Portal hypertension, a condition that results from cirrhosis or intrahepatic liver disease, is characterized by an elevated portal pressure, portosystemic shunting, an intense intestinal vasodilation and decreased vasoconstrictor responsiveness. Previous work has established that elevations in plasma glucagon and nitric oxide (NO) contribute to the vasodilation, both by a direct effect and by interfering with vasoconstrictor function. Now, it has become clear that other important participants in this process include cAMP and cGMP, which appear to relax vascular smooth muscle by altering the Ca2+ sensitivity of the contractile machinery, i.e., actin and myosin. This latter mechanism is the focus of this application. The applicant suggests three means by which this process might occur. First, activation of Gs signaling pathway is proposed, leading to an increase in cAMP and thus activation of PKA. Second, failure of the small GTP binding protein Rho A to activate in response to GI and Gq coupled vasoconstrictor is suggested, as this event would interfere with post-receptor signal transduction, thus limiting the vasoconstrictive effect of a specific agonist. Third, it is suggested that the myosin-associated protein telokin, which affects the Ca2+ sensitivity of MLCK, may be altered, leading to reduced contractile efficiency. These possibilities will be tested in four specific aims. The work will utilize a novel method of gene transfer developed in the applicant's laboratory. Thus, the applicant has demonstrated the ability to insert cDNA constructs into intact blood vessels in situ by electroporation. The vessels are harvested days later and studied in vitro. Using this method, the applicant proposes to study the proposed mechanisms by insertion of dominant negative or constitutively active constructs of several key players (e.g., G proteins, PKA and Rho A) to specifically alter the transduction pathways within the cells without the application of exogenous pharmacological probes.
Showing the most recent 10 out of 22 publications
