The relationship between alpha 1 adrenergic receptor occupation and response will be examined in situ and in vitro. The relationship linking alpha 1 adrenergic receptor occupation and regulation of intracellular Ca2+ concentration will in examined intact BC3H-1 cells during phasic and prolonged hormonal stimulation. Plasma membrane vesicles and permeabilized cell preparations will be used to study regulation of phosphoinositide metabolism by guanine nucleotide-dependent regulatory proteins (G- proteins) and to examine the mechanisms by which the inositol phosphates and diacylglycerol/protein kinase C pathways control Ca2+ cycling at the plasma membrane and endoplasmic reticulum, respectively. Additional studies will investigate the relationship between agonist occupancy and receptor response at the molecular level. The receptor will be detergent solubilized from DDT-1 smooth muscle cells and purified using derivatives of the antagonist prazosin as ligands for affinity chromatography. Endogenous G proteins will also be identified and purified DDT-1 cells. Novel fluorescent prazosin analogs will be developed as probes of the receptor's agonist/antagonist binding site. Functional interactions between the receptor G proteins will be studied in detergent solubilized and membrane reconstituted systems using fluorescence spectroscopy as well as radioligand binding techniques. These studies will ultimately progress toward reconstitution of the biochemical entities involved in alpha 1 receptor signalling in a defined lipid environment. Studies in this proposal will offer insight into the mechanisms by which vascular smooth muscle cells respond to the sympathetic neurotransmitter norepinephrine through its actions at the post junctional alpha 1-adrenergic receptor. This process is likely to be an important determinant of vascular tone in both normal and pathophysiological states.
