The overall goal of this project is to understand the alpha-1- adrenergic receptor mechanisms that underlie the arterial supersensitivity to norepinephrine (NE) which develops during aldosterone-salt hypertension in the rat (AHR). The approach integrates pharmacological analysis with biochemical and biophysical measure of responses in aortic smooth muscle. The functional measures will focus on dose and time dependent changes in membrane 42K and 45Ca fluxes and in contraction. The biochemical endpoints include inositol phosphate (IP, IP2, IP3), phosphatidic acid (PA), diacylglycerol (DAG) and prostaglandins (6-keto PGF1alpha, PGE2). Radioligand binding of 125I-HEAT will also be analyzed. The hypothesis to be tested is that the increase in sensitivity to NE in aorta from AHR involves the linage between receptor occupancy and the activation of phospholipase C (PLC) as measured by the production of inositol phosphates (IP's), PA and DAG.
The specific aims deal with the determination of: 1) steady state dose responses to NE and the production of IP's in aortas from control (C) and AHR. It is hypothesized that the dose dependent production of IP's will be shifted to the left (supersensitivity) in AHR and the shift will be similar for functional response (contraction, 45Ca efflux, 42K efflux). 2) the dose dependent as well as timed response to NE and the production of PA and DAG in aorta from C and AHR. It is hypothesized that the dose dependent production of PA and DAG will exhibit a shift to the left in AHR which will be similar to the functional responses (contraction, 45Ca influx, 42 K efflux). 3) the dose dependent relations between NE and production of PG's by aorta and functional responses during inhibition of PG synthesis. It is hypothesized the NE dependent PG synthesis is shifted to the left in AHR but does not underlie the super-sensitivity of the functional endpoints. 4) the relation between receptor occupancy and production of IP's after selective alpha-receptor inactivation by a non-competitive antagonist, dibenamine. The interaction will also be determined between NE and serotonin (5HT). It is hypothesized that little receptor reserve exists for the production of IP's in C while contraction and 42K efflux will exhibit more efficient coupling. It is further hypothesized the NE and 5HT operate through different receptors but through the same effector system (phospholipase C). 5) cellular Ca by means of microspectrofluorescence techniques applied to the single cells loaded with the Ca probe, fura-2. It is hypothesized that the supersensitivity of functional responses in whole arteries from AHR will be paralleled by receptor coupled changes in cellular Ca.
