed studies is to investigate in detail the specific characteristics and regulatory mechanisms of calcium channels in freshly-isolated VSM cells from several different types of vessels in the rat (small mesenteric artery, aorta, portal vein). Since blood vessels from different vascular beds have different degrees of excitability, the proportion of various ion channels and/or their regulatory processes may differ. Macroscopic and microscopic (single-channel) Ca2+ current (ICa) will be recorded using whole-cell voltage clamp and patch clamp techniques. Intracellular agents will be delivered through the use of the intracellular perfusion technique. A possible role of phosphorylation, mediated by various protein kinases (PK), in regulating ICa will be explored. Specific aims include: (1) Assessing the role of cAMP- and cGMP-dependent PK systems (PK-A and PK- G) and the effect of phosphorylation and dephosphorylation on ICa. Techniques will include the application of cyclic nucleotide analogs, PK-A (cat subunit) and PK-G, and phosphatases (PPases), as well as the use of PK and PPase inhibitors. (2) The role of PK-C in the modulation of ICa, will be explored. Responses to exogenous PK-C activators (i.e. DAG, OAG), to purified PK-C, and to PK inhibitors will be determined. (3) The role of Ca2+-calmodulin-dependent PK (Ca/CaM- PKII), will be investigated using specific activators and inhibitors of this PK. The influence of intracellular levels of calcium on Ca2+ channel activity will also be studied. (4) The role of metabolism and intracellular levels of ATP on ICa will be further studied through the use of stable ATP analogs, phosphorylation blockers and metabolic poisons. (Preliminary data showed that lowering intracellular ATP levels inhibited Ca2+ channel activity and ICa.) (5) The modulation of ICa by G-proteins and G-protein gating will be examined. The results of these studies should provide comprehensive information on the complex mechanisms involved in calcium channel regulation in VSM cells, and should help to define how calcium influx, cellular excitability, and contraction are altered by important vasoactive substances.