In vivo PTH provokes apical NHE3 and NaPi2 redistribution and Na-K-ATPase inhibition

and Alicia A. McDonough. In vivo PTH provokes apical NHE3 and NaPi2 redistribution and Na-K-ATPase inhibition. Am. J. Physiol. 276 ( Renal Physiol. 45): F711–F719, 1999. —The aim of this study was to test the hypothesis that in vivo adminis-tration of parathyroid hormone (PTH) provokes diuresis/ natriuresis through redistribution of proximal tubule apical sodium cotransporters (NHE3 and NaPi2) to internal stores and inhibition of basolateral Na-K-ATPase activity and to determine whether the same cellular signals drive the changes in apical and basolateral transporters. PTH-(1–34) (20 U), which couples to adenylate cyclase (AC), phospholipase C (PLC), and phospholipaseA 2 (PLA 2 ), or [Nle 8,18 ,Tyr 34 ]PTH-(3– 34) (10 U), which couples to PLC and PLA 2 but not AC, were given to anesthetized rats as an intravenous bolus followed by low-dose infusion (1 U·kg 2 1 ·min 2 1 for 1 h). Renal cortex membranes were fractionated on sorbitol density gradients. urine and lithium clearance (C Li ) 2.8 6 0.3-fold. With this diuresis/natriuresis, 25% of NHE3 and 18% of NaPi2 immunoreactivity redistributed from apical membranes to higher density fractions containing intracellular membrane markers, and basolateral Na-K-ATPase activity decreased 25%. [Nle 8,18 ,Tyr 34 ]PTH-(3–34) failed to increase V or C Li or to provoke redistribution of NHE3 or NaPi2, but it did inhibit Na-K-ATPase activity 25%. We conclude that in vivo PTH stimulates natriuresis/diuresis associated with internalization of apical NHE3 and NaPi2 and inhibition of Na-K-ATPase activity, that cAMP-protein kinase A stimulation is necessary for the natriuresis/diuresis and NHE3 and NaPi2 internalization, and that Na-K-ATPase inhibition is not secondary to depressed apical Na 1 transport. detection of the Na/H exchanger NHE3 isoform, monoclonal cell culture superna- tant (19F5), generously provided by D. Biemesderfer and P. Aronson (Yale) (4), was used without dilution on blots and detected with an ECL enhanced chemiluminescence kit from Amersham. For all blots, autoradiographic signals were quantiﬁed with a Bio-Rad Imaging Densitometer with Molecular Analyst software. Multiple exposures of autoradiograms were analyzed to ensure that signals were within the linear range of the ﬁlm. Quantitation and statistical analysis. Data are expressed as means 6 SE. ANOVA was applied to determine whether there was a signiﬁcant effect of treatment on the overall fractionation pattern of a given parameter. Treatment was one repeated factor, and fraction was another repeated factor. If the interaction between treatment and fraction was found to be signiﬁcant ( P , 0.05), then it was concluded that the treatment had a signiﬁcant effect. If so, the location of the difference in the pattern was assessed by one-tailed Student’s t -test for paired samples, and differences were regarded as signiﬁcant at P , 0.05, after applying an adjustment for multiple comparisons. A one-tailed t -test was applied in all cases, because we were testing the hypotheses that there was an increase in urine production and lithium clearance and a decrease in sodium transporter activity and/or abundance in surface membranes with PTH treatment.