This proposal will investigate a potential regulating mechanism of the physiological activity of vasoactive peptides that contain proline residues. Prolyl-peptide bonds exist in two interconverting configurations, i.e, as cis and trans isomers. Converting enzymes, peptidases and receptors have been shown to have isomeric preferences for peptides in in vitro studies; for example, several peptidases preferentially cleave trans isomers of prolyl-peptides bonds. Nevertheless, there have been few studies of the influence of cis and trans isomers of prolyl-peptide bonds on metabolism or activity of vasoactive peptides in biological systems. Therefore, the proposed study will examine the preferences of lung peptidases and receptors for cis and trans isomers of proline containing vasoactive peptides in vitro and in the intact perfused lung. The lung is an ideal model system for these studies. It is the most efficient fixed enzyme reactor known, capable of metabolizing physiological concentrations of many peptides in a single pass through the capillary bed. Since cis-trans isomerization is significantly slower (tens to hundreds of seconds) that the time course of a single pass through the lungs (two to three seconds), isomeric specificities of lung peptidases are readily apparent under the appropriate conditions. Whether lung peptidases are specific for cis or trans isomers of different vasoactive peptides will be determined from progress curves for peptide metabolism in the lung and in vitro. The data from theses curves will be interpreted using mathematical models that represent hypothesized kinetic processes. Preferential metabolism of trans isomers of peptides in the lung causes the venous effluent to be highly enriched for cis isomers. We will take advantage of this bioreactor system to study the isomeric preferences of receptor activation by vasoactive peptides. The importance of understanding the functional roles of cis and trans isomers of prolyl-peptide bonds in vasoactive peptides is highlighted by the recent discovery of a ubiquitous family of peptidyl-prolyl cis-trans isomerases. These enzymes, which are also the immunophilin binding proteins for the immunosuppressive drugs cyclosporine A and FK506, catalyze cis-trans isomerization of prolyl-peptide bonds. In our proposal, we will use these isomerases primarily as tools to study the isomeric specificities of lung peptidases and receptors for vasoactive peptides. Coincidently, our results may also provide insights into a mechanism of cardiovascular toxicity of cyclosporine A. In immunosuppression and inflammation, immunophilin activity may be compromised in such a way as to alter normal isomerization kinetics of vasoactive substances. This could lead to imbalances in the normal rates of metabolism or activation of these peptides.
