D. Kikeri

Atrial natriuretic peptides inhibit conductive sodium uptake by rabbit inner medullary collecting duct cells.

The inner medullary collecting duct (IMCD) effects net sodium reabsorption under the control of volume regulatory hormones, including atrial natriuretic peptides (ANP). These studies examined the mechanisms of sodium transport and its regulation by ANP in fresh suspensions of IMCD cells. Sodium uptake was inhibited by amiloride but insensitive to furosemide, bu-metanide, and hydrochlorthiazide. These results are consistent with uptake mediated by a sodium channel or Na+/H+ exchange. To determine the role of sodium channels, cells were hyperpolarized by preincubation in high potassium medium followed by dilution into potassium-free medium. Membrane potential measurements using the cyanine dye, Di(S)-C3-5 verified a striking hyperpolarization of IMCD cells using this protocol. Hyperpolarization increased the apparent initial rate of sodium uptake fourfold. Amiloride and ANP inhibited potential-stimulated sodium uptake 73% and 65%, respectively; the two agents together were not additive. Addition of 5 mM sodium to hyperpolarized cells resulted in a significant amiloride-sensitive depolarization. Half-maximal inhibition of potential-driven sodium uptake occurred at 3 X 10(-7) M amiloride, and 5 X 10(-11) M ANP. We conclude that sodium enters IMCD cells via a conductive, amiloride-sensitive sodium channel, which is regulated by ANP. ANP inhibition of luminal sodium entry in the IMCD appears to contribute to the marked natriuretic effect of this hormone in vivo.

Mechanism of pH‐Independent Transport of NH+4 by Mouse Renal Medullary Thick Limbs

The medullary thick ascending limb of Henle (MAL) actively absorbs ammonium in the absence of a favorable transepithelial pH gradient.I4 To define the mechanisms involved in pH-independent transport of NH:, intracellular pH (pHi) was measured using BCECF in both the isolated perfused mouse MAL (IP-MAL) and suspensions of mouse MAL tubules (S-MAL). In nonbicarbonate HEPES-buffered Ringer’s solution pH 7.4, the steady-state pH, was 7.16 ? 0.04 in IP-MAL ( n = 10) and 7.43 ? 0.01 in S-MAL ( n = 12). In IP-MAL, the addition of 20 mM NH4CI to the medium bathing the luminal surface resulted in a striking acidification of pHi to 6.43 2 0.08, whereas the addition of 20 mM NH4CI to the medium bathing the basolateral side of the tubule led to rapid alkalinization of pHi from 7.09 ? 0.08 to 7.4 ? 0.16. Thus the basolateral membranes of MAL cells are highly NH3 permeable, while apical membranes are highly NH; permeable. The addition of NH4C1 in concentrations greater than 0.5 mM to the media bathing SMAL or tlo both apical and basolateral media bathing IP-MAL led to significant acidification of pHi, indicating that overall cell membrane NH; entry was greater than entry of NH3. NH,Cl-induced acidification was also observed in S-MAL in the preserice of COJHCOT-buffered media, and in HEPES-buffered media, the stilbene DIDS (0.2-0.5 mM) did not inhibit or attenuate the NH4C1-induced acidification response. Thus, bicarbonate exit pathways do not participate in the acidification response. In IP-MAL, the acidification response to luminal NH4CI was completely inhibited by the combination of 10 mM BaCI2 and 0.1 mM furosemide, whereas in SMAL either Ba2+ or bumetanide (0.1 mM) partially inhibited ammonium-induced acidification, indicating that both apical K+ channels and Na : K : 2C1 cotransporters mediate luminal NH: entry into MAL cells; however, the major fraction of NH; entry was via the Ba2+-sensitive pathway. The decrement in pHi observed with the luminal 20 mM NH: addition in IP-MAL was approximately two times greater than that in S-MAL at the same NH4Cl concentration. This was the result of a very low permeability of the apical cell membrane to NH3 as demonstrated by the lack of cell alkalinization in IP-MAL with luminal addition of NH4Cl when NH: entry pathways were completely blocked. Recovery of pHi in S-MAL following NH; withdrawal was inhibited more than 85% by either sodium removal or amiloride (0.1 mM), but not by Ba2+ or furosemide. Thus, NH: exit from MAL cells requires Na : H exchange. Moreover, because this cation cannot exit cells by K+ channels or Na : K : 2C1 cotransport, NH; transport by these pathways is apparently rectified (i.e., they can mediate the entry, but not exit, of NH:). The Na : H exchanger required for NH: exit was localized to the apical plasma membrane in the IP-MAL; apical but not basolateral amiloride inhibited pH, recovery after NH: withdrawal.