TPK gene products mediate cAMP-independent thermotolerance in Saccharomyces cerevisiae.

Incubation of Saccharomyces cerevisiae with the plant cytokinin N6-(delta 2-isopentenyl)adenine (2iP) resulted in an induction of thermotolerance similar to that induced by sublethal temperatures. Intracellular cAMP levels did not change significantly either during incubation at a sublethal temperature or in the presence of 2iP or ethanol. This suggested that stress-induced thermotolerance is triggered by a mechanism independent of cAMP activation. However, measurement of stress-induced thermotolerance in two mutant strains (tpk1, tpk2, TPK3; tpk1, TPK2, tpk3) each deficient in two of the catalytic subunits of the cAMP-dependent protein kinase (cAPK), revealed that sublethal heat induces thermotolerance by a mechanism part-mediated by the catalytic subunits of cAPK. In contrast, 2iP and ethanol induced thermotolerance by a mechanism fully dependent on the catalytic subunits of cAPK for expression. Therefore, this implies there must be an alternative novel mechanism, other than cAMP, for activating cAPK during stress. Sublethal heating resulted in large increases in intracellular trehalose levels which correlated with the induction of thermotolerance. However, incubation in 2iP or ethanol had no significant effect. This suggests trehalose synthesis is either coincidental with heat stress or that different stress factors induce thermotolerance by alternative mechanisms. Incubation with protein synthesis inhibitors reduced the levels of trehalose synthesized during sublethal heating, suggesting that synthesis of trehalose-6-phosphate synthase during heat stress could be accounting for the increased trehalose levels.     

Molecular events associated with acquisition of heat tolerance by the yeast Saccharomyces cerevisiae

Abstract: The heat shock response is an inducible protective system of all living cells. It simultaneously induces both heat shock proteins and an increased capacity for the cell to wisthstand potentially lethal temperatures (an increased thermotolerance). This has lead to the suspicion that these two phenomena must be inexorably linked. However, analysis of heat shock protein function in Saccharomyces cerevisiae by molecular genetic techniques has revealed only a minority of the heat shock proteins of this organism having appreciable influences on thermotolerance. Instead, physiological perturbations and the accumulation of trehalose with heat stress may be more important in the development of thermotolerance during a preconditioning heat shock. Vegetative S. cerevisiae also acquires thermotolerance through osmotic dehydration, through treatment with certain chemical agents and when, due to nutrient limitation, it arrests growth in the GI phase of the cell cycle. There is evidence for the activities of the cAMP-dependent protein kinase and plasma membrane ATPase being very important in thermotolerance determination. Also, intracellular water activity and trehalose probably exert a strong influence over thermotolerance through their effects on stabilisation of membranes and intracellular assemblies. Future investigations should address the unresolved issue of whether the different routes to thermotolerance induction cause a common change to the physical state of the intracellular environment, a change that may result in an increased stabilisation of cellular structures through more stable hydrogen bonding and hydrophobic interactions.     

Inducible pH homeostasis and the acid tolerance response of Salmonella typhimurium.

The acid tolerance response (ATR) is an adaptive system triggered at external pH (pHo) values of 5.5 to 6.0 that will protect cells from more severe acid stress (J. Foster and H. Hall, J. Bacteriol. 172:771-778, 1990). Correlations between the internal pH (pHi) of adapted versus unadapted cells at pHo of 3.3 indicate that the ATR system produces an inducible pH-homeostatic function. This function serves to maintain the pHi above 5 to 5.5. Below this range, cells rapidly lose viability. Development of this pH homeostasis mechanism was sensitive to protein synthesis inhibitors and operated only to augment the pHi at pHo values below 4. In contrast, classical constitutive pH homeostasis was insensitive to protein synthesis inhibitors and was efficient only at pHo values above 4. Physiological studies indicated an important role for the Mg(2+)-dependent proton-translocating ATPase in affording ATR-associated survival during exposure to severe acid challenges. Along with being acid intolerant, cells deficient in this ATPase did not exhibit inducible pH homeostasis. We speculate that adaptive acid tolerance is important to Salmonella species in surviving acid encounters in both the environment and the infected host.     

Rise in intracellular pH is concurrent with 'start' progression of Saccharomyces cerevisiae

Intracellular pH (pHi) was determined during arrest and recovery of temperature sensitive-cell division cycle mutants of Saccharomyces cerevisiae. In all mutants, pHi decreased during arrest; but when the mutants were released from arrest a rapid increase in pHi ensued in only cdc28- and cdc37-arrested cells. Both of these mutations cause arrest at 'start', the sole regulatory point in the S. cerevisiae cell cycle. In cells with cdc4 or cdc7 mutations, which arrest past start, pHi remained constant and exhibited a decrease, respectively, upon recovery of growth. The activity of plasma membrane ATPase decreased during the first 30 min of recovery of cdc28-arrested cells, concomitant with the rise in pHi. During the same period, there was no significant change in activity in cdc4-bearing cells, whereas an increase was observed for cdc7-bearing cells. Increase in pHi may be used as a specific signal by S. cerevisiae for start traversal and commitment to a new cycle.     

Activation of plasma membrane ATPase of Saccharomyces cerevisiae by octanoic acid.

Plasma membrane ATPase activity of Saccharomyces cerevisiae IGC 3507III grown in the presence of the lipophilic acid octanoic acid [4-50 mg l-1 (0.03-0.35 mM), pH 4.0] was 1.5-fold higher than that in cells grown in its absence. The Km for ATP, the pH profile and the sensitivity to orthovanadate of the basal and the activated forms of the membrane ATPase were identical. This activation was closely associated with a decrease in the biomass yield and an increase in the ethanol yield, and was rapidly reversed in vivo after removal of the acid. However, the activated level was preserved when membranes were extracted and subjected to manipulations which eliminated or decreased octanoic acid incorporation in the plasma membrane. The activity of the basal plasma membrane ATPase in the total membrane fraction was slightly increased by incubation at pH 6.5 with octanoic acid at 100 mg l-1 or less (2.4 mg acid form plus 97.6 mg octanoate ion l-1). However, destruction of the permeability barrier between the enzyme and its substrate could not explain the in vivo activation. A role for plasma membrane ATPase activation in the regulation of the intracellular pH (pHi) of cells grown with octanoic acid was not proven.     

The Na+/H+ exchange system in glial cell lines. Properties and activation by an hyperosmotic shock

The properties of the Na+/H+ exchange system in the glial cell lines C6 and NN were studied from 22Na+ uptake experiments and measurements of the internal pH (pHi) using intracellularly trapped biscarboxyethyl-carboxyfluorescein. In both cell types, the Na+/H+ exchanger is the major mechanism by which cells recover their pHi after an intracellular acidification. The exchanger is inhibited by amiloride and its derivatives. The pharmacological profile (ethylisopropylamiloride greater than amiloride greater than benzamil) is identical for the two cell lines. Both Na+ and Li+ can be exchanged for H+. Increasing the external pH increases the activity of the exchanger in the two cell lines. In NN cells the external pH dependence of the exchanger is independent of the pHi. In contrast, in C6 cells, changing the pHi value from 7.0 to 6.5 produces a pH shift of 0.6 pH units in the external pH dependence of the exchanger in the acidic range. Decreasing pHi activates the Na+/H+ exchanger in both cell lines. Increasing the osmolarity of the external medium with mannitol produces an activation of the exchanger in C6 cells, which leads to a cell alkalinization. Mannitol action on 22Na+ uptake and the pHi were not observed in the presence of amiloride derivatives. Mannitol produces a modification of the properties of interaction of the antiport with both internal and external H+. It shifts the pHi dependence of the system to the alkaline range and the external pH (pHo) dependence to the acidic range. It also suppresses the interdependence of pHi and pHo controls of the exchanger's activity. NN cells that possess an Na+/H+ exchange system with different properties do not respond to mannitol by an increased activity of the Na+/H+ exchanger. The action of mannitol on C6 cells is unlikely to be mediated by an activation of protein kinase C.     

Role of Na+/H+ exchange in the control of intracellular pH and cell membrane conductances in frog skin epithelium

Ion-sensitive microelectrodes and current-voltage analysis were used to study intracellular pH (pHi) regulation and its effects on ionic conductances in the isolated epithelium of frog skin. We show that pHi recovery after an acid load is dependent on the operation of an amiloride-sensitive Na+/H+ exchanger localized at the basolateral cell membranes. The antiporter is not quiescent at physiological pHi (7.1-7.4) and, thus, contributes to the maintenance of steady state pHi. Moreover, intracellular sodium ion activity is also controlled in part by Na+ uptake via the exchanger. Intracellular acidification decreased transepithelial Na+ transport rate, apical Na+ permeability (PNa) and Na+ and K+ conductances. The recovery of these transport parameters after the removal of the acid load was found to be dependent on pHi regulation via Na+/H+ exchange. Conversely, variations in Na+ transport were accompanied by changes in pHi. Inhibition of Na+/K+ ATPase by ouabain produced covariant decreases in pHi and PNa, whereas increases in Na+ transport, occurring spontaneously or after aldosterone treatment, were highly correlated with intracellular alkalinization. We conclude that cytoplasmic H+ activity is regulated by a basolateral Na+/H+ exchanger and that transcellular coupling of ion flows at opposing cell membranes can be modulated by the pHi-regulating mechanism.     

Use of flow cytometry and SNARF to calibrate and measure intracellular pH in NS0 cells.

BACKGROUND: Two calibration methods have been proposed for determining the relation between the fluorescence ratio of a pH-sensitive fluorescent indicator and intracellular pH (pHi). The first method uses nigericin to clamp pHi to external pH (pHe) and the second is the null point method. We compared these different calibration methods, solution conditions, and temperatures by using flow cytometry and the fluorescent dye 1,5- (and-6)-carboxy seminaphtorhodafluor-1-acetoxymethyl ester with an NS0 cell line. METHODS: The nigericin method was performed in glucose solutions supplemented with KCl and 2-(N-morpholino)ethane sulphonic acid plus tris(hydroxymethyl)aminomethane (solution 1A), a mixture of K2HPO4/KH2PO4 in glucose-solution supplemented solutions (solution 2A), or bicarbonate buffered growth medium supplemented with K2HPO4/KH2PO4 (solution 2B); this allowed a range of pHe values to be used. The effect of temperature (22 degrees C or 37 degrees C) on the nigericin calibration curve was also investigated. The null point method was performed by using a series of solutions with a mixture of weak acid and base with a known pHi response. RESULTS: Using solution 1A as the calibration solution resulted in acidic values of pHi for cells cultured in medium as compared with the values achieved with solution 2A. Using solution 2B did not affect the calibration curve. For the temperatures considered in this study, there was no affect on the calibration curve, but temperature did affect the pHi value of cells in phosphate buffered saline. The pseudo-null point method used with flow cytometry resulted in a calibration curve that was significantly different (P<0.05) from that achieved using the nigericin method. CONCLUSIONS: Our data indicates that the choice of calibration solution can affect the reported pHi value; therefore, careful choice of solution is important.     

Improvement of lactic acid production in Saccharomyces cerevisiae by cell sorting for high intracellular pH

Yeast strains expressing heterologous L-lactate dehydrogenases can produce lactic acid. Although these microorganisms are tolerant of acidic environments, it is known that at low pH, lactic acid exerts a high level of stress on the cells. In the present study we analyzed intracellular pH (pHi) and viability by staining with cSNARF-4F and ethidium bromide, respectively, of two lactic-acid-producing strains of Saccharomyces cerevisiae, CEN.PK m850 and CEN.PK RWB876. The results showed that the strain producing more lactic acid, CEN.PK m850, has a higher pHi. During batch culture, we observed in both strains a reduction of the mean pHi and the appearance of a subpopulation of cells with low pHi. Simultaneous analysis of pHi and viability proved that the cells with low pHi were dead. Based on the observation that the better lactic-acid-producing strain had a higher pHi and that the cells with low pHi were dead, we hypothesized that we might find better lactic acid producers by screening for cells within the highest pHi range. The screening was performed on UV-mutagenized populations through three consecutive rounds of cell sorting in which only the viable cells within the highest pHi range were selected. The results showed that lactic acid production was significantly improved in the majority of the mutants obtained compared to the parental strains. The best lactic-acid-producing strain was identified within the screening of CEN.PK m850 mutants.     

Photosensitized irreversible binding of estrone to protein via a hydroperoxide intermediate: an explanation of (photo-) allergic side-effects of estrogens

Recent studies have established that polypeptide growth factors cause an elevation of the cytoplasmic pH (pHi) in cultured mammalian cells by stimulating Na+/H+ exchange. We show that vanadate, previously found to act as a mitogen for a number of cells, reversibly activates Na+/H+ exchange at micromolar concentrations in A431 cells, leading to a large increase of pHi. The stimulation of Na+/H+ exchange by vanadate is not due to inhibition of the Na+/K+ ATPase and is unrelated to possible effects of vanadate on cAMP levels. Elevation of pHi by vanadate and by epidermal growth factor (EGF) both display similar kinetics, and both EGF and vanadate stimulate the rate of pHi recovery following an acute acid load, suggesting that vanadate stimulates Na+/H+ exchange by a mechanism similar to that of polypeptide growth factor stimulation. Thus, stimulation of Na+/H+ exchange may be a common property not only of polypeptide growth factors but also of other, chemically unrelated mitogens.     

Thimerosal induces calcium mobilization, fructose 2,6-bisphosphate synthesis and cytoplasmic alkalinization in rat thymus lymphocytes

The effect of thimerosal on intracellular calcium ([Ca2+]i), pH (pHi) and fructose 2,6-bisphosphate (Fru 2,6-P2) in thymus lymphocytes was investigated. The effect of thimerosal on cell growth was also examined. Thimerosal produced a dose-dependent increase in [Ca2+]i, pHi and in the level of fructose 2,6-bisphosphate. Thimerosal was, however, unable to produce cell proliferation and inhibited [3H]thymidine incorporation when cells were challenged with PHA and costimulator. In the absence of external calcium, thimerosal produced only a slight increase in [Ca2+]i. In Na(+)-containing buffer, thimerosal induced an initial acidification (0.05 +/- 0.01 pH units), followed by an alkalinization of 0.08 pH units/min, whereas in Na(+)-free media, pHi decreased 0.2 +/- 0.02 units and this acidification was maintained for more than 40 min. When external calcium was removed the initial acidification was unchanged and no further increase in pHi was observed. Polymyxin B, an inhibitor of protein kinase C, did not modify the initial thimerosal-induced acidification although pH returned to basal levels after 10 min. It was concluded that alkalinization induced by thimerosal is probably due to activation of the Na+/H+ exchanger and that changes in internal Ca2+, pH and metabolic rate are not sufficient to induce cellular proliferation. The mechanism by which thimerosal inhibits thymocyte proliferation remains to be clarified.     

Yeast thermotolerance does not require protein synthesis.

Heat shock at 37 degrees C induces synthesis of stress (heat shock) proteins in Saccharomyces cerevisiae and also induces thermotolerance. Amino acid analogs that are powerful inducers of stress protein synthesis failed to induce thermotolerance, suggesting that the stress proteins do not play a causal role in acquired thermotolerance at 37 degrees C. This suggestion was confirmed by the observation that protein synthesis was not required for the induction of thermotolerance at 37 degrees C.     

pH regulation in the vertebrate central nervous system: microelectrode studies in the brain stem of the lamprey

Studies of intracellular pH (pHi) in nervous tissue are summarized and recent investigation of intracellular and extracellular pH (pHo) in the isolated brain stem of the lamprey is reviewed. In the lamprey, pHi regulation was studied in single reticulospinal neurons using double-barrel ion-selective microelectrodes (ISMs). In nominally HCO3(-)-free HEPES-buffered media, acute acid loading was followed by a spontaneous recovery of pHi requiring 10-20 min and was associated with a prolonged rise in intracellular Na+. The recovery of pHi was blocked by 1-2 mM amiloride. Amiloride also caused a small rise in pHo. Substitution of external Na+ caused a slow intracellular acidification and extracellular alkalinization. Return of external Na+ reversed these effects. Transition from HEPES to HCO3(-)-buffered media increased the rate of acid extrusion during recovery of pHi. Recovery in HCO3(-)-buffered media was inhibited by 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid and was slowed after exposure to Cl(-)-free media. Following inhibition of acid extrusion by amiloride, transition to HCO3- media restored pHi recovery. These data indicate that lamprey neurons recover from acute acid loads by both Na+-H+ exchange and an independent HCO3(-)-dependent mechanism. Evidence for HCO3(-)-dependent acid extrusion in other vertebrate cells and the protocols of pHi studies using ISMs are discussed.     

Intracellular pH and Na fluxes in barnacle muscle with evidence for reversal of the ionic mechanism of intracellular pH regulation

The ion transport mechanism that regulates intracellular pH (pHi) in giant barnacle muscle fibers was studied by measuring pHi and unidirectional Na+ fluxes in internally dialyzed fibers. The overall process normally results in a net acid extrusion from the cell, presumably by a membrane transport mechanism that exchanges external Na+ and HCO-3 for internal Cl- and possibly H+. However, we found that net transport can be reversed either by lowering [HCO-3]o and pHo or by reducing [Na+]o. This reversal (acid uptake) required external Cl-, was stimulated by raising [Na+]i, and was blocked by SITS. When the transporter was operating in the net forward direction (acid extrusion), we found a unidirectional Na+ influx of approximately 60 pmol . cm-2 . s-1, which required external HCO-3 and internal Cl- and was stimulated by cyclic AMP and blocked by SITS or DIDS. These properties of the Na+ influx are all shared with the net acid extrusion process. We also found that under conditions of net forward transport, the pHi-regulating system mediated a unidirectional Na+ efflux, which was significantly smaller than the simultaneous Na+ influx. These data are consistent with a reversible transport mechanism which, even when operating in the net forward direction, mediates a small amount of reversed transport. We also found that the ouabain-sensitive Na+ efflux was sharply inhibited by acidic pHi, being totally absent at pHi values below approximately 6.8.     

Relationship between cytoplasmic pH and proliferation during exponential growth and cellular quiescence

Flow cytometry was used to measure intracellular pH (pHi) on an individual cell basis during exponential and plateau phases of growth. In all three cell lines examined a range of pHi values was associated with exponential growth. When cells from the extremes of the pHi distribution were sorted using a fluorescence-activated cell sorter and then restained for cellular DNA content, it was found that the higher pHi values were associated with enrichment of the S, G2, and M phases of the cell cycle, with a corresponding increase in the percentage of G1 cells at the lower pH1 range, suggesting cell-cycle dependence of pHi. It has been shown previously (I. W. Taylor and P. Hodson, 1984, J. Cell Physiol. 121, 517) that PMC-22 human melanoma cells are capable of entering a distinct pH-dependent quiescent state in response to the acidification of the growth medium which occurs naturally during growth to plateau phase. Simultaneous measurement of pHi and external pH showed that under these conditions pHi was maintained at control values down to an external pH of approximately 6.5, below which cytoplasmic acidification took place. This fall in pHi coincided with the onset of the transition to quiescence. Individual quiescent cells (defined by failure to incorporate bromodeoxyuridine during a 24-h exposure) could not be identified as such on the basis of a low pHi, suggesting that the probability of cell cycling is reduced by lowering pHi. Those cells which remained in cycle showed a markedly reduced rate of DNA synthesis, but a cell-cycle phase distribution similar to that in exponential growth, indicating that prolongation of all cell-cycle phases is an additional factor influencing overall population growth. The external pH at which both of these effects on cell proliferation kinetics took place in vitro is similar to that which occurs regionally within solid tumors, suggesting that pH effects could play a significant role in determining tumor cell growth in vivo.     

Temperature-dependent induction of thermotolerance by ethanol

Ethanol (1 M) cytotoxicity in asynchronous Chinese hamster ovary cells was strongly temperature dependent, yielding families of cell survival curves between 34 and 39 degrees C that were similar to those obtained at hyperthermic temperatures in medium without ethanol. Below 36 degrees C, survival curves were biphasic, indicating the development of thermotolerance during ethanol exposures. At room temperature (22 degrees C) ethanol was completely nontoxic with incubation periods up to 6 h. A comparison of survival curves with and without ethanol showed that the major effect of ethanol was an effective temperature shift of circa 6.5 degrees C, i.e., the cell survival curve at 37 degrees C in 1 M ethanol was equivalent to that at 43.6 degrees C in medium without ethanol. In addition to the effective temperature shift, ethanol also resulted in sensitization to "heat" with a temperature dependence that was similar to the stepdown heating effect. When thermotolerance was induced with acute ethanol exposures (25 min, 37 degrees C or 60 min, 35.5 degrees C), the kinetics and the magnitude of tolerance were similar to those after isotoxic conditioning treatments with heat alone (10 min, 45 degrees C). In contrast, equimolar ethanol at 22 degrees C did not induce thermotolerance. These data provide a rationale for conflicting results in the literature regarding thermotolerance induction by ethanol. Both heat sensitization and the induction of thermotolerance are interpreted as the effect of ethanol on the solution properties of intracellular water. These solvent alterations reduce the temperature necessary to elicit cytotoxicity and the development of thermotolerance.     

Relief from alkaline load in two-cell stage mouse embryos by bicarbonate/chloride exchange

Mouse embryos at the two-cell stage are able to recover from an alkaline load. We found that this recovery is mediated by sodium-independent bicarbonate/chloride exchange: intracellular pH (pHi) recovery from alkaline load is inhibited by the anion exchange inhibitor 4,4'-diisothiocyanostilbene disulfonic acid, lack of bicarbonate, or lack of chloride. The dependence of the pHi recovery on extracellular chloride concentration exhibits Michaelis-Menten kinetics. Furthermore, uptake of chloride is inhibited in a dose-dependent manner by extracellular bicarbonate. The Km for external chloride was found to be about 3 mM, with a Ki for external bicarbonate of about 2 mM. The exchanger is active above approximately pH 7.15. These results demonstrate that mouse embryos at the two-cell stage possess a sodium-independent bicarbonate/chloride exchange mechanism that is similar to that found in other mammalian cells. This bicarbonate/chloride exchanger appears to be the sole pHi-regulatory mechanism in the two-cell stage mouse embryo, since our previous results have shown that there are apparently no specific mechanisms active in these cells for relieving acid loads.     

Fluorescence emission spectroscopy of 1,4-dihydroxyphthalonitrile. A method for determining intracellular pH in cultured cells.

We have developed new methodology for measuring intracellular pH (pHi) in cultured cell monolayers and epithelia by analyzing the emission spectra of the trapped fluorescent pH probe, 1,4-dihydroxyphthalonitrile (1,4-DHPN). This compound is unique since both its acid and base forms possess different fluorescence emission characteristics that can be used to quantitate pHi. The fluorescence difference spectrum between an acid and alkaline solution of 1,4-DHPN has a maximum at 455 nm and a minimum at 512 nm. By determining the ratio of the intensity at these two wavelengths as a function of pH, a calibration curve was constructed. Since the two intensities are determined simultaneously, the measurement is independent of dye concentration, bleaching, and intensity fluctuation of the excitation source. Furthermore, analysis of the emission spectra permitted the detection of light scattering, binding effects, and chemical modification of the probe. A microspectrofluorometer was constructed to analyze low light level emission spectra from intracellular 1,4-DHPN. The instrument consists of a modified Leitz inverted microscope (E. Leitz, Inc., Rockleigh, NJ) with a Ploem illuminator adapted for broadband excitation and objective focusing capability. The emission spectra were collected by focusing the fluorescence from the cell onto the entrance slit of an imaging monochromator, which was scanned by a SIT camera interfaced with a computer. This permitted the acquisition of fluorescence emission spectra extending from 391-588 nm in approximately 33 ms. pHi measured in the cultured toad kidney epithelial cell line, A6, was 7.49 +/- 0.04 (n = 12) with an external pH of 7.6. A6 cells were found to regulate pHi in response to both acute acid and alkali loads and maintained pHi relatively constant over a wide range of external pH values. The technique described in this report overcomes several of the difficulties encountered with other fluorescent pH probes where excitation spectroscopy is required to monitor pH.     

Regulation of mitochondrial K+/H+ antiport activity by hydrogen ions

The effect of matrix pH (pHi) on the activity of the mitochondrial K+/H+ antiport has been studied using the fluorescence of 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) to monitor pHi and passive swelling in K+ acetate to follow antiport activity. Heart mitochondria suspended in hypotonic K+ acetate in the absence of respiration show an initial delta pH of -0.4 (interior acid) that decays slowly. Addition of A23187 to deplete matrix Mg2+ results in a further acid shift in pHi followed by equilibration of delta pH. This equilibration appears to depend on K+/H+ antiport and is slow at acid pHi but very rapid when the matrix is alkaline. Swelling of Mg(2+)-depleted mitochondria in K+ acetate is multiphasic with a slow initial rate, a period of maximum swelling, and a final period in which the rate declines. At constant external pH (pH0), the initial rate of swelling is faster with increasing pHi and the time to the onset of the maximum swelling rate decreases. The maximum swelling rate is initiated at pHi 7.4 when pH0 is 7.8 and at pHi 7.1 when pH0 is 7.4. The maximum rate of swelling increases linearly with increasing pH0 in the range from 7.0 to 8.2. This rate also shows a linear relationship to the value of pHi at the time the maximum rate is attained. Dixon plots of the reciprocal of the maximum swelling rate vs [H+]0 suggest that external [H+] is a noncompetitive inhibitor of K+ entry on the antiport. It is concluded that K+/H+ antiport in Mg(2+)-depleted heart mitochondria can be regulated by matrix [H+] (see Beavis, A. D., and Garlid, K. D. (1990) J. Biol. Chem. 265, 2538-2545), but that this antiport is also sensitive to external [H+] or to delta pH when it acts in the direction of K+ uptake.