Available evidence indicates that there are two types of Na(+)-H+ exchangers, type A (housekeeping type) and type B (epithelial or apical type), in mammalian cells. We have recently reported, using isolated membrane vesicles, that these two types can be differentiated by their relative sensitivities to inhibition by clonidine and cimetidine [Kulanthaivel, Leibach, Mahesh, Cragoe & Ganapathy (1990) J. Biol. Chem. 264, 1249-1252]. The present study was undertaken to determine whether this approach is also effective in cultured cells. The JAR human placental choriocarcinoma cell line and the opossum kidney (OK) cell line, when grown as confluent monolayer cultures on an impermeable plastic support, express Na(+)-H+ exchanger activity which is measurable by determining Na+ uptake into the cells from the culture medium. The JAR cell Na(+)-H+ exchanger was found to be about 100 times more sensitive to inhibition by dimethylamiloride than the OK cell Na(+)-H+ exchanger. Inhibition studies with clonidine and cimetidine were able to differentiate between these two exchangers very clearly. Cimetidine was 18 times more potent than clonidine in inhibiting the JAR cell Na(+)-H+ exchanger. In contrast, clonidine was at least 8 times more potent than cimetidine in inhibiting the Na(+)-H+ exchanger of the OK cell. The results show that the JAR cell expresses the type A Na(+)-H+ exchanger, whereas the OK cell expresses the type B Na(+)-H+ exchanger. This approach also proved to be very effective in correctly identifying the type of Na(+)-H+ exchanger in a third cell line (HeLa). It is concluded that the relative susceptibility to inhibition by clonidine and cimetidine offers an easy and efficient means of differentiating between the two types of Na(+)-H+ exchangers in cultured cells.

The Na+ content of poliovirus-infected HeLa S3 cells increased during the late phase of virus replication, after virus inhibition of host cell protein synthesis and in coincidence with late viral functions. Guanidine hydrochloride blocked the rise in Na+ content, whereas the antiguanidine agent choline fully reversed the guanidine block. Expression of one or more late viral functions was essential for Na+ accumulation to occur because accumulation was inhibited by cycloheximide or guanidine added to the infected culture during the late phase. Increased permeability of infected cell membrane rather than inhibition of cellular Na+-K+ adenosine triphosphatase activity appears to be primarily responsible for Na+ accumulation by virus-infected cells.

During the early period after poliovirus infection of HeLa cells, cellular Na+/K+ ATPase activity is transiently activated. We investigated the possibility that Na+/K+ ATPase activation is a consequence of Na+/H+ antiporter activation. Increased uptake of the weak organic acid 5,5-dimethyloxazolidine-2,4-dione by infected cells around 2 h after infection suggested cytoplasmic alkalinization equivalent to pH 7.7 during the biosynthetic phase of viral replication. Consistent with the involvement of Na+/H+ antiporter activation in this phenomenon, it was found to be [Na+]-dependent and inhibited by 5-(N-ethyl-N-isopropyl)amiloride (EIPA). However, the pH increase was not associated with an increase in amiloride-sensitive Na+ uptake by infected cells predicted by this mechanism. By contrast, the alkalinization could be abolished with the anion-exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), implicating an anion-exchange mechanism, such as Cl-/HCO3- exchange, in this process. In addition to abolishing virus-induced intracellular alkalinization, both EIPA and DIDS moderately inhibited viral replication. Manipulation of intracellular pH with nigericin in the incubation medium revealed that maximum viral replication required a pH of about 7.7 and that replication was significantly inhibited even at pH 7.3. Thus, the pH increase in infected cells appeared to be physiologically relevant. These findings represent the first demonstration of a biologically meaningful pH increase in cells infected with a lytic virus.

To better understand the significance of 22Na' accumulation by poliovirusinfected HeLa cells (C. N. Nair, J. W. Stowers, and B. Singfield, J. Virol. 31:184, 1979), measurements of cellular Na+, K+, and Clcontents, volume, and density were carried out at intervals after infection. In addition, the rates of 'Na'

Human rhinovirus type 14 contained polyadenylated RNA. Virus growth in HeLa cells was inhibited by cordycepin or polyuridilic acid and stimulated by polyadenylic acid. Polyadenylic acid also reversed cordycepin inhibition of virus-induced cytopathology of infected HeLa cells.