As the first and rate limiting enzyme of the renin-angiotensin system, renin has an important role in the control of cardiovascular homeostasis. Definition of the molecular mechanisms regulating renin gene expression and the subsequent intracellular events of synthesis, sorting, trafficking, processing, storage and finally release of the active renin is essential for understanding the renin-angiotensin system. Most of the molecular mechanisms regulating intracellular handling of renin proximal to its secretion site remain to be defined. In the normal adult kidney, renin is synthesized by the juxtaglomerular (JG) cells. However, during early development, renin is expressed in arcuate and interlobular arteries. If an adult animal is subjected to converting enzyme inhibition, there is a recruitment of renin gene expressing and releasing cells along the preglomerular vessels, resembling the situation in the immature animal. We hypothesize that the ability of smooth muscle cells (SMCs) from the renal arterial tree to turn on and off the renin gene is mediated by tissue specific transecting factors binding to the 5' flanking region of the renin gene. We propose to isolate and characterize those factors and to identify the Cis regulatory sequences involved in the response. Co-transfection studies of native and mutated renin promoter expression constructs with transecting factors will allow us to assess the functional significance of sequences involved. The molecular mechanisms responsible for the intracellular processing of renin have not been investigated in detail. Mutations of the pre-, or pro- and renin sequences of the renin gene will be produced and introduced into AT-T20 cells and, using EM microscopy, the intracellular trafficking or renin will be investigated. These studies will determine whether specific sequences within the renin gene are responsible for intracellular sorting of the renin molecule. Similarly, using antibodies directed against specific domains (pre, pro, renin) of the molecule, we will determine the specific organelles where renin is processed. Additional studies will examine the relationship of renin gene transcription (measured by intervening sequence probes) to renin release from individual cells detected by the reverse hemolytic plaque assay. The cell membrane and cytosolic events leading to renin release will be investigated in response to angiotensin II, beta-receptor agonists and antagonists and changes in the calcium concentration. The proposed experiments will increase our understanding of the molecular processes regulating renin biosynthesis, trafficking and release.
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