Intracellular pH modulates the availability of vascular L-type Ca2+ channels

L-type Ca2+ channel currents were recorded from myocytes isolated from bovine pial and porcine coronary arteries to study the influence of changes in intracellular pH (pHi). Whole cell ICa fell when pHi was made more acidic by substituting HEPES/NaOH with CO2/bicarbonate buffer (pHo 7.4, 36 degrees C), and increased when pHi was made more alkaline by addition of 20 mM NH4Cl. Peak ICa was less pHi sensitive than late ICa (170 ms after depolarization to 0 mV). pHi-effects on single Ca2+ channel currents were studied with 110 mM BaCl2 as the charge carrier (22 degrees C, pHo 7.4). In cell-attached patches pHi was changed by extracellular NH4Cl or through the opened cell. In inside-out patches pHi was controlled through the bath. Independent of the method used the following results were obtained: (a) Single channel conductance (24 pS) and life time of the open state were not influenced by pHi (between pHi 6 and 8.4). (b) Alkaline pHi increased and acidic pHi reduced the channel availability (frequency of nonblank sweeps). (c) Alkaline pHi increased and acidic pHi reduced the frequency of late channel re- openings. The effects are discussed in terms of a deprotonation (protonation) of cytosolic binding sites that favor (prevent) the shift of the channels from a sleepy to an available state. Changes of bath pHo mimicked the pHi effects within 20 s, suggesting that protons can rapidly permeate through the surface membrane of vascular smooth muscle cells. The role of pHi in Ca2+ homeostases and vasotonus is discussed.     

Properties of single voltage-gated proton channels in human eosinophils estimated by noise analysis and by direct measurement

Voltage-gated proton channels were studied under voltage clamp in excised, inside-out patches of human eosinophils, at various pHi with pHo 7.5 or 6.5 pipette solutions. H+ current fluctuations were observed consistently when the membrane was depolarized to voltages that activated H+ current. At pHi < or = 5.5 the variance increased nonmonotonically with depolarization to a maximum near the midpoint of the H+ conductance-voltage relationship, gH-V, and then decreased, supporting the idea that the noise is generated by H+ channel gating. Power spectral analysis indicated Lorentzian and 1/f components, both related to H+ currents. Unitary H+ current amplitude was estimated from stationary or quasi-stationary variance, sigmaH2. We analyze sigmaH2 data obtained at various voltages on a linearized plot that provides estimates of both unitary conductance and the number of channels in the patch, without requiring knowledge of open probability. The unitary conductance averaged 38 fS at pHi 6.5, and increased nearly fourfold to 140 fS at pHi 5.5, but was independent of pHo. In contrast, the macroscopic gH was only 1.8-fold larger at pHi 5.5 than at pHi 6.5. The maximum H+ channel open probability during large depolarizations was 0.75 at pHi 6.5 and 0.95 at pHi 5.5. Because the unitary conductance increases at lower pHi more than the macroscopic gH, the number of functional channels must decrease. Single H+ channel currents were too small to record directly at physiological pH, but at pHi < or = 5.5 near Vthreshold (the voltage at which gH turns on), single channel-like current events were observed with amplitudes 7-16 fA.     

The effects of external pH on calcium channel currents in bullfrog sympathetic neurons.

We have investigated the effects of external pH (pHo) on whole-cell calcium channel currents in bullfrog sympathetic neurons. The peak inward current increased at alkaline pHo and decreased at acidic pHo. We used tail currents to distinguish effects of pHo on channel gating and permeation. There were large shifts in the voltage dependence of channel activation (approximately 40 mV between pHo and 9.0 and pHo 5.6), which could be explained by binding of H+ to surface charge according to Gouy-Chapman theory. To examine the effects of pHo on permeation, we measured tail currents at 0 mV, following steps to + 120 mV to maximally activate the channels. Unlike most previous studies, we found only a approximately 10% reduction in channel conductance from pHo 9.0 to pHo 6.4, despite a approximately 25 mV shift of channel activation. At lower pHo the channel conductance did decrease, which could be described by binding of H+ to a site with pKa = 5.1. In some cells, there was a separate slow decrease in conductance at low pHo, possibly because of changes in internal pH. These results suggest that changes in current at pHo > 6.4 result primarily from a shift in the voltage dependence of channel activation. A H(+)-binding site can explain a rapid decrease in channel conductance at lower pHo. The surface charge affecting gating has little effect on the local ion concentration near the pore, or on the channel conductance.     

Control of intracellular pH. Predominant role of oxidative metabolism, not proton transport, in the eukaryotic microorganism Neurospora

Recessed-tip microelectrodes were used to measure internal pH (pHi) in the fungus Neurospora, and to examine the response of pHi to several kinds of stress: changes of extracellular pH (pHo), inhibition of the principal proton pump in the plasma membrane, and inhibition of respiration. Under control conditions, at pHo = 5.8, pHi in Neurospora is 7.19 +/- 0.04. Changes of pHo between 3.9 and 9.3 affect pHi linearly but with a slope of only approximately 0.1 unit pHi per unit pHo, stable pHi being reached within 3 min of changed pHo. Despite a postulated high passive permeability of the Neurospora membrane to protons (Slayman, 1970), neither active nor passive H+ transport appears critical to pHi because (alpha) specific inhibition of the proton pump by orthovanadate has little effect on pHi, and (b) cytoplasmic acidification produced by respiratory blockade is unaffected by the size or direction of proton gradient. To convert measured changes in pHi into net proton fluxes, intracellular buffering capacity (beta i) was measured by the weak acid/weak base technique. At pHi = 7.2, beta i was (-) 35 mmol H+ (liter cell water)-1 (pH unit)-1, but beta i increased substantially in both the acid and alkaline directions, which suggests that amino acid side chains are the principal source of buffer.     

Intracellular pH is a major factor in the induction of tolerance to acid and other stresses in Lactococcus lactis.

This study demonstrates that exposure of log-phase Lactococcus lactis subsp. cremoris 712 cells to mildly acid conditions induces resistance to normally lethal intensities of environmental stresses such as acid, heat, NaCl, H2O2, and ethanol. The intracellular pH (pHi) played a major role in the induction of this multistress resistance response. The pHi was dependent on the extracellular pH (pHo) and on the specific acid used to reduce the pHo. When resuspended in fresh medium, cells were able to maintain a pH gradient even at pHo values that resulted in cell death. Induction of an acid tolerance response (ATR) coincided with an increase in the ability of cells to resist change to an unfavorable pHi; nevertheless, a more favorable pHi was not the sole reason for the increased survival at acid pHo. Cells with an induced ATR survived exposure to a lethal pHo much better than did uninduced cells with a pHi identical to that of the induced cells. Survival following lethal acid shock was dependent on the pHi during induction of the ATR, and the highest survival was observed following induction at a pHi of 5.9, which was the lowest pHi at which growth occurred. Increased acid tolerance and the ability to maintain a higher pHi during lethal acid stress were not acquired if protein synthesis was inhibited by chloramphenicol during adaptation.     

Intracellular pH controls cell membrane Na+ and K+ conductances and transport in frog skin epithelium

We determined the effects of intracellular respiratory and metabolic acid or alkali loads, at constant or variable external pH, on the apical membrane Na+-specific conductance (ga) and basolateral membrane conductance (gb), principally due to K+, in the short-circuited isolated frog skin epithelium. Conductances were determined from the current-voltage relations of the amiloride-inhibitable cellular current pathway, and intracellular pH (pHi) was measured using double barreled H+-sensitive microelectrodes. The experimental set up permitted simultaneous recording of conductances and pHi from the same epithelial cell. We found that due to the asymmetric permeability properties of apical and basolateral cell membranes to HCO3- and NH+4, the direction of the variations in pHi was dependent on the side of addition of the acid or alkali load. Specifically, changing from control Ringer, gassed in air without HCO3- (pHo = 7.4), to one containing 25 mmol/liter HCO3- that was gassed in 5% CO2 (pHo = 7.4) on the apical side caused a rapid intracellular acidification whereas when this maneuver was performed from the basolateral side of the epithelium a slight intracellular alkalinization was produced. The addition of 15 mmol/liter NH4Cl to control Ringer on the apical side caused an immediate intracellular alkalinization that lasted up to 30 min; subsequent removal of NH4Cl resulted in a reversible fall in pHi, whereas basolateral addition of NH4Cl produced a prolonged intracellular acidosis. Using these maneouvres to change pHi we found that the transepithelial Na+ transport rate (Isc), and ga, and gb were increased by an intracellular alkalinization and decreased by an acid shift in pHi. These variations in Isc, ga, and gb with changing pHi occurred simultaneously, instantaneously, and in parallel even upon small perturbations of pHi (range, 7.1-7.4). Taken together these results indicate that pHi may act as an intrinsic regulator of epithelial ion transport.     

Relationship between acid tolerance, cytoplasmic pH, and ATP and H+-ATPase levels in chemostat cultures of Lactococcus lactis.

The acid tolerance response (ATR) of chemostat cultures of Lactococcus lactis subsp. cremoris NCDO 712 was dependent on the dilution rate and on the extracellular pH (pHo). A decrease in either the dilution rate or the pHo led to a decrease in the cytoplasmic pH (pHi) of the cells, and similar levels of acid tolerance were observed at any specific pHi irrespective of whether the pHi resulted from manipulation of the growth rate, manipulation of the pHo, or both. Acid tolerance was also induced by sudden additions of acid to chemostat cultures growing at a pHo of 7.0, and this induction was completely inhibited by chloramphenicol. The end products of glucose fermentation depended on the growth rate and the environmental pHo of the cultures, but neither the spectrum of end products nor the total rate of acid production correlated with a specific pHi. The rate of ATP formation was not correlated with pHi, but a good correlation between the cellular level of H+-ATPase and pHi was observed. Moreover, an inverse correlation between the cytoplasmic levels of ATP and pHi was established. Each pHi below 6. 6 was characterized by unique levels of ATR, H+-ATPase, and ATP. High levels of H+-ATPase also coincided with high levels of acid tolerance of cells in batch cultures induced with sublethal levels of acid. We concluded that H+-ATPase is one of the ATR proteins induced by acid pHi through growth at an acid pHo or a slow growth rate.     

Influence of unsaturated fatty acids in chloroplasts. Shift of the pH optimum of electron flow and relations to deltapH, thylakoid internal pH and proton uptake.

Linolenic acid (C18:3) is the main endogenous unsaturated fatty acid of thylakoid membrane lipids, and seems in its free form to exert significant effects on the structure and function of photosynthetic membranes. In this investigation the effect of linolenic acid was studied at various pH values on the electron flow rate in isolated spinach chloroplasts and related to deltapH, the proton pump and the pH of the inner thylakoid space (pHi). The deltapH and pHi were estimated from the extent of the fluorescence quenching of 9-aminoacridine. Linolenic acid caused a shift (approximately one unit) of the pH optimum for electron flow toward acidity in the following systems: (a) photosystems II + I (from H2O to NADP+ or to 2,6-dichlorophenolindophenol) coupled or non-coupled; (b) photosystem II (from H2O to 2,6-dichlorophenolindophenol in the presence of dibromothymoquinone). In photosystem I conditions (phenazine methosulphate), the deltapH of the control increased as a function of external pHo with a maximum around pH 8.8. When linolenic acid was added, the deltapH dropped, but its optimum was shifted toward more acidic pHo. The same phenomena were also observed in photosytems II + I (from H2O to ferricyanide) and in photosystem II conditions (from H2O to ferricyanide in the presence of dibromothymoquinone). However, the deltapH was smaller and the sensitivity of the proton gradient toward linolenic acid was eventually higher than for photosystem I electron flow activity. The proton pump which might be considered as a measure of the internal buffering capacity of thylakoids was optimum at pHo, 6.7 in the controls. An addition of linolenic acid diminished the proton pump and shifted its optimum toward higher pHo. As a consequence, pHi increased when pHo was raised. At the optimal pHo 8.6 to 9, pHi were 5 to 5.5. Additions of increasing concentrations of linolenic acid displaced the curves toward higher pHi. A decrease of pHo was therefore required to maintain the pHi in the range of 5-5.5 for maximum electron flow. In conclusion, the electron flow activity seems to be delicately controlled by the proton pump (buffer capacity), deltapH, pHi and pHo. Fatty acids damage the membrane integrity in such a way that the subtile equilibrium between the factors is disturbed.     

Regulation of intracellular pH in LLC-PK1 cells studied using 31P-NMR spectroscopy

31P-NMR spectroscopy was used to monitor intracellular pH (pHi) in a suspension of LLC-PK1 cells, a renal epithelial cell line. The regulation of intracellular pH (pHi) was studied during intracellular acidification with 20% CO2 or intracellular alkalinization with 30 mM NH4Cl. The steady-state pHi in bicarbonate-containing Ringer's solution (pHo 7.40) was 7.14 +/- 0.04 and in bicarbonate-free Ringer's solution (pHo 7.40) 7.24 +/- 0.04. When pHo was altered in nominally HCO3(-)-free Ringer's, the intracellular pHi changed to only a small extent between pHo 6.6 and pHo 7.6; beyond this range pHi was linearly related to pHo. Below pHo 6.6 the cell was capable of maintaining a delta pH of 0.2 pH unit (inside more alkaline), above pH 7.6 a delta pH of 0.4 unit could be generated (inside more acid). During exposure to 20% CO2 in HCO3(-)-free Ringer's solution, pHi dropped initially to 6.9 +/- 0.05, the rate of realkalinisation was found to be 0.071 pH unit X min-1. After removal of CO2 the pHi increased by 0.65 and the rate of reacidification was 0.056 pH unit X min-1. Exposure to 30 mM NH4Cl caused a raise of pHi by 0.48 pH unit and an initial rate of re-acidification of 0.063 pH unit X min-1, after removal of NH4Cl the pHi fell by 0.58 pH unit below the steady-state pHi, followed by a subsequent re-alkalinization of 0.083 pH unit X min-1. Under both experimental conditions, the pHi recovery after an intracellular acidification, introduced by exposure to 20% CO2 and by removal of NH4+, was found to be inhibited by 53% and 63%, respectively, in the absence of sodium and 60% and 72%, respectively, by 1 mM amiloride. These studies indicate that 31P-NMR can be used to monitor steady-state intracellular pH as well a pHi transients in suspensions of epithelial cells. The results support the view that LLC-PK1 cells use an Na+-H+ exchange system to readjust their internal pH after acid loading of the cell.     

Intracellular pH recovery from lactic acidosis of single skeletal muscle fibers

Intracellular pH (pHi), measured with H+-selective microelectrodes, in quiescent frog sartorius muscle fibres was 7.29 +/- 0.09 (n = 13). Frog muscle fibres were superfused with a modified Ringer solution containing 30 mM HEPES buffer, at extracellular pH (pHo) 7.35. Intracellular pH decreased to 6.45 +/- 0.14 (n = 13) following replacement of 30 mM NaCl with sodium lactate (30 mM MES, pHo 6.20). Intracellular pH recovery, upon removal of external lactic acid, depended on the buffer concentration of the modified Ringer solution. The measured values of the pHi recovery rates was 0.06 +/- 0.01 delta pHi/min (n = 5) in 3 mM HEPES and was 0.18 +/- 0.06 delta pHi/min (n = 13) in 30 mM HEPES, pHo 7.35. The Na+-H+ exchange inhibitor amiloride (2 mM) slightly reduced pHi recovery rate. The results indicate that the net proton efflux from lactic acidotic frog skeletal muscle is mainly by lactic acid efflux and is limited by the transmembrane pH gradient which, in turn, depends on the extracellular buffer capacity in the diffusion limited space around the muscle fibres.     

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.     

Regulation of intracellular pH in human neutrophils

The intracellular pH (pHi) of isolated human peripheral blood neutrophils was measured from the fluorescence of 6-carboxyfluorescein (6-CF) and from the equilibrium distribution of [14C]5,5-dimethyloxazolidine -2,4-dione (DMO). At an extracellular pH (pHo) of 7.40 in nominally CO2-free medium, the steady state pHi using either indicator was approximately 7.25. When pHo was suddenly raised from 7.40 to 8.40 in the nominal absence of CO2, pHi slowly rose by approximately 0.35 during the subsequent hour. A change of similar magnitude in the opposite direction occurred when pHo was reduced to 6.40. Both changes were reversible. Intrinsic intracellular buffering power, determined by using graded pulses of CO2 or NH4Cl, was approximately 50 mM/pH over the pHi range of 6.8-7.9. The course of pHi obtained from the distribution of DMO was followed during and after imposition of intracellular acid and alkaline loads. Intracellular acidification was brought about either by exposing cells to 18% CO2 or by prepulsing with 30 mM NH4Cl, while pHo was maintained at 7.40. In both instances, pHi (6.80 and 6.45, respectively) recovered toward the control value at rates of 0.029 and 0.134 pH/min. These rates were reduced by approximately 90% either by 1 mM amiloride or by replacement of extracellular Na with N-methyl-D-glucamine. Recovery was not affected by 1 mM SITS or by 40 mM alpha-cyano-4-hydroxycinnamate (CHC), which inhibits anion exchange in neutrophils. Therefore, recovery from acid loading is probably due to an exchange of internal H for external Na. Intracellular alkalinization was achieved by exposing the cells to 30 mM NH4Cl or by prepulsing with 18% CO2, both at a constant pHo 7.40. In both instances, pHi, which was 7.65 and 7.76, respectively, recovered to the control value. The recovery rates (0.033 and 0.077 pH/min, respectively) were reduced by 80-90% either by 40 mM CHC or by replacement of extracellular Cl with p-aminohippurate (PAH). SITS, amiloride, and ouabain (0.1 mM) were ineffective.(ABSTRACT TRUNCATED AT 400 WORDS)     

The voltage-activated hydrogen ion conductance in rat alveolar epithelial cells is determined by the pH gradient

Voltage-activated H+ currents were studied in rat alveolar epithelial cells using tight-seal whole-cell voltage clamp recording and highly buffered, EGTA-containing solutions. Under these conditions, the tail current reversal potential, Vrev, was close to the Nernst potential, EH, varying 52 mV/U pH over four delta pH units (delta pH = pHo - pHi). This result indicates that H+ channels are extremely selective, PH/PTMA > 10(7), and that both internal and external pH, pHi, and pHo, were well controlled. The H+ current amplitude was practically constant at any fixed delta pH, in spite of up to 100-fold symmetrical changes in H+ concentration. Thus, the rate-limiting step in H+ permeation is pH independent, must be localized to the channel (entry, permeation, or exit), and is not bulk diffusion limitation. The instantaneous current- voltage relationship exhibited distinct outward rectification at symmetrical pH, suggesting asymmetry in the permeation pathway. Sigmoid activation kinetics and biexponential decay of tail currents near threshold potentials indicate that H+ channels pass through at least two closed states before opening. The steady state H+ conductance, gH, as well as activation and deactivation kinetic parameters were all shifted along the voltage axis by approximately 40 mV/U pH by changes in pHi or pHo, with the exception of the fast component of tail currents which was shifted less if at all. The threshold potential at which H+ currents were detectably activated can be described empirically as approximately 20-40(pHo-pHi) mV. If internal and external protons regulate the voltage dependence of gH gating at separate sites, then they must be equally effective. A simpler interpretation is that gating is controlled by the pH gradient, delta pH. We propose a simple general model to account for the observed delta pH dependence. Protonation at an externally accessible site stabilizes the closed channel conformation. Deprotonation of this site permits a conformational change resulting in the appearance of a protonation site, possibly the same one, which is accessible via the internal solution. Protonation of the internal site stabilizes the open conformation of the channel. In summary, within the physiological range of pH, the voltage dependence of H+ channel gating depends on delta pH and not on the absolute pH.     

Pyranine as a sensitive pH probe for liposome interiors and surfaces. pH gradients across phospholipid vesicles.

Pyranine is shown to be a convenient and sensitive probe for reporting pH values, pHi, at the interior of anionic and at the outer surface of cationic liposomes. It is well shielded from the phospholipid headgroups by water molecules in the interior of anionic liposomes, but it is bound to the surface of cationic liposomes. Hydrogen ion concentrations outside the liposomes, 'bulk pH values', pHo, were measured by a combination electrode. While pHi = pHo for neutral, pHi less than pHo for anionic and pHi greater than pHo for cationic liposomes prepared in 5.0 . 10(-3) M phosphate buffers. pKa values for the ionization of pyranine were 7.22 +/- 0.04 and 6.00 +/- 0.05 in water and at the external surface of cationic liposomes. The surface potential for cationic liposomes containing dipalmitoyl-DL-alpha-phosphatidylcholine, cholesterol and octadecylamine in the molar ratio of 1.00 : 0.634 : 1.01, were calcuated to be +72.2 mV. Proton permeabilities were measured for single and multicompartment anionic liposomes. Transfer of anionic liposomes prepared at a given pH to a solution of different pH resulted in a pH gradient if sodium phosphate or borate were used as buffers. In the presence of sodium acetate proton equilibration is promptly established.     

pH regulation in adult rat carotid body glomus cells. Importance of extracellular pH, sodium, and potassium [published erratum appears in J Gen Physiol 1993 Jan;101(1):following 144]

The course of intracellular pH (pHi) was followed in superfused (36 degrees C) single glomus (type I) cells of the freshly dissociated adult rat carotid body. The cells had been loaded with the pH-sensitive fluorescent dye 2',7'-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein. The high K(+)-nigericin method was used for calibration. The pHi of the glomus cell at pHo 7.40, without CO2, was 7.23 +/- 0.02 (n = 70); in 5% CO2/25 mM HCO3-, pHi was 7.18 +/- 0.08 (n = 9). The pHi was very sensitive to changes in pHo. Without CO2, delta pHi/delta pHo was 0.85 (pHo 6.20-8.00; 32 cells), while in CO2/HCO3- this ratio was 0.82 irrespective of whether pHo (6.80-7.40; 14 cells) was changed at constant PCO2 or at constant [HCO3-]o. The great pHi sensitivity of the glomus cell to pHo is matched only by that of the human red cell. An active Na+/H+ exchanger (apparent Km = 58 +/- 6 mM) is present in glomus cells: Na+ removal or addition of the amiloride derivative 5-(N,N-hexamethylene)-amiloride induced pHi to fall by as much as 0.9. The membrane of these cells also contains a K+/H+ exchanger. Raising [K+]o from 4.7 to 25, 50, or 140 mM reversibly raised pHi by 0.2, 0.3, and 0.6, respectively. Rb+ had no effect, but in corresponding concentrations of Tl+ alkalinization was much faster than in K+. Reducing [K+]o to 1.5 mM lowered pHi by 0.1. These pHi changes were shown not to be due to changes in membrane voltage, and were even more striking in the absence of Na+. Intrinsic buffering power (amount of strong base required to produce, in the nominal absence of CO2, a small pHi rise) increased from 3 to approximately 21 mM as pHi was lowered, but remained nearly unchanged below pHi 6.60. The fitted expression assumed the presence of one "equivalent" intracellular buffer (pK 6.41, 41 mM). The exceptional pHi sensitivity to pHo suggests that the pHi of the glomus cell is a link in the chemoreceptor's response to external acidity.     

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.     

Na(+)-dependent Cl-HCO3 exchange in the squid axon. Dependence on extracellular pH.

Intracellular pH (pHi) in squid giant axons recovers from acid loads by means of a Na(+)-dependent Cl-HCO3 exchanger, the actual mechanism of which might be exchange of: (i) external Na+ and HCO3- for internal Cl- and H+, (ii) Na+ plus two HCO3- for Cl-, (iii) Na+ and CO3= for Cl-, or (iv) the NaCO3- ion pair for Cl-. Here we examine sensitivity of transport to changes of extracellular pH (pHo) in the range 7.1-8.6. We altered pHo in four ways, using: (i) classical "metabolic" disturbances in which we varied [HCO3-]o, [NaCO3-]o, and [CO3=]o at a fixed [CO2]o; (ii) classical "respiratory" disturbances in which we varied [CO2]o, [NaCO3-]o, and [CO3=]o at a fixed [HCO3-]o; (iii) novel mixed-type acid-base disturbances in which we varied [HCO3-]o and [CO2]o at a fixed [CO3=]o and [NaCO3-]o; and (iv) a second series of novel mixed-type disturbances in which we varied [CO2]o, [CO3=]o, and [Na+]o at a fixed [HCO3-]o and [NaCO3-]o. Axons (initial pHi approximately 7.4) were internally dialyzed with a pH 6.5 solution containing 400 mM Cl- but no Na+. After pHi, measured with a glass microelectrode, had fallen to approximately 6.6, dialysis was halted. The equivalent acid extrusion rate (JH) was computed from the rate of pHi recovery (i.e., increase) in the presence of Na+ and HCO3-. When pHo was varied by method (i), which produced the greatest range of [CO3=]o and [NaCO3-]o values, JH increased with pHo in a sigmoidal fashion; the relation was fitted by a pH titration curve with a pK of approximately 7.7 and a Hill coefficient of approximately 3.0. With method (ii), which produced smaller changes in [CO3=]o and [NaCO3-]o, JH also increased with pHo, though less steeply. With method (iii), which involved changes in neither [CO3=]o nor [NaCO3-]o, JH was insensitive to pHo changes. Finally, with method (iv), which involved changes in neither [HCO3-] nor [NaCO3-]o, but reciprocal changes in [CO3=]o and [Na+]o, JH also was insensitive to pHo changes. We found that decreasing pHo from 8.6 to 7.1 caused the apparent Km for external HCO3- ([Na+]o = 425 mM) to increase from 1.0 to 26.7 mM, whereas Jmax was relatively stable. Decreasing pHo from 8.6 to 7.4 caused the apparent Km values for external Na+ ([HCO3-]o = 48 mM) to increase from 8.6 to 81 mM, whereas Jmax was relatively stable.(ABSTRACT TRUNCATED AT 400 WORDS)     

The relation between intracellular pH and rate of protein synthesis in sea urchin eggs and the existence of a pH-independent event triggered by ammonia

The relation between rate of protein synthesis and intracellular pH (pHi) was investigated in the eggs of the sea urchin Strongylocentrotus purpuratus. Increasing external pH (pHo) resulted in raising pHi of eggs and also in increased rate of protein synthesis. Similarly, at constant pHo, adding various concentrations of NH4Cl to eggs caused graded increases of both pHi and protein synthesis. Using various concentrations of NH4Cl at a low pHo and incubating eggs at high pHo, we compared protein synthesis under similar pHi conditions and this revealed that at least half the increased protein synthesis stimulated by NH4Cl is independent of induced rise of pHi, as also seems to be chromosome condensation which was never observed in eggs incubated at high pHoS. The additional pH-independent event triggered by NH4Cl does not appear related to elevated free Ca2+, since protein synthesis and chromosome condensation do not require external Ca2+ and no increases of free Ca2+ sufficient to activate the Ca2+-calmodulin-mediated enzyme NAD kinase occurred. Monensin disrupts intravesicular pH gradients but does not stimulate protein synthesis, indicating that this local effect, also promoted by NH4Cl, is not involved in ammonia-induced increase of protein synthesis. Using two other amines which have low pKa values, benzocaine and tricaine, we observed 2-fold increases in protein synthesis rates, even though pHi was lowered. While the exact nature of the pH-independent event(s) triggered by NH4Cl, and possibly by other amines, remains unidentified, its possible involvement in normal mitosis is stressed.     

Properties of the pH-sensitive site that controls the lambda max of Limulus metarhodopsin

A pH-sensitive site controls the lambda max of Limulus metarhodopsin. The properties of this site were examined using intracellular recordings of the early receptor potential (ERP) as a pigment assay. ERPs recorded over a range of extracellular pHs indicate that the apparent pK of the site is in the range of 8.3-8.6. Several lines of evidence indicate that the site responds directly to changes in extracellular pH (pHo) rather than to changes in intracellular pH(pHi) that follow as a secondary result of changing pHo : (a) the effect of changing pHo was rapid (less than 60 s); (b) when pHo was raised, the simultaneous rise in pHi, as measured with phenol red, was relatively small; (c) raising pHi by intracellular injection of pH 10 glycine buffer did not affect the site; and (d) the effect of changing pH0 could not be blocked by increasing the intracellular pH buffering capacity. It is concluded that the pH-sensitive site on metarhodopsin is on the extracellular surface of the plasma membrane.     

Role of bicarbonate in the regulation of intracellular pH in the mammalian ventricular myocyte.

Bicarbonate is important for pHi control in cardiac cells. It is a major part of the intracellular buffer apparatus, it is a substrate for sarcolemmal acid-equivalent transporters that regulate intracellular pH, and it contributes to the pHo sensitivity of steady-state pHi, a phenomenon that may form part of a whole-body response to acid/base disturbances. Both bicarbonate and H+/OH- transporters participate in the sarcolemmal regulation of pHi, namely Na(+)-HCO3-cotransport (NBC), Cl(-)-HCO3- exchange (i.e., anion exchange, AE), Na(+)-H+ exchange (NHE), and Cl(-)-OH- exchange (CHE). These transporters are coupled functionally through changes of pHi, while pHi is linked to [Ca2+]i through secondary changes in [Na+] mediated by NBC and NHE. Via such coupling, decreases of pHo and pHi can ultimately lead to an elevation of [Ca2+]i, thereby influencing cardiac contractility and electrical rhythm. Bicarbonate is also an essential component of an intracellular carbonic buffer shuttle that diffusively couples cytoplasmic pH to the sarcolemma and minimises the formation of intracellular pH microdomains. The importance of bicarbonate is closely linked to the activity of the enzyme carbonic anhydrase (CA). Without CA activity, intracellular bicarbonate-dependent buffering, membrane bicarbonate transport, and the carbonic shuttle are severely compromised. There is a functional partnership between CA and HCO3- transport. Based on our observations on intracellular acid mobility, we propose that one physiological role for CA is to act as a pH-coupling protein, linking bulk pH to the allosteric H+ control sites on sarcolemmal acid/base transporters.