The longterm objective of the proposed research is to explore the possibility that the benzodiazepines (BZD) may exert direct tubular effects on the transport function of the thick ascending limb of Henle's loop (TALH), and to examine the cellular mechanisms of action of BZD. This is prompted by recent studies which revealed a major class of BZD binding sites that are broadly distributed in many non-neuronal tissues including the kidney (where they are predominantly localized to the TALH). The physiologic significance of these binding sites remains largely unknown but it has been proposed that they may represent functional receptors for interacting with putative BZD-like endogenous ligands which may then exert modulatory effects on cell function. Moreover, ligand interaction studies in some tissues suggest that these receptors may regualte calcium-ion gating across cells membranes. Furthermore, preliminary evidene suggests that BZD increases urinary sodium excretion, inhibit ouabain-sensitive oxygen consumption by medullary TALH and lower cytosolic calcium concentration. Studies are designed to assess the effects of BZD on TALH function and to explore the mode of action including the possibility that these effects are due to modulation of cell- calcium homeostasis. Several tools of investigation are applied in the study protocol. These include: 1) Monintoring oxygen consumption in cells or tubules from the TALH; 2) Isolated perfused tubules; 3) Clearance studies; 4) Measurement of net calcium tracer uptake by TALH cells; and 5) Monitoring cytosolic calcium concentration. In addition, the novel technique of immunodissection will be utilized to isolate a pure cell suspension of cortical TALH. Ouabain-sensitive oxygen consumption (an index of solute transport) and isolated perfused tubules will examine whether BZD-modulation of TALH transport activity is consisten with activation of non-neuronal BZD receptors. Clearance studies should provide the integrated response on whole kidney function. The monitoring cytosolic calcium concentration using fluorescent indicators and by measuring 45Ca-isotope influx and efflux across cells. Additional experiments will examine the possible sites of action at the membrane level and will assess any role of autacoid- mediation.
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