REGULATION OF ION CHANNELS IN VASCULAR SMOOTH MUSCLE

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The ion channels in the membrane of vascular smooth muscle cells play an important role in excitation-contraction coupling and in setting vasomotor tone. The activity of these ion channels is controlled by neurotransmitters such as norepinephrine, vasoactive hormones such as angiotensin-II, an clinically useful drugs such as Ca2+ antagonists. The ion channels may be regulated either directly or indirectly by these substances, and the regulatory processes may include the formation or release of intracellular messengers, such as Ca2+, ATP, G-proteins, cyclic AMP, cyclic GMP, inositol phosphates (IP3/IP4), and diacylglycerol. In some disease states, the properties and/or regulation of the ion channels may be altered. Since blood vessels from different regions have different degrees of excitability,k the proportion of various ion channels and/or their regulatory processes may differ also. The objective of this study is to investigate in detail the specific characteristics and regulatory mechanisms for the ion channels, including the various types of Ca2+ and K+ channels, in freshly isolated and cultured vascular smooth muscle cells. Macroscopic and microscopic (single-channel) currents will be recorded using whole-cell voltage clamp and patch-clamp techniques, respectively. The influence of intracellular levels of Ca2+, ATP, and cyclic nucleoties on the activity of Ca2+ and K+ channels will be determined by intracellular perfusion of these substances. A possible role of phosphorylation mediated by various protein kinases (i.e., cyclic nucleotide-dependent, Ca2+/calmodulin-dependent, and Ca2+/phospholipid-dependent) in regulating ion channel activity and cellular excitability will be explored. The mechanism of action of vasoactive substances will be investigated. The possible role of phosphoinositide metabolism (i.e., IP3, IP4, and diacyclglycerol formation) in the electrophysiological actions of agonists such as angiotensin-II will be assessed. The possible gating of ion channels by G-proteins will also be investigated. The results of these studies should provide comprehensive information about how ion channels in vascular smooth muscle function and are regulated and should help to clarify how cellular excitability is altered by important vasoactive substances.