K +/NH 4+ antiporter: a unique ammonium carrying transporter in the kidney inner medulla

Abstract

The mechanism of NH 4 + transport in inner medulla is not known. The purpose of these experiments was to study the process that is involved in ammonium (NH 4 +) transport in cultured inner medullary collecting duct (mIMCD-3) cells. Cells grown on coverslips were exposed to NH 4 + and monitored for pH i changes by the use of the pH-sensitive dye BCECF. The rate of cell acidification following the initial cell alkalinization was measured as an index of NH 4 + transport. The rate of NH 4 + transport was the same in the presence or absence of sodium in the media (0.052±0.003 vs 0.048±0.004 pH/min, P>0.05), indicating that NH 4 + entry into the cells was independent of sodium. The presence of ouabain, bumetanide, amiloride, barium, or 4,4′-di-isothiocyanostilbene-2-2′-disulfonic acid (DIDS) did not block the NH 4 +-induced cell acidification, indicating lack of involvement of Na +:K +-ATPase, Na +:K +:2 Cl − transport, Na +:H + exchange, K + channel, or Cl −/base exchange, respectively, in NH 4 + transport. The NH 4 +-induced cell acidification was significantly inhibited in the presence of high external [K +] as compared to low external [K +] (0.018±0.001 vs. 0.049±0.003 pH/min for 140 mM K + vs. 1.8 mM K + in the media, respectively, P<0.001). Inducing K + efflux by imposing an outward K + gradient caused intracellular acidification by ∼0.3 pH unit in the presence but not the absence of NH 4 +. This K + efflux-induced NH 4 + entry increased by extracellular NH 4 + in a saturable manner with a Km of ∼5 mM, blocked by increasing extracellular K + and was not inhibited by barium. The K + efflux-coupled NH 4 + entry was electroneutral as monitored by the use of cell membrane potential probe 3,3′-dipropylthiadicarbocyanine. These results are consistent with the exchange of internal K + with external NH 4 + in a 1:1 ratio. The K +-NH 4 + antiporter was inhibited by verapamil and Schering 28080 in a dose-dependent manner, was able to work in reverse mode, and did not show any affinity for H + as a substrate, indicating that it is distinct from other NH 4 +-carrying transporters. We conclude that a unique transporter, a potassium-ammonium (K +/NH 4 +) antiport, is responsible for NH 4 + transport in renal inner medullary collecting duct cells. This antiporter is sensitive to verapamil and Schering 28080, is electroneutral, and is selective for NH 4 + and K + as substrates. The K +/NH 4 + antiporter may play a significant role in acid-base regulation by excretion of ammonium and elimination of acid.