The specific aim of this research is to learn what molecular signaling events regulate the contraction of smooth muscle cells from dog colon. This project proposes that the changes occurring in a sub-set of colonic disease, are in large part, specific to the muscularis. The working hypothesis that will be tested is that the targets for catecholamines in colonic circular muscle are, in addition to beta2-adrenergic receptors, two distinct alpha-adrenergic receptors located in two distinct cellular compartments of smooth muscle. Furthermore, the two compartments are interactive. The alpha1 receptor, when stimulated, augments acetylcholine induced contractions by activating phospholipase C through a GTP-binding protein leading to the formation of inositol trisphosphate. The alpha2 receptor also contributes to a contractile response in the muscle by decreasing the beta-adrenergic receptor mediated increase in cyclic AMP formation through a GTP-dependent inhibition of adenylate cyclase and y activation of aracadonate formation secondary to accelerate Na+/H+ exchange. The plan to accomplish the stated goals o the research is; 1) to isolate smooth muscle cells from regions of doe proximal colon circular muscle and study them in suspensions and primary culture: 2) to conduct direct radioligand binding experiments that will establish or refute the presence of the proposed cadre of receptors on these cells and measure their numbers and regulation of agonist affinity; 3) to determine the molecular consequences of stimulation of these receptors by determining what inositol phosphate metabolites are formed when alpha1 and alpha 2 receptors are stimulated, and whether these patterns are additive or distinct from the muscarinic receptor mediated changes in the same smooth muscle cells; and 4) whether the stimulation of the alpha2 receptor on the smooth muscle cell is coupled to the activation of phospholipase A2 and aracadonate release through acceleration of Na+/H= exchange; and 5) in order to establish the role of these second messengers in the physiology of smooth muscle cells, Digital Imaging Microscopy using two technologically advanced systems (Meridian ACAS 470 and Tracor Fluoroplex III) will be conducted using intracellular dyes for Ca+2 (Fura 2 and INDO 1) and pH (BCECF) in individual cells. Because the details of the coupling of these receptors to generation of cellular messengers, such as cyclic sugar phosphates and aracadonate metabolites, will be explored in colonic smooth muscle, the research is likely to yield unique information about smooth muscle in general as well as information about the colonic alpha1 and alpha2 receptors that have not been discovered previously. Finally, where available, the project will study normal human colonic muscle and the changes that occur in disease.
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