We examined the effect of shrinkage on Na(+)-H+ exchange in single muscle fibers at intracellular pH (pHi) values of 6.8, 7.2, and 7.6 using pH microelectrodes and internal dialysis. Under normotonic conditions (975 mosmol/kgH2O) at pHi 6.8, the amiloride-sensitive acid-extrusion rate (JAmil/s) averaged 17 microM/min. Exposure to hypertonic solutions (1,600 mosmol/kgH2O) increased JAmil/s to 304 microM/min at pHi 6.8. At pHi approximately 7.2 and 7.6, hypertonicity increased JAmil/s from approximately 0 to approximately 172 microM/min (pHi 7.2) and approximately 0 to approximately 90 microM/min (pHi 7.6). Thus, under normotonic conditions, Na(+)-H+ exchange activity is slight at pHi approximately 6.8 and virtually nil at higher pHi values. Shrinkage stimulated Na(+)-H+ exchange, more at low pHi values. We also examined the Cl- dependence of the Na(+)-H+ exchanger's response to shrinkage. Our results indicate that shrinkage-induced activation of Na(+)-H+ exchange requires Cl-, specifically intracellular Cl-. These results establish that shrinkage is both pHi dependent and requires intracellular Cl-.

In typical physiological solutions, CO2 is in equilibrium with HCO3- and H+ (CO2 + H2O<==>HCO3- +H+). Because one cannot independently alter CO2 and HCO3- concentrations and pH, it is impossible to distinguish between the effects of CO2 and HCO3- on physiological processes. Here we describe a continuous-flow, rapid-mixing approach for generating out-of-equilibrium CO2/HCO3- solutions with a physiological pH and CO2 (but little HCO3-), or pH and HCO3- (but little CO2). We have exploited these out-of-equilibrium solutions to introduce HCO3- exclusively to either the outside or inside of a squid giant axon, and verify the presence of a new K/HCO3 cotransporter. The out-of-equilibrium approach could be useful in a variety of applications for independently controlling CO2 and HCO3- concentrations and pH.

We used microelectrodes to monitor the recovery (i.e., decrease) of intracellular pH (pHi) after using internal dialysis to load squid giant axons with alkali to pHi values of 7.7, 8.0, or 8.3. The dialysis fluid (DF) contained 400 mM K + but was free of Na + and C1-. The artificial seawater (ASW) lacked Na +, K +, and CI-, thereby eliminating effects of known acid-base transporters on pHi. Under these conditions, halting dialysis unmasked a slow phi decrease caused at least in part by acid-base transport we refer to as "base effiux." Replacing K § in the DF with either NMDG § or TEA + significandy reduced base effiux and made membrane voltage (Vm) more positive. Base effiux in K+-dialyzed axons was stimulated by decreasing the pH of the ASW (pHo) from 8 to 7, implicating transport of acid or base. Although postdialysis acidifications also occurred in axons in which we replaced the K + in the DF with Li +, Na +, Rb +, or Cs +, only with Rb + was base efflux stimulated by low pHo. Thus, the base effiuxes supported by K § and Rb § appear to be unrelated mechanistically to those observed with Li § Na § or Cs § The combination of 437 mM K § and 12 mM HCO~ in the ASW, which eliminates the gradient favoring a hypothetical K+/HCO~ effiux, blocked pHi recovery in K+-dialyzed axons. However, the pHi recovery was not blocked by the combination of 437 mM Na +, veratridine, and CO2/HCO~ in the ASW, a treatment that inverts electrochemical gradients for H § and HCOg and would favor passive H § and HCOg fluxes that would have alkalinized the axon. Similarly, the recovery was not blocked by K § alone or HCOg alone in the ASW, nor was it inhibited by the K-H pump blocker Sch28080 nor by the Na-H exchange inhibitors amiloride and hexamethyleneamiloride. Our data suggest that a major component of base effiux in alkali-loaded axons cannot be explained by metabolism, a H + or HCO~ conductance, or by a K-H exchanger. However, this component could be mediated by a novel K/HCO~ cotransporter.

Many cells respond to shrinkage by stimulating specific ion transport processes (e.g., Na-H exchange). However, it is not known how the cell senses this volume change, nor how this signal is transduced to an ion transporter. We have studied the activation of Na-H exchange in internally dialyzed barnacle muscle fibers, measuring intracellular pH (pHi) with glass microelectrodes. When cells are dialyzed to a pHi of approximately 7.2, Na-H exchange is active only in shrunken cells. We found that the shrinkage-induced stimulation of Na-H exchange, elicited by increasing medium osmolality from 975 to 1,600 mosmol/kgH2O, is inhibited approximately 72% by including in the dialysis fluid 1 mM guanosine 5'-O-(2-thiodiphosphate). The latter is an antagonist of G protein activation. Even in unshrunken cells, Na-H exchange is activated by dialyzing the cell with 1 mM guanosine 5'-O-(3-thiotriphosphate), which causes the prolonged activation of G proteins. Activation of Na-H exchange is also elicited in unshrunken cells by injecting cholera toxin, which activates certain G proteins. Neither exposing cells to 100 nM phorbol 12-myristate 13-acetate nor dialyzing them with a solution containing 20 microM adenosine 3',5'-cyclic monophosphate (cAMP) (or 50 microM dibutyryl cAMP) plus 0.5 mM 3-isobutyl-1-methylxanthine substantially stimulates the exchanger. Thus our data suggest that a G protein plays a key role in the transduction of the shrinkage signal to the Na-H exchanger via a pathway that involves neither protein kinase C nor cAMP.

We used microelectrodes to determine whether the K/HCOa cotransporter tentatively identified in the accompanying paper (Hogan, E. M., M. A.Cohen, and W. F. Boron. 1995.Journal of GeneralPhysiology. 106:821--844) can mediate an increase in the intracellular pH (pHi) of squid giant axons. An 80-min period of internal dialysis increased pHi to 7.7, 8.0, or 8.3; the dialysis fluid was free of K +, Na +, and C1-. Our standard artificial seawater (ASW), which also lacked Na § K § and CI-, had a pH of 8.0. Halting dialysis unmasked a slow pHi decrease. Subsequently introducing an ASW containing 437 mM K § and 0.5% COj12 mM HCOg had two effects: (a) it caused membrane potential (Vm) to become very positive, and (b) it caused a rapid pHi decrease, because of COz influx, followed by a slower plateau-phase pHi increase, presumably because of inward cotransport of K § and HCO~ ("base influx"). Only extracellular Rb § substituted for K § in producing the plateau-phase pHi increase in the presence of CO2/ HCO~. Mean fluxes with Na +, Li +, and Cs § were not significantly different from zero, even though Vm shifts were comparable for all monovalent cations tested. Thus, unless K § or Rb § (but not Na § Li § or Cs § somehow activates a conductive pathway for H +, HCO~, or both, it is unlikely that passive transport of H +, HCO~, or both makes the major contribution to the pHi increase in the presence of K + (or Rb +) and CO2/HCO3. Because exposing axons to an ASW containing 437 mM K +, but no COz/HCO~, produced at most a slow pHi increase, K-H exchange could not make a major contribution to base influx. Introducing an ASW containing COJHCO~, but no K + also failed to elicit base influx. Because we observed base influx when the ASW and DF were free of Na § and CI-, and because the disulfonic sdlbene derivatives SITS and DIDS failed to block base influx, Na +-dependent CI-HCOa exchange also cannot account for the results. Rather, we suggest that the most straightforward explanation for the pHi increase we observed in the simultaneous presence of K + and CO2/HCOg is the coupled uptake of K + and HCO~.