The mechanism of NH4+ transport in inner medulla is not known. The purpose of these experiments was to study the process that is involved in ammonium (NH4+) transport in cultured inner medullary collecting duct (mIMCD-3) cells. Cells grown on coverslips were exposed to NH4+ 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 NH4+ transport. The rate of 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 NH4+ 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 NH4+-induced cell acidification, indicating lack of involvement of Na+:K+-ATPase, Na+:K+:2Cl(-) transport, Na+:H+ exchange, K+ channel, or transport. The NH4+-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 similar to 0.3 pH unit in the presence but not the absence of NH4+. This K+ efflux-induced entry increased by extracellular NH4+ in a saturable manner with a Km of similar to 5 mM, blocked by increasing extracellular K+ and was not inhibited by barium. The K+ efflux-coupled NH4+ entry was electroneutral as monitored by the use of cell membrane potential probe 3,3'-dipropylthiadicarbocyanine. These results are consistent with the exchange ofinternal K+ with external NH4+ in a 1:1 ratio. The K+-NH4+ 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 NH4+-carrying transporters. We conclude that a unique transporter, a potassium-ammonium (K+/NH4+) antiport, is responsible for NH4+ transport in renal inner medullary collecting duct cells. This antiporter is sensitive to verapamil and Schering 28080, is electroneutral, and is selective for NH4+ as substrates. The K+/NH4+ antiporter may play a significant role in acid-base regulation by excretion of ammonium and elimination of acid.
