NO 3 2-induced pH Changes in Mammalian Cells Evidence for an NO 3 2H 1 Cotransporter

The effect of NO 3 2 on intracellular pH (pH i ) was assessed microfluorimetrically in mammalian cells in culture. In cells of human, hamster, and murine origin addition of extracellular NO 3 2 induced an intracellular acidification. This acidification was eliminated when the cytosolic pH was clamped using ionophores or by perfusing the cytosol with highly buffered solutions using patch-pipettes, ruling out spectroscopic artifacts. The NO 3 2 induced pH change was not due to modulation of Na 1 /H 1 exchange, since it was also observed in Na 1 /H 1 antiport-deficient mutants. Though NO 3 2 is known to inhibit vacuolar-type (V) H 1 -ATPases, this effect was not responsible for the acidification since it persisted in the presence of the potent V-ATPase inhibitor bafilomycin A 1 . NO 3 2 /HCO 3 2 exchange as the underlying mechanism was ruled out because acidification occurred despite nominal removal of HCO 3 2 , despite inhibition of the anion exchanger with disulfonic stilbenes and in HEK 293 cells, which seemingly lack anion exchangers (Lee, B.S., R.B. Gunn, and R.R. Kopito. 1991. J. Biol. Chem. 266:11448– 11454). Accumulation of intracellular NO 3 2 , measured by the Greiss method after reduction to NO 2 2 , indicated that the anion is translocated into the cells along with the movement of acid equivalents. The simplest model to explain these observations is the cotransport of NO 3 2 with H 1 (or the equivalent counter-transport of NO 3 2 for OH 2 ). The transporter appears to be bi-directional, operating in the forward as well as reverse directions. A rough estimate of the fluxes of NO 3 2 and acid equivalents suggests a one-to-one stoichiometry. Accordingly, the rate of transport was unaffected by sizable changes in transmembrane potential. The cytosolic acidification was a saturable function of the extracellular concentration of NO 3 2 and was accentuated by acidification of the extracellular space. The putative NO 3 2 -H 1 cotransport was inhibited markedly by ethacrynic acid and by a -cyano-4-hydroxycinnamate, but only marginally by 4,4 9 -diisothiocyanostilbene-2,2 9 disulfonate or by p -chloromercuribenzene sulfonate. The transporter responsible for NO 3 2 -induced pH changes in mammalian cells may be related, though not identical, to the NO 3 2 -H 1 cotransporter described in Arabidopsis and Aspergillus . The mammalian cotransporter may be important in eliminating the products of NO metabolism, particularly in cells that generate vast amounts of this messenger. By cotransporting NO 3 2 with H 1 the cells would additionally eliminate acid equivalents from activated cells that are metabolizing actively, without added energetic investment and with minimal disruption of the transmembrane potential, inasmuch as the cotransporter is likely electroneutral. key words: nitrate • proton • ion transport • pH regulation i n t r o d u c t i o n Nitric oxide has been recently recognized as an important second messenger in a variety of cell types. The generation of NO from l -arginine is catalyzed by NOsynthase in response to a number of stimuli including bacterial lipopolysaccharide, tumor necrosis factora , and g -interferon (see Kiechle and Malinski, 1993; Anggard, 1994; Weinberg et al., 1995 for review). NO is an unstable intermediate compound which, in aerobic aqueous solutions such as the cytosol, is rapidly metabolized primarily to nitrite (NO 2 2 ) and to a lesser extent to nitrate (NO 3 2 ). In the presence of oxidizing species such as oxyhemoproteins, NO 2 2 is rapidly converted to the more stable NO 3 2 (Ignarro et al., 1993; Veszelovszky et al., 1995). Lipopolysaccharide and cytokine-induced formation of NO, NO 2 2 , and NO 3 2 in vitro has been extensively reported in neutrophils, primary macrophages, and monocyte/macrophage cell lines (Iyengar et al., 1987; Miwa et al., 1987; Schmidt et al., 1989; Wright et al., 1989). In animal models, increased production of NO 3 2 has been reported in sepsis (Ohshima et al., 1994; Oudenhoven et al., 1994; Wright et al., 1992), glomerulonephritis (Sever et al., 1992), and graft rejection (Tanaka et al., 1995; Winlaw et al., 1994; Drobyski et al., 1994). In humans, acute graft vs. host disease, bacterial and viral meningitis, acute gastroenteritis, and sepsis have been associated with increased NO 3 2 production (Jungersten et al., 1993; Shi et al., 1993; Milstien et al., 1994; Weiss et al., 1995). Address correspondence to Dr. Sergio Grinstein, Division of Cell Biology, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada. Fax: 416-813-5028; E-mail: sga@resunix.ri. sickkids.on. ca