[Arginine8]-vasopressin (AVP) is a peptide hormone which is released from the posterior pituitary gland in response to a decrease in blood pressure or an increase in plasma osmolality. AVP is both a potent vasoconstrictor and a mitogen for vascular smooth muscle. These effects of AVP are important not only for physiological regulation of blood flow and pressure, but may also play a role in diseases, such as hypertension and atherosclerosis, which involve alterations in vascular smooth muscle contractility and proliferation, respectively. The long term objectives of this project are to understand the molecular mechanisms involved in these responses of arterial smooth muscle to AVP. Both contraction and proliferation of arterial smooth muscle cells involve an increase in cytosolic Ca2+ concentration ([Ca2+]i). In A7r5 rat aortic smooth muscle cells, AVP binds to a single class of vasopressin receptors of the V1a subtype. However, AVP has two distinct effects on [Ca2+]i in A7r5 cells: low concentrations of AVP (10-500 pM) stimulate oscillations in [Ca2+]i (Ca2+ spikes); higher concentrations (0.5-100 nM) inhibit Ca2+ spiking, but elicit a biphasic elevation of [Ca2+]i resulting from release of intracellular Ca2+ stores and increased Ca2+ influx across the plasma membrane. These two effects involve different mechanisms. AVP-stimulated Ca2+ spiking results from action potentials that depend on Ca2+ entry through L-type voltage-sensitive Ca2+ channels. The release of intracellular Ca2+ by higher concentrations of AVP is attributed to inositol trisphosphate produced as a result of activation of phospholipase C. The proposed research project will examine the signal transduction mechanisms involved in AVP-stimulated Ca2+ spiking. Preliminary studies suggest that activation of phospholipase D (PLD) is required for stimulation of Ca2+ spiking by AVP. [Ca2+]i measurements with fura-2 or indo-1 and electrophysiological methods will be used to examine the effects of AVP on whole cell membrane currents and membrane potential to test the hypothesis that AVP increases Ca2+ spiking activity by inhibiting delayed rectifier K+ currents, resulting in membrane depolarization and activation of voltage-sensitive Ca2+ channels. The involvement of PLD and the non-receptor tyrosine kinase, PYK2, in K+ channel inhibition will also be investigated using immunoprecipitation techniques. AVP-stimulated PYK2 phosphorylation and Ca2+ spiking are prevented by protein kinase C (PKC) inhibitors. Additional experiments will examine the role of specific PKC isoforms in the AVP signaling pathway and their relationship to PLD activation. Finally, the effects of AVP and other hormones on [Ca2+]i and membrane currents will be examined in freshly isolated smooth muscle cells from rat mesenteric arteries.
