It recently has been shown that epithelial Na+ channels are controlled by a receptor for intracellular Na+ a G protein (Go) and a ubiquitin-protein ligase (Nedd4). by raised [Na+]i acting via a Na+ Veliparib receptor and Go. This inhibition entails ubiquitination but does not involve the ubiquitin protein ligase Nedd4. We conclude that control of membrane transport systems by intracellular Na+ receptors may provide a general mechanism for regulating intracellular Na+ concentration. oocytes is usually lost when the expressed channels contain mutations known to cause the autosomal dominant form of hypertension Liddle’s syndrome Pdpn (13). The mechanisms by which intracellular Na+ acts in these systems are however not yet known (12 14 These findings suggest that opinions control by intracellular Na+ of epithelial Na+ channels may be a phenomenon of general physiological significance in absorptive epithelia and they raise the question of whether other epithelial Na+ transport systems also might be controlled by a similar mechanism. One Na+-dependent transporter that could be expected to be subject to opinions regulation by intracellular Na+ is the Na+-H+ exchanger in the secretory (endpiece) cells of salivary glands. The endpieces of salivary glands secrete Na+ Cl? and HCO3? by a mechanism relying on the transport of Na+ across the basolateral membrane by transporters such as Na+-H+ exchangers and Na+-K+-2Cl? and Na+-HCO3? cotransporters (15-18). The onset of secretion by salivary endpiece cells is usually accompanied by a dramatic increase in the activity of these transporters (15-17 19 and at maximum secretory rates the intracellular Na+ content in the secretory cells can be calculated to turn over every 15 sec (18). It is clear that to maintain a relatively stable intracellular composition during secretion requires that these basolateral Na+-dependent transporters be subject to opinions regulation and in fact intracellular Na+ concentration has been observed to oscillate during secretion in a manner suggestive of the presence of such a opinions mechanism (15). Nevertheless despite the considerable work that has been done around the mechanisms that activate the basolateral transporters at the onset of salivary secretion (19-25) no work has been carried out on these hypothetical inhibitory opinions systems. In the present Veliparib paper we investigate whether the Na+-H+ exchanger in the secretory cells of the mouse mandibular Veliparib gland is usually subject to opinions regulation by intracellular Na+. MATERIALS AND METHODS Cell Preparation. Male Quackenbush strain mice were killed by cervical dislocation and the mandibular glands were removed finely minced and incubated for 12 min in a physiological salt answer made up of 1 mg/ml collagenase (Worthington type IV). The cell suspension then was dispersed by trituration and washed with new Na+-rich bath answer made up of 145 mM NaCl 5.5 mM KCl 1.2 mM MgCl2 7.5 mM Na-Hepes 7.5 mM H-Hepes 1 mM CaCl2 and 10 mM glucose; the pH was adjusted to 7.4 with NaOH. The cells were filtered through a 75-μm nylon mesh and kept on ice until required. Veliparib Patch-Clamp Techniques. We used a technique based on that of Demaurex and coworkers (26) in which the whole-cell patch-clamp technique is used to control cytosolic composition while the pH-sensitive dye BCECF [2′ 7 All experiments were performed at 22°C. RESULTS Veliparib AND Conversation We used a technique explained by Demaurex and coworkers (26) in which the whole-cell configuration of the patch-clamp technique is used to control cytosolic composition while the pH-sensitive dye BCECF is used to measure pHi. The cells were bathed initially in a zero Na+ answer so that they would be unable to oppose the acid load imposed by the pipette answer. The bath answer then was changed to one made up of 155 mM Na+ so as to activate the Na+-H+ exchanger and cause pHi to recover toward normal levels (Fig. ?(Fig.11and.