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In vitro experiments showed that the connecting tubule (CNT) sends a signal that dilates the afferent arteriole (Af-Art) when Na+ reabsorption in the CNT lumen increases. We call this process CNT glomerular feedback (CTGF) to differentiate it from tubuloglomerular feedback (TGF), which is a cross talk between the macula densa (MD) and the Af-Art. In TGF, the MD signals the Af-Art to constrict when NaCl transport by the MD is enhanced by increased luminal NaCl. CTGF is mediated by CNT Na+ transport via epithelial Na+ channels (ENaC). However, we do not know whether CTGF occurs in vivo or whether it opposes the increase in Af-Art resistance caused by TGF. We hypothesized that CTGF occurs in vivo and opposes TGF. To test our hypothesis, we conducted in vivo micropuncture of individual rat nephrons, measuring stop-flow pressure (PSF) as an index of glomerular filtration pressure. To test whether activation of CTGF opposes TGF, we used benzamil to block CNT Na+ transport and thus CTGF. CTGF inhibition with the ENaC blocker benzamil (1 μM) potentiated the decrease in PSF at 40 and 80 nl/min. Next, we tested whether we could augment CTGF by inhibiting NaCl reabsorption in the distal convoluted tubule with hydrochlorothiazide (HCTZ, 1 mM) to enhance NaCl delivery to the CNT. In the presence of HCTZ, benzamil potentiated the decrease in PSF at 20, 40, and 80 nl/min. We concluded that in vivo CTGF occurs and opposes the vasoconstrictor effect of TGF. [ABSTRACT FROM AUTHOR]

Phenamil, an analog of amiloride, has previously been shown to bind specifically to sodium channels in toad bladder (J.L. Garvin et al., J. Membrane Biol. 87:45-54, 1985). In this paper, 3H-phenamil was used to measure sodium channel density in both isolated epithelial cells and intact bladders. From the specific binding to intact bladders, a channel density of 455 +/- 102 channels/micron2 was calculated. No correlation between specific binding and the magnitude of irreversible inhibition of short-circuit current was found. Pretreatment of intact bladders with 1 mg/ml trypsin reduced specific binding to isolated cells by 82 +/- 5%. In isolated cells, neither aldosterone nor vasopressin had any significant effect on specific phenamil binding. It is inferred that phenamil binds to both open and closed channels which may be either in the mucosal membrane or in the submembrane space. Finally, and rather surprisingly, we found that 3H-phenamil binds irreversibly to the basolateral membrane at concentrations as low as 4 X 10(-7) M. Therefore, care must be used in interpreting binding studies with amiloride or its analog at such concentrations. [ABSTRACT FROM AUTHOR]

Several new amiloride analogues and two reported photoaffinity analogues were tested for irreversible inhibition of short-circuit current, Isc, in toad bladder. Bromoamiloride, a photoaffinity analogue, induced 40% irreversible inhibition at 500 microM after irradiation with ultraviolet light greater than or equal to 320 nm. Iodoamiloride caused no irreversible inhibition. Of the new analogues tested, only 3,5-diamino-6-chloro-N-[(phenylamino) amino-methylene] pyrazinecarboxamide, phenamil, irreversibly inhibited Isc at concentrations of 0.05 to 5 microM when added to the mucosal solution. Irreversible inhibition of Isc by phenamil may be attributed to specific blockage of the mucosal sodium channels, which depended on: 1) time of exposure; 2) mucosal pH; 3) mucosal sodium concentration. For example, 5 microM phenamil irreversibly inhibited Isc by 38% in 103 mM Na at pH 8.6 and nearly 75% in 30 mM Na at pH 6.4 after a 40-min exposure. Irreversible inhibition occurred in two phases with time constants of less than or equal to 10 min and approximately 140 min. Due to its irreversible nature, phenamil may be used to measure channel density. [ABSTRACT FROM AUTHOR]