The first step in active transepithelial Na+ transport across the mammalian cortical collecting tubule (CCT) is mediated by the amiloride- sensitive, high selectivity Na+ channel in the apical (luminal) membrane. This Na+ channel is the primary site for mineralcorticoid action, and as such provides discretionary control of total body Na+ homeostasis. Therefore, investigating this channel protein's regulatory mechanisms is essential to our understanding of the physiology of fluid and electrolyte balance. The mechanisms for controlling Na+ reabsorption can be thought of as forming a threshold hierarchy. The first level involves systemic hormonal modulation at an organismic level. Hormonal agents interact with specific receptors at the basolateral (serosal) cell surface to activate the second level of regulation. Intracellular second messenger cascades transduce the hormonal signal from the basolateral to the apical membrane where the primary control of Na+ transport occurs. The third level involves molecular events confined to the local microenvironment of the apical membrane itself. Membrane-bound regulatory elements, activated by cystolic second messengers, directly modulate the activity of apical amiloride-sensitive, high selectivity Na+ channel molecules. Unfortunately, the geometry and morphology of renal tubules makes unequivocal studies of the ion channels responsible for Na+ transport difficult. Fortunately, patch clamp techniques allow resolution of the picoampere currents conducted by individual Na+ channels. """"""""Excised"""""""" patches allow direct access to the intracellular surface of the apical membrane. Thus, it is now possible for us to study the modulation of individual Na+ channels at the proximal site of regulation; that is, apical membrane deliminated events. Likely candidates for such local control include apical membrane phospholipid metabolites such as eicosanoids from phospholipase A2, or inositol polyphosphates and diacylglycerol from phospholipase C. These metabolites could modulate Na+ channels directly or indirectly via activation of other membrane- associated regulatory components (ie., protein kinase C or guanine nucleotide-binding proteins). Despite the large body of literature describing the properties of macroscopic Na+ reabsorption in isolated perfused rabbit distal nephron, previous patch clamp studies of isolated and cultured rabbit CCT have failed to identify the amiloride-inhibitable, highly selective Na+ channels implied from the results of whole tissue measurements and predicted by the Koeford-Johnsen and Ussing model of transepithelial Na+ transport. Based on our experience with the A6 amphibian distal nephron cell line we felt that rabbit CCT primary cultures, grown on permeable supports in the presence of aldosterone, might provide an excellent model for studying apical membrane-deliminated regulation of Na+ selective channels.
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