The juxtaglomerular apparatus (JGA) of the mammalian kidney is an anatomic structure which connects the macula densa cells of the distal nephron with the glomerular vascular pole. It has been established that changes in NaCl concentration at the macula densa cells initiate a chain of events which alters afferent arteriolar tone and renin secretion. In order to better understand possible interactions between the different cellular components of the JGA the intrinsic properties of these cells need to be known. In its first part this proposal aims at investigating the secretory and contractile responses of afferent arteriolar cells to angiotensin II and adenosine, two agents which exert profound modulating effects at the level of the JGA and which have been noted to affect preglomerular vessels in a synergistic fashion. The second part of this application will utilize a highly sensitive technique for assessment of mRNA levels to study regulation of gene expression in cells of the JGA.
The specific aims of part 1 are to describe the extent of the mutual dependency of the vasoconstrictor effects of angiotensin and adenosine and to understand the mechanisms for the synergism. The degree of synergism will be studied by examining a) whether and to what extent the vasoconstriction and the inhibition of renin secretion produced by adenosine is potentiated by angiotensin II and inhibited by angiotensin II antagonists, and conversely, whether and to what extent the same responses produced by angiotensin II are potentiated by adenosine and inhibited by adenosine antagonists, and b) whether the afferent constriction and inhibition of renin release elicited by increased NaCl at the macula densa depend upon the availability of both agonists. The mechanisms of the synergistic interaction will be studied by examining a) a possible facilitating interaction at the level of the respective receptors, b) a possible potentiating effect in generating modulating phosphoinositide metabolites, c) a possible potentiating effect on Ca release from intracellular stores or Ca entry, d) effects on transmembrane ion currents which enhance cell activation, e) effects on intracellular levels of cyclic nucleotides, and f) effects on the generation of phospholipase A dependent products.
A specific aims of part 2 are to use the polymerase chain reaction (PCR) to assess mRNA levels in single JGA preparations. In these studies we will a) optimize techniques for monitoring recovery or amplification efficiency at each step of the PCR method applied to single isolated JGA specimen, b) assess the response pattern of mRNA for a set of housekeeping genes and define the response of these mRNA species to manipulations of the renin- angiotensin system, c) study the effect of angiotensin II, adenosine, and cyclic nucleotides on renin mRNA in single JGA, and d) examine the role of glucose-6-phosphate dehydrogenase in macula densa cells by assessing in parallel the levels of renin and G6PDH mRNA in the JGA.
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