In response to osmotic perturbation, the Amphiuma red blood cell regulates volume back to "normal" levels. After osmotic swelling, the cells lose K, Cl, and osmotically obliged H2O (regulatory volume decrease [RVD] ). After osmotic shrinkage, cell volume is regulated as a result of Na, Cl, and H2O uptake (regulatory volume increase [RVI] ). As previously shown (Cala, 1980 alpha), ion fluxes responsible for volume regulation are electroneutral, with alkali metal ions obligatorily counter-coupled to H, whereas net Cl flux is in exchange for HCO3. When they were exposed to the Ca ionophore A23187, Amphiuma red blood cells lost K, Cl, and H2O with kinetics (time course) similar to those observed during RVD. In contrast, when cells were osmotically swollen in Ca-free media, net K loss during RVD was inhibited by approximately 60%. A role for Ca in the activation of K/H exchange during RVD was suggested from these experiments, but interpretation was complicated by the fact that an increase in cellular Ca resulted in an increase in the membrane conductance to K (GK). To determine the relative contributions of conductive K flux and K/H exchange to total K flux, electrical studies were performed and the correspondence of net K flux to thermodynamic models for conductive vs. K/H exchange was evaluated. These studies led to the conclusion that although Ca activates both conductive and electroneutral K flux pathways, only the latter pathways contribute significantly to net K flux. On the basis of observations that A23187 did not activate K loss from cells during RVI (when the Na/H exchange was functioning) and that amiloride inhibited K/H exchange by swollen cells only when cells had previously been shrunk in the presence of amiloride, I concluded that Na/H and K/H exchange are mediated by the same membrane transport moiety.

After osmotic perturbation, the red blood cells of Amphiuma exhibited a volume-regulatory response that returned cell volume back to or toward control values. After osmotic swelling, cell-volume regulation (regulatory volume decrease; RVD) resulted from net cellular loss of K, Cl, and osmotically obliged H2O. In contrast, the volume-regulatory response to osmotic shrinkage (regulatory volume increase; RVI) was characterized by net cellular uptake of Na, Cl, and H2O. The net K and Na fluxes characteristic of RVD and RVI are increased by 1-2 orders of magnitude above those observed in studies of volume-static control cells. The cell membrane potential of volume-regulating and volume-static cells was measured by impalement with glass microelectrodes. The information gained from the electrical and ion-flux studies led to the conclusion that the ion fluxes responsible for cell-volume regulation proceed via electrically silent pathways. Furthermore, it was observed that Na fluxes during RVI were profoundly sensitive to medium [HCO3] and that during RVI the medium becomes more acid, whereas alkaline shifts in the suspension medium accompany RVD. The experimental observations are explained by a model featuring obligatorily coupled alkali metal-H and Cl-HCO3 exchangers. The anion- and cation-exchange pathways are separate and distinct yet functionally coupled via the net flux of H. As a result of the operation of such pathways, net alkali metal, Cl, and H2O fluxes proceed in the same direction, whereas H and HCO3 fluxes are cyclic. Data also are presented that suggest that the ion-flux pathways responsible for cell-volume regulation are not activated by changes in cell volume per se but by some event associated with osmotic perturbation, such as changes in intracellular pH.

In Amphiuma red blood cells, the Na/H exchanger has been shown to play a central role in the regulation of cell volume following cell shrinkage (Cala, P. M. 1980. Journal of General Physiology. 76:683-708.) The present study was designed to evaluate the existence of pH regulatory Na/H exchange in the Amphiuma red blood cell. The data illustrate that when the intracellular pHi was decreased below the normal value of 7.00, Na/H exchange was activated in proportion to the degree of acidification. Once activated, net Na/H exchange flux persisted until normal intracellular pH (6.9-7.0) was restored, with a half time of approximately 5 min. These observations established a pHi set point of 7.00 for the pH-activated Na/H exchange of Amphiuma red blood cell. This is in contrast to the behavior of osmotically shrunken Amphiuma red blood cells in which no pHi set point could be demonstrated. That is, when activated by cell shrinkage the Na/H exchange mediated net Na flux persisted until normal volume was restored regardless of pHi. In contrast, when activated by cell acidification, the Na/H exchanger functioned until pHi was restored to normal and cell volume appeared to have no effect on pH-activated Na/H exchange. Studies evaluating the kinetic and inferentially, the molecular equivalence of the volume and pHi-induced Amphiuma erythrocyte Na/H exchanger(s), indicated that the apparent Na affinity of the pH activated cells is four times greater than that of shrunken cells. The apparent Vmax is also higher (two times) in the pH activated cells, suggesting the involvement of two distinct populations of the transporter in pH and volume regulation. However, when analyzed in terms of a bisubstrate model, the same data are consistent with the conclusion that both pH and volume regulatory functions are mediated by the same transport protein. Taken together, these data support the conclusion that volume and pH are regulated by the same effector (Na/H exchanger) under the control of as yet unidentified, distinct and cross inhibitory volume and pH sensing mechanisms.

Exposure of Amphiuma red blood cells to millimolar concentrations of N-ethylmaleimide (NEM) resulted in net K loss. In order to determine whether net K loss was conductive or was by electroneutral K/H exchange or KCl cotransport, studies were performed evaluating K flux in terms of the thermodynamic forces to which K flux by the above pathways should couple. The direction and magnitude of the NEM-induced net K flux did not correspond with the direction and magnitude of the forces relevant to K conductance or electroneutral KCl cotransport. Both the magnitude and direction of the NEM-activated K flux responded to the driving force for K/H exchange. We therefore conclude that NEM-induced K loss, like that by osmotically swollen Amphiuma red blood cells, is by an electroneutral K/H exchanger. In addition to the above studies, we evaluated the kinetic behavior of the volume- and NEM-induced K/H exchange flux pathways in media where Cl was replaced by SCN, NO3, para-aminohippurate (PAH), or gluconate. The anion replacement studies did not permit a distinction between K/H exchange and KCl cotransport, since, depending upon the anion used as a Cl replacement, partial inhibition or stimulation of volume-activated K/H exchange fluxes was observed. In contrast, all anions used were stimulatory to the NEM-induced K loss. Since, on the basis of force-flow analysis, both volume-and NEM-induced K loss are K/H exchange, it was necessary to reevaluate assumptions (i.e., anions serve as substrates and therefore probe the translocation step) associated with the use of anion replacement as a means of flux route identification. When viewed together with the force-flow studies, the Cl replacement studies suggest that anion effects upon K/H exchange are indirect. The different anions appear to alter mechanisms that couple NEM exposure and cell swelling to the activation of K/H exchange, as opposed to exerting direct effects upon K and H translocation.

In adult hearts, ischemic preconditioning (PC) has been shown to decrease ischemia-induced changes in intracellular pH (pHi) and [Ca] ([Ca]i) and decrease associated injury. These results are consistent with the interpretation that PC decreases the stimulus for Na uptake via Na/H exchange, thereby decreasing intracellular Na (Nai) accumulation, and thus decreasing the change in force driving Na/Ca exchange, which otherwise contributes to ischemia-induced increases in [Ca]i. Given documented age-related differences in myocardial responses to ischemia, we tested the hypothesis that in newborn hearts, PC will diminish intracellular [H], Nai, and [Ca]i during ischemia/reperfusion. NMR was used to measure pHi, Nai, [Ca]i, ATP, and PCr in isolated newborn (4-7 days) rabbit hearts Langendorff-perfused with Krebs-Henseleit solution equilibrated with 95% O2/5% CO2 at 36+/-1 degrees C. Control hearts were perfused 30 min before initiating 40 min global ischemia followed by 40 min reperfusion. PC hearts were treated the same except four 5-min intervals of ischemia each followed by 10 min of perfusion which preceded global ischemia. At end ischemia, pHi was higher in PC than control hearts (6.31+/-0.03 v 5.83+/-0.05; P<0.05). Similarly, PC diminished Nai-accumulation during ischemia and reperfusion (P<0.05). Control Nai rose from 16.2+/-2.6 to 108.8+/-10.3 (mEq/kg dry weight) and recovered to 55.2+/-10.1 and the corresponding values for PC hearts were 25.6+/-6.2, 70.0+/-7.9 and 21.9+/-5.2. PC also improved [Ca]i recovery during reperfusion (P<0.05). Control [Ca]i rose from 418+/-43 to 1100+/-78 (nm/l) and recovered to 773+/-63, whereas in PC hearts the values were 382+/-40, 852+/-136 and 371+/-45, respectively. In addition, PC decreased coronary resistance during reperfusion (P<0.05) as reflected by lower perfusion pressures under constant flow conditions (65.9+/-1.5 v 56. 1+/-4.1 mmHg at end of reperfusion). Finally, PC improved recovery of left-ventricular developed pressure (LVDP-43.8+/-12.0 v 17.2+/-3. 0% of control; P<0.05) and diminished CK release (607+/-245 v 2432+/-639 IU/g dry weight; P<0.05) during reperfusion. The results are consistent with the hypothesis.

Numerous studies suggest that in adult hearts myocardial ischemic injury is in part the result of proton stimulation of Na/H exchange which increases intracellular Na (Nai) and thus leads to increases in intracellular Ca concentration ( [Ca]i) due to changes in Na/Ca exchange flux. Corollary to the hypothesis, inhibition of Na/H exchange diminishes Na and Ca accumulation and improves heart function after ischemia. To test this hypothesis and its corollary in newborn hearts, NMR spectroscopy was used to measure intracellular pH (pHi), Nai, [Ca]i, and high energy phosphates in isolated, 4-7-day-old rabbit hearts, Langendorff-perfused with Krebs-Henseleit solution at pH 7.4+/-0.5 equilibrated with 95% O2/5% CO2 at 36+/-1 degrees C. Control hearts were perfused for 30 min before initiating 40 min of global ischemia followed by 40 min of reperfusion. In a second group of hearts ethylisopropylamiloride (EIPA-10 microM) was added to the perfusate 20 min before global ischemia to inhibit Na/H exchange. After 15 min ischemia, pHi in EIPA-treated hearts (6.41+/-0.04) was higher than that of the control hearts (6.20+/-0.08; P<0.05). EIPA also limited the increase in Nai and [Ca]i during ischemia and improved Nai and [Ca]i recovery during reperfusion (P<0.05). Nai (mEq/kg dry weight) rose from 18. 1+/-3.2 to 110.6+/-14.0 and recovered to 53.3+/-12.3 in the control group. The corresponding Nai values for EIPA-treated hearts were 16. 2+/-2.4, 39.6+/-9.6 and 12.6+/-3.5, respectively. In control hearts [Ca]i (nM/l) rose from 332+/-42 to 1157+/-89 and recovered to 842+/-55, whereas in EIPA-treated hearts the values were 255+/-32, 616+/-69 and 298+/-34, respectively. EIPA also preserved cellular ATP during ischemia and reperfusion and diminished inorganic phosphate during reperfusion (P<0.05). Finally, EIPA treatment improved recovery of left ventricular developed pressure (68.2+/-8.9 v 16.2+/-3.6% of control) and limited myocardial injury as indicated by decreased total creatine kinase release during reperfusion (348+/-132 v 2432+/-639 IU/g dry weight). Thus, as in adults, the results from newborn hearts are consistent with the hypothesis.

In this study, we test the hypothesis that in newborn hearts (as in adults) hypoxia and acidification stimulate increased Na(+) uptake, in part via pH-regulatory Na(+)/H(+) exchange. Resulting increases in intracellular Na(+) (Na(i)) alter the force driving the Na(+)/Ca(2+) exchanger and lead to increased intracellular Ca(2+). NMR spectroscopy measured Na(i) and cytosolic Ca(2+) concentration ([Ca(2+)](i)) and pH (pH(i)) in isolated, Langendorff-perfused 4- to 7-day-old rabbit hearts. After Na(+)/K(+) ATPase inhibition, hypoxic hearts gained Na(+), whereas normoxic controls did not [19 +/- 3.4 to 139 +/- 14.6 vs. 22 +/- 1.9 to 22 +/- 2.5 (SE) meq/kg dry wt, respectively]. In normoxic hearts acidified using the NH(4)Cl prepulse, pH(i) fell rapidly and recovered, whereas Na(i) rose from 31 +/- 18.2 to 117.7 +/- 20.5 meq/kg dry wt. Both protocols caused increases in [Ca](i); however, [Ca](i) increased less in newborn hearts than in adults (P < 0.05). Increases in Na(i) and [Ca](i) were inhibited by the Na(+)/H(+) exchange inhibitor methylisobutylamiloride (MIA, 40 microM; P < 0.05), as well as by increasing perfusate osmolarity (+30 mosM) immediately before and during hypoxia (P < 0.05). The data support the hypothesis that in newborn hearts, like adults, increases in Na(i) and [Ca](i) during hypoxia and after normoxic acidification are in large part the result of increased uptake via Na(+)/H(+) and Na(+)/Ca(2+) exchange, respectively. However, for similar hypoxia and acidification protocols, this increase in [Ca](i) is less in newborn than adult hearts.

In the context of the "pump-leak" hypothesis (37), changes in myocardial intracellular Na (Nai) during ischemia and reperfusion have historically been interpreted to be the result of changes in Na efflux via the Na-K pump. We investigated the alternative hypothesis that changes in Nai during ischemia are the result of changes in the Na "leak" rather than changes in the pump. More specifically, we hypothesize that the increase in Nai during ischemia is in part the result of increased Na uptake mediated by Na/H exchange. Furthermore, we present data consistent with the interpretation that the Na-K-2Cl cotransporter is active (or, alternatively, displaced from equilibrium) during ischemia and may contribute an additional Na efflux pathway during reperfusion. Thus inhibition of Na efflux via Na-K-2Cl cotransport during ischemia and reperfusion could result in increased Nai and therefore decreased force driving Ca efflux via Na/Ca exchange and ultimately increased intracellular Ca concentration ([Ca]i). Nai (in meq/kg dry wt) and [Ca]i (in nM) were measured in isolated Langendorff-perfused rabbit hearts using nuclear magnetic resonance spectroscopy. Except, during the 65 min of ischemia, hearts were perfused with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Krebs-Henseleit solution equilibrated with 100% O2 at 23 degrees C and pH 7.4 +/- 0.05. During ischemia, Nai rose from 16.6 +/- 0.3 to 62.9 +/- 5.1 (delta Nai approximately 46) meq/kg dry wt and decreased during subsequent reperfusion (mean +/- SE, n = 3 hearts). To measure Na uptake ("leak") in the absence of efflux via the Na-K pump, in all of the protocols described below, the perfusate was nominally K-free solution containing 1 mM ouabain for 10 min before ischemia and during the 30-min reperfusion. After K-free perfusion, Nai rose from 20.2 +/- 0.5 to 79.1 +/- 5.3 (delta Nai approximately 59) meq/kg dry wt (n = 3) during ischemia and decreased during K-free reperfusion. When amiloride (1 mM) was added to the K-free perfusate to inhibit Na/H exchange, Nai rose from 16.3 +/- 0.9 to 44.7 +/- 5.1 (delta Nai approximately 28) meq/kg dry wt (n = 3) during ischemia; i.e., amiloride decreased Na uptake. When bumetanide (20 microM) was added to the nominally K-free perfusate to inhibit Na-K-2Cl contransport, Nai rose from 22.5 +/- 3.9 to 83.8 +/- 13.9 (delta Nai approximately 61 meq/kg dry wt (n = 3) during ischemia and did not decrease during reperfusion; i.e., bumetanide inhibited Na recovery during reperfusion (P < 0.05 compared with bumetanide free). For the same protocol, the presence of bumetanide resulted in increased [Ca]i during ischemia and reperfusion (P < 0.05); these increases in [Ca]i are interpreted to be the result of increased Nai. Thus the results are consistent with the hypotheses.