Alkaline Load Research Articles

Cl −/base exchange and cellular pH regulation in enterocytes isolated from chick small intestine

Intracellular pH (pH i) and Cl −/base exchange activity have been examined in isolated chicken enterocytes, both in the presence and absence of 25 mM HCO 3 −/5% CO 2. Intracellular pH was measured with BCECF, a pH-sensitive car☐ylfluorescein derivative. Under resting conditions pH i was 7.17 in Hepes and 7.12 in HCO 3 −-buffered solutions. Cells became more alkaline upon withdrawal of Cl −. Cells depleted of Cl − acidified upon reinstatement of Cl −. These changes were faster in the presence of HCO 3 − than in its absence. After an alkaline load (removal of HCO 3 − from the medium) pH i decreases towards base line in the presence of Cl −, but not in its absence. The Cl −-dependent pH i changes were prevented by H 2DIDS and were unaffected by Na +. The Cl −-induced recovery from an alkaline load exhibited simple saturation kinetics, with an apparent K m of 12.5 mM Cl − and maximum velocity of ≈ 0.20pH units min −1. The Cl −/base exchange is functional under resting conditions, as shown by cell alkalinization on exposure to 0.5 mM H 2DIDS, both in the presence and in the absence of HCO 3 −. It is concluded that Cl −/base exchange participates in setting the resting intracellular pH in isolated chicken enterocytes and helps recover from alkaline loads. The exchange operates both in the presence and in the absence of bicarbonate.

Effects of pHi on Na(+)-H+, Na(+)-dependent, and Na(+)-independent C1(-)-HCO3-exchangers in vascular smooth muscle.

The mechanisms that control intracellular pH (pHi) in vascular smooth muscle are not fully understood. We reported that pHi in primary cultured vascular smooth muscle cells from canine femoral artery is 7.26, a value maintained via HCO3- influx by the Na(+)-dependent C1(-)-HCO3-exchanger but not via H+ efflux by the Na(+)-H+ exchanger [A. M. Kahn, E. J. Cragoe, Jr., J. C. Allen, R. D. Halligan, and H. Shelat. Am. J. Physiol 259 (Cell Physiol. 28): C134-C143, 1990]. To explain these findings, in the present study, we determined the pHi activity profile of these two transport systems. Although both were active at acidic pHi, Na(+)-H+ exchange activity was very low at and above pHi 7.0, while Na(+)-dependent C1(-)-HCO3-exchange activity maintained near-maximal activity up to pHi 7.26 but fell to undetectable levels by pHi 7.4. A Na(+)-independent C1(-)-HCO3-exchanger was present, which mediated HCO3-efflux after an acute alkaline load. The activity of this system was negligible at pHi 7.2 and was stimulated at alkaline pHi. In conclusion, the pHi of these vascular smooth muscle cells at rest is maintained via HCO3-influx by the Na(+)-dependent C1(-)-HCO3-exchanger. After acute acidic or alkaline loads, correction of pHi is mediated by activation of the normally quiescent Na(+)-H+ and Na(+)-independent C1(-)-HCO3-exchangers, respectively. All three acid-base transport systems have pHi set points that protect the cell from overcorrecting pHi after a disturbance in either direction.

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.

Cl(-)-HCO3- exchanger in isolated rat hepatocytes: role in regulation of intracellular pH

In rat hepatocytes, basolateral Na(+)-H+ exchange and Na(+)-HCO3- cotransport function as acid extruders. To assess mechanisms of acid loading, intracellular pH (pHi) recovery from an alkaline load was analyzed in short-term cultured rat hepatocyte monolayers using the pH-sensitive dye BCECF. Electrophysiological techniques were also used to assess the role of the membrane potential (Vm). Cells were alkaline loaded by suddenly reducing external CO2 and HCO3- (from 10% and 50 mM, respectively, to 5% and 25 mM) at constant pHo. After this maneuver, pHi rapidly rose by 0.13 +/- 0.03 pH units (pHu) and recovered to baseline at an initial rate of 0.026 +/- 0.009 pHu/min. Intracellular buffering power was estimated from the dependence of pHi on [NH4+]o and varied between 70 and 10.5 mM/pHu in a pHi range of 6.5-7.6. Initial pHi recovery corresponded to a rate of OH- efflux (JOH) of 1.76 +/- 0.71 mM/min and was blocked by 0.5 mM DIDS (0.003 +/- 0.002; JOH = 0.18 +/- 0.06) or by 1 mM H2DIDS (0.001 +/- 0.002; JOH = 0.26 +/- 0.08) and by removal of [Cl-]o (0.003 +/- 0.007; JOH = 0.28 +/- 0.07). The dependence of JOH on [Cl-]o exhibited saturation kinetics with an apparent Km for [Cl-]o of 5.1 mM. pHi recovery was Na+ independent and was not inhibited by substitution of Na+ with NMDG (0.045 +/- 0.09; JOH = 2.94 +/- 0.59). During an alkaline load, cell Vm hyperpolarized from -33.4 +/- 1.8 to -43.4 +/- 2.8 mV, mainly due to an increase in K+ conductance by a factor of 2.8 +/- 0.3.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-cell stage mouse embryos appear to lack mechanisms for alleviating intracellular acid loads.

Mouse embryos at the two-cell stage, like other cells, can recover from an intracellular acid-load. Our previous work has shown, surprisingly, that there is no contribution to this recovery by Na+/H+ antiport activity. Here we show that the recovery similarly is not affected by inhibition of other known intracellular pH (pHi) regulatory mechanisms. Specifically, the recovery is unaffected by lack of external Na+, inhibition of anion exchange, or lack of bicarbonate, which eliminates the Na(+)-dependent HCO3-/Cl- exchanger as a possible mechanisms. These conditions also eliminate any possible Na+,HCO3- cotransporter operating to relieve acid-loading. Recovery is unaffected similarly by nonspecific inhibitors of H(+)-ATPase activity. These observations lead to the conclusion that recovery from acid-load is a passive process in the two-cell mouse embryo. Similarly, the mean base-line pHi (6.84) is not dependent on known pHi regulatory mechanisms. The embryos exhibit a marked intracellular alkalinization when exposed to Cl(-)-free medium in the presence of bicarbonate. This response is eliminated by an inhibitor of anion exchange and by lack of bicarbonate, but is independent of Na+. These results indicate that there is probably a Na(+)-independent HCO3-/Cl- exchanger active in these cells, presumably functioning to alleviate alkaline loads.

Cl − permeability of the basolateral membrane of the Rana esculenta epithelium: activation of Cl −/HCO 3− exchange by alkaline intracellular pH

We have investigated Cl − transport mechanism(s) located in the basolateral membranes of the frog skin epithelium and in particular activation of Cl −/HCO 3 − exchange following an alkaline load. We found that 87% of the total 36Cl uptake by the epithelial cells occurs across the basolateral membranes ( J Cl − b) and submitting the epithelium to an alkaline load (HCO 3 −-Ringer solution, pH 8.1) increased J Cl − b. Intracellular Cl − activity ( a Cl − i), measured with ion-sensitive microelectrodes, increased when the Ringer solution bathing the basolateral membranes was changed from a Ringer solution equilibrated in air (pH 7.4) to one containing CO 2/HCO 3 − (pH 7.4). pH i recovery following an alkaline load was dependent on Cl − since it did not occur in serosal Cl −-free media, indicating the presence of a Cl −-dependent regulatory mechanism. Acid loading of the epithelial cells (5% CO 2, HCO 3 −-free Ringer) produced no change in J Cl − b but stimulated an amiloride-sensitive 22Na uptake across the basolateral membranes of the epithelium, compatible with an activation of a Na +/H + exchanger, previously described in this tissue. J Cl − b was partially blocked by SITS (5 · 10 −4 mmol/l), niflumic acid (5 · 10 −5 mmol/l), furosemide or bumetanide. Simultaneous addition of furosemide and niflumic acid produced an inhibition of J Cl − b which was not different with furosemide alone. Substitution of Na + by choline had no effect on J Cl − b and furosemide did not block the 22Na + uptake, suggesting that J Cl − b is not a Na +-dependent process (cotransport). We conclude that a significant Cl − permeability at the basolateral membranes of the epithelial cells is due to the presence of a Cl −/HCO 3 − exchanger which is essential for the recovery of pH i following an alkaline load.

Mechanism of intestinal secretion. Effect of serotonin on rabbit ileal crypt and villus cells.

To determine the mechanism of action of an intestinal secretagogue, serotonin, we have isolated crypt and villus cells and demonstrated Na:H and Cl:HCO3 exchange activity using the intracellular pH-sensitive fluorescent dye, 2,7-bis (carboxy-ethyl)-5,6-carboxy-fluorescein. Serotonin alkalinized both crypt and villus cells. Alkalinization in villus cells was HCO3 dependent and Na independent. In contrast, alkalinization in crypt cells was HCO3 independent and Na dependent. In villus cells, recovery from an alkaline load induced by Cl removal, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid or propionate pulse, known to occur via the Cl:HCO3 exchange, is inhibited by serotonin. In contrast, in crypt cells, recovery from an acid load induced by Na removal, amiloride and NH4Cl pulse, known to occur via Na:H exchange, is stimulated by serotonin. These data suggest that serotonin is inhibiting Cl:HCO3 exchange in villus cells and stimulating Na:H exchange in crypt cells. These effects of serotonin would be expected to inhibit coupled Na and Cl absorption by villus cells and stimulate HCO3 secretion by crypt cells in the intact ileum.

Hypoventilation in a Dialysis Patient with Severe Metabolic Alkalosis: Treatment by Hemodialysis

A patient with end-stage renal disease (ESRD) developed metabolic alkalosis and alkalemia from protracted vomiting. As a result of the absence of the alkali excretory capacity in this patient with ESRD, the alkaline load accumulated rapidly. Once the amount of acid lost from vomiting exceeded the amount of acid gained from metabolism, alkalemia supervened. The initial arterial blood gas on room air revealed hypercarbia, hypoxia and alkalemia. Her serum bicarbonate was greater than 50 mEq/l. Compensatory hypoventilation occurred. In this report, the extent of compensatory hypoventilation in the setting of metabolic alkalosis in patients treated for ESRD and therapeutic approaches to this problem will be discussed. Treatment was aimed at correcting the primary disorder, namely metabolic alkalosis. Conventional bicarbonate dialysis was shown to be effective in improving acid-base homeostasis in this patient.

Intracellular pH recovery from alkalinization. Characterization of chloride and bicarbonate transport by the anion exchange system of human neutrophils.

The nature of the intracellular pH-regulatory mechanism after imposition of an alkaline load was investigated in isolated human peripheral blood neutrophils. Cells were alkalinized by removal of a DMO prepulse. The major part of the recovery could be ascribed to a Cl-/HCO3- counter-transport system: specifically, a one-for-one exchange of external Cl- for internal HCO3-. This exchange mechanism was sensitive to competitive inhibition by the cinnamate derivative UK-5099 (Ki approximately 1 microM). The half-saturation constants for binding of HCO3- and Cl- to the external translocation site of the carrier were approximately 2.5 and approximately 5.0 mM. In addition, other halides and lyotropic anions could substitute for external Cl-. These ions interacted with the exchanger in a sequence of decreasing affinities: HCO3- greater than Cl approximately NO3- approximately Br greater than I- approximately SCN- greater than PAH-. Glucuronate and SO4(2-) lacked any appreciable affinity. This rank order is reminiscent of the selectivity sequence for the principal anion exchanger in resting cells. Cl- and HCO3- displayed competition kinetics at both the internal and external binding sites of the carrier. Finally, evidence compatible with the existence of an approximately fourfold asymmetry (Michaelis constants inside greater than outside) between inward- and outward-facing states is presented. These results imply that a Cl-/HCO3- exchange mechanism, which displays several properties in common with the classical inorganic anion exchanger of erythrocytes, is primarily responsible for restoring the pHi of human neutrophils to its normal resting value after alkalinization.

Absorbance signals from resting frog skeletal muscle fibers injected with the pH indicator dye, phenol red.

Singly dissected twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with the pH indicator dye, phenol red. Dye-related absorbances in myoplasm, denoted by A0(lambda) and A90(lambda), were estimated as a function of wavelength lambda (450 nm less than or equal to lambda less than or equal to 640 nm) with light polarized parallel (0 degrees) and perpendicular (90 degrees) to the fiber axis respectively. At all lambda, A0(lambda) was slightly greater than A90(lambda), indicating that some of the phenol red molecules were bound to oriented structures accessible to myoplasm. The phenol red "isotropic" signal, [A0(lambda) + 2A90(lambda)]/3, a quantity equal to the average absorbance of all the dye molecules independent of their orientation, had a spectral shape that was red-shifted by approximately 10 nm in comparison with in vitro dye calibration curves measured in 140 mM KCl. The red-shifted spectrum also indicates that some phenol red molecules were bound in myoplasm. A quantitative estimate of indicator binding was obtained from measurements of the dye's apparent diffusion constant in myoplasm, denoted by Dapp. The small value of Dapp, 0.37 x 10(-6) cm2 s-1 (at 16 degrees C), can be explained if approximately 80% of the dye was bound to myoplasmic sites of low mobility. To estimate the apparent myoplasmic pH, denoted by pHapp, the isotropic absorbance of phenol red was fitted by in vitro calibration spectra. pHapp was found to be independent of dye concentration (0.2-2 mM), but varied widely (range, 6.8-7.5; mean value, 7.17) among fibers judged from functional characteristics to be normal. When fibers were subjected to acid or alkaline loads by exposure to Ringer's solution containing, respectively, dissolved CO2 or NH3, the changes in pHapp were in agreement with those expected from pH micro-electrode studies. It is concluded that in spite of the several indications for the presence of bound phenol red inside muscle cells, the pHapp signal from the indicator is useful for monitoring changes in myoplasmic pH in response to physiological and pharmacological manipulations.

The Regulation of Intracellular pH Estimated By 31P-Nmr Spectroscopy in the Anterior Byssus Retractor Muscle of Mytilus Edulis L

ABSTRACT The regulation of the intracellular pH (pHi) in the anterior byssus retractor muscle (ABRM) of Mytilus edulis L. during an acidic and alkaline load was studied by 31P-NMR spectroscopy. Under aerobic conditions, a total intracellular buffer capacity of 26.5±0.7mmol l−1 pH unit−1 (N=3) was estimated. In the absence of serotonin, active acid extrusion was by a Na+-independent, primary active transport mechanism coupled to anion transport. Base equivalents appear to be extruded by a C1−/HCO3− exchanger. Serotonin (10−5 moll−1) induced an increase in pHi through additional activation of a Na+/H+ exchanger.

Intracellular pH regulates Na(+)-independent Cl(-)-base exchange in JTC-12 (proximal tubule) cells

The role of an anion exchange pathway in the regulation of intracellular pH (pHi) under alkaline load and steady-state conditions and the modulation of this transporter by pHi was investigated in confluent monolayers of cloned JTC-12 cells, derived from monkey kidney proximal tubule. Regulation of pHi was fluorometrically monitored using the pH-sensitive probe, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Monolayers in which pHi was rapidly elevated by removal of HCO3(-)-CO2 from the bathing medium demonstrated an absolute requirement for Cl- to recover toward base-line pHi. The recovery process proceeded in the absence of Na+, was inhibited 80% by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and was unaffected by 10(-4) M amiloride. When extracellular pH (pHo) was serially lowered from 7.4 to 6.7, recovery from an alkaline load induced by removal of HCO3(-)-CO2 from the medium occurred only when pHi was elevated above approximately 7.25. Below pHi approximately 7.25 no recovery toward initial pHi was observed. When pHi was elevated above approximately 7.25 with pHo maintained at 6.7, the recovery process ceased at pHi approximately 7.25 despite favorably oriented Cl- and OH- chemical gradients. Consistent with these observations, removal of Cl- from the medium of cells buffered with 25 mM HCO3(-)-5% CO2 at pHo 7.4 (in the absence of Na+) resulted in reversible elevation of pHi, whereas in a solution buffered to pHo 6.7 with 5 mM HCO3(-)-5% CO2, removal of Cl- failed to elevate pHi. Under steady-state conditions in the presence of 25 mM HCO3(-)-5% CO2 at pHo 7.4, pHi was 7.40 +/- 0.02 and reversibly decreased to 7.23 +/- 0.01 on removal of Na+ (in the presence of amiloride) from the bathing medium, indicating that the Cl(-)-base exchanger is operative under basal conditions and functions as a base extruder. In summary the JTC-12 cell possesses a Na(+)-independent Cl(-)-base exchange mechanism that is operative under alkaline load and steady-state conditions. pHi but not pHo modulates the activity of this transport pathway, and below pHi approximately 7.25 the exchanger is quiescent.

Intracellular pH dependence of buffer capacity and anion exchange in the parietal cell

When parietal cells (PC) are stimulated with histamine, the anion exchanger rate increases three to five times to compensate for alkaline loading induced by H+-K+ adenosinetriphosphatase (ATPase) and to provide Cl for acid secretion. It has been hypothesized that this increased activity is caused by the increase in intracellular pH (pHi) that often occurs in stimulated PC (from 7.1 to a maximum of 7.3). The dependence of the anion exchanger on pHi was studied using microspectrofluorimetry of the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solutions were used because the anion exchanger can transport OH- (or HCO3) in exchange for Cl- even with [HCO3]o = 200 microM. It was found that when solutions were changed either from NaCl to Cl- free or Cl- free to NaCl, rates of change of pHi (delta pH/delta t) were strongly dependent on pHi: nearly 0 at pHi 6.6 and 1.25 pH/min at pHi 8.0. To convert these pHi changes into anion flux rates, the intrinsic buffer capacity (beta i) was determined over the same pHi range by making small changes of [NH4]o to determine the resulting changes of [NH4]i and pHi (i.e., beta i = delta[NH4]i/delta pHi) in PC that had been pretreated with 1 mM amiloride and 200 microM [H2]4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) [to block Na+-H+ and Cl- -OH-(HCO3-) exchange]. beta i was also strongly dependent on pHi: at pHi 6.5 beta i = 48 mM/pH, and this decreased as pHi increased; at pHi 7.75 beta i = 8 mM/pH. The derived anion fluxes (i.e., JOH = beta i x delta pH/delta t) were roughly linearly related to pHi between 6.6 (JOH near 0) and 8.1 (JOH = 13 mM/min). Between pHi 7.1 and 7.3, the range normally observed during stimulation of PC, rates of anion exchange increased by 75%. This pHi sensitivity cannot explain the 300-500% increase in anion exchanger activity observed during secretagogue stimulation of PC.

PH regulation in hepatoma cells: roles for Na-H exchange, Cl-HCO3 exchange, and Na-HCO3 cotransport

Regulation of intracellular pH (pHi) was studied in Fu5, a rat hepatoma cell line that maintains a variety of differentiated functions. Microspectrofluorimetry of the pH-sensitive dye 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) was used to measure pHi in 10-15 cells growing on cover glasses that were mounted in a flow-through chamber on the stage of a microscope. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solutions, pHi was 7.14, and intrinsic buffer capacity was inversely related to pHi. Amiloride (0.1 mM) caused pHi to decrease by 0.33 pH units in 4 min. Recovery from an acid load (using either NH4 prepulse technique or Na-free solutions) was completely blocked by amiloride. In HCO3-CO2-buffered solutions, pHi was 7.15, and buffer capacity was relatively insensitive to pHi between pHi of 6.6 and 7.2. Amiloride caused pHi to decrease by only 0.09 units. Recovery from an acid load was Na dependent, occurred in Cl-free solutions, and was totally blocked by the combination of amiloride plus 0.5 mM dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2DIDS); recovery occurred when either amiloride or H2DIDS was removed. Removal of external Cl caused a rapid, H2DIDS-blockable alkalinization that was faster in HCO3-CO2 than in HEPES. The apparent Km for Clout for relaxation of Cl-free alkalinization was 4.5 mM. Rate of HCO3 transport during Cl-free treatment increased at alkaline resting pHi. It is concluded that Fu5 cells have two Na-dependent base-loading mechanisms and an acid-loading Cl-HCO3 exchanger. In solutions containing HCO3-CO2, the Na-H exchanger accounts for approximately 40% of recovery from an acid load, and a Na-HCO3 cotransporter accounts for the remainder. Recovery from an alkaline load appears to occur through the activity of the Cl-HCO3 exchanger.

Cl-/base exchange in rat mesangial cells: Regulation of intracellular pH

The present study was designed to determine whether rat glomerular mesangial cells possess Cl- -dependent intracellular pH (pHi) regulatory processes. Rat glomerular mesangial cells were grown to confluence on glass coverslips. Intracellular pH (pHi) was measured with BCECF. Steady state pHi in HCO3- containing solutions was 7.08 +/- 0.03 (N = 13). When extracellular Cl- was acutely removed, pHi increased at a rate of 0.57 +/- 0.03 pH/min units (N = 8), P less than 0.001. DIDS (0.5 mM) significantly decreased the rate of increase in pHi to 0.34 +/- 0.04 pH/min, P less than 0.01. Na+ removal and amiloride (1 mM) did not alter the increase in pHi induced by Cl- removal. Steady state pHi in the absence of Cl- was significantly increased above control, 7.39 +/- 0.02 (N = 7), P less than 0.001. Following the acute alkalinization of pHi by CO2 removal, pHi recovered at a rate of 0.07 +/- 0.01 pH/min (N = 9). In the absence of Cl-, the pHi recovery rate was significantly decreased to 0.01 +/- 0.008 pH/min (N = 5), P less than 0.01. DIDS (0.5 mM) significantly decreased the rate of pHi recovery to 0.02 +/- 0.01 pH/min (N = 5), P less than 0.01. Na+ removal and amiloride (1 mM) had no effect on the rate of pHi recovery following acute alkaline loading.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytoplasmic pH regulation and chloride/bicarbonate exchange in avian osteoclasts.

Osteoclasts resorb bone by first attaching to the bone surface and then secreting protons into an isolated extracellular compartment formed at the cell-bone attachment site. This secretion of protons (local acidification) is required to solubilize bone hydroxyapatite crystals and for activity of bone collagen-degrading acid proteases. However, the large quantity of protons required, 2 mol/mol of calcium, would result in an equal accumulation of cytosolic base equivalents. This alkaline load must be corrected to maintain cytosolic pH within physiologic limits. In this study, we have measured cytoplasmic pH with pH-sensitive fluorescent compounds, while varying the extracellular ionic composition of the medium, to determine the nature of the compensatory mechanism used by osteoclasts during bone resorption. Our data show that osteoclasts possess a chloride/bicarbonate exchanger that enables them to maintain normal intracellular pH in the face of a significant proton efflux. This conclusion follows from the demonstration of a dramatic cytoplasmic acidification when osteoclasts that have been incubated in bicarbonate-containing medium are transferred into bicarbonate-free medium. This acidification is absolutely dependent on and proportional to medium [Cl-]. Furthermore, acidification is inhibited by the classic inhibitor of red cell anion exchange, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, and by diphenylamine-2-carboxylate, an inhibitor of chloride specific channels. However, the acidification process is neither energy nor sodium dependent. The physiologic importance of chloride/bicarbonate exchange is demonstrated by the chloride dependence of recovery from an endogenous or exogenous alkaline load in osteoclasts. We conclude that chloride/bicarbonate exchange is in large part responsible for cytoplasmic pH homeostasis of active osteoclasts, showing that these cells are similar to renal tubular epithelial cells in their regulation of intracellular pH.

Bicarbonate reabsorption by the kidney of the newborn rabbit.

Bicarbonate reabsorption by the immature kidney in response to acute acid-base changes was assessed in 50 anesthetized newborn rabbits before the end of nephrogenesis. The normal newborn rabbit (age 5-12 days) is in a state of hypochloremic metabolic alkalosis (PHCO3-, 31.9 +/- 0.6 mmol/l; PCl-, 83.1 +/- 1.0) and excretes a hypertonic (Uosmol = 578 +/- 41 mosmol/kgH2O), alkaline (UpH = 7.40 +/- 0.15) urine containing 50 +/- 9 mmol/l Cl- and 13 +/- 4 mmol/l Na+. The alkalosis is probably generated by an alkaline load contained in the mother's milk and maintained by a state of chloride wasting and volume contraction. In this alkalotic model, bicarbonate reabsorption, expressed per milliliter glomerular filtration rate (GFR), correlates positively with arterial CO2 pressure (PaCO2). The ability of the immature kidney to reclaim filtered bicarbonate in response to an elevation of the plasma carbon dioxide tension remains unlimited up to PaCO2 of 110 mmHg (y = 20.7 + 0.15 x, r = 0.82, P less than 0.001). Hypercapnia is associated with a marked fall in GFR, so that the positive correlation between bicarbonate reabsorption and PaCO2 vanishes when the bicarbonate reabsorption rate is expressed in absolute terms. Bicarbonate reabsorption is strongly dependent on the filtered load during both acutely induced metabolic acidosis and alkalosis. The acid-base state of the newborn rabbit is in sharp contrast with that of most animal species, and the renal handling of bicarbonate as a function of GFR does not show signs of tubular immaturity.

Pathogenesis of abnormal acid–base balance in the young spontaneously hypertensive rat

1. We have previously reported reduced blood pH and plasma bicarbonate in young Okamoto-Aoki spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto rats (WKY). Acid loading with 1.5% (w/v) NH4Cl as the sole drinking fluid produced identical falls in blood pH, the difference remaining significant. 2. The ability of SHR to excrete acid and alkaline loads was compared with that of WKY under metabolic cage conditions. The effects of such manipulations on urinary sodium, potassium, calcium and phosphate excretion were also determined. 3. No difference was found in the ability to excrete an acid load or to reduce urine pH. Neither total urinary ammonium ion nor titratable acid differed significantly between the strains under either baseline or acid-loading conditions. 4. Baseline urinary bicarbonate excretion was not significantly different between strains but intraperitoneal administration of NaHCO3 at 2.0 mmol/kg body weight resulted in enhanced excretion in the SHR (SHR vs WKY: 625.2 +/- 71.5 vs 381.8 +/- 40.6 mumol 24 h-1 kg-1 body weight, P less than 0.01, mean +/- SEM). 5. No difference in urinary sodium or potassium excretion was observed between SHR and WKY, but basal calcium and phosphate excretion were reduced in SHR (P less than 0.05). 6. Increased urinary bicarbonate excretion in the presence of significantly reduced plasma bicarbonate suggests reduced tubular reabsorption of bicarbonate, which may contribute to the mild metabolic acidosis in young SHR.

Evidence for Na/H exchange and Cl/HCO3 exchange in A10 vascular smooth muscle cells.

In the present study we used the pH sensitive absorbance of 5(and6)-carboxy-4',5'-dimethylfluorescein to investigate intracellular pH (pHi) regulation in A10 vascular smooth muscle cells: (1) The steady state pHi in A10 cells averaged 7.01 +/- 0.1 (mean +/- SEM, n = 26) at an extracellular pH of 7.4 (28 mM HCO3/5% CO2). (2) Removal of extracellular sodium led to an intracellular acidification of 0.36 +/- 0.07 pH-units (mean +/- SEM, n = 8). (3) pHi-Recovery after an acute intracellular acid load (by means of NH4Cl-prepulse) was reversibly blocked by 1 mM amiloride and was dependent on the presence of sodium. The velocity of pHi recovery increased with increasing sodium concentrations with an apparent Km for external sodium of about 30 mM and a Vmax of about 0.35 pH units/min. These findings are compatible with a Na/H exchanger being responsible for pHi recovery after an acid load. (4) Removal of extracellular chloride induced an intracellular alkalinization of 0.23 +/- 0.03 pH-units (mean +/- SEM, n = 10). The alkalinization was dependent on the presence of extracellular bicarbonate. (5) Removal of chloride during pHi recovery from an alkaline load (imposed by acetate prepulse) stopped and reversed pHi backregulation. Chloride removal had no effect in the absence of bicarbonate or in the presence of 10(-4) M DIDS, suggesting that the effects were mediated by a Cl/HCO3 exchanger. In conclusion we have demonstrated evidence for a Na/H exchanger and a Cl/HCO3 exchanger in A10 vascular smooth muscle cells.

Characterization of Na+-linked and Na+-independent Cl-/HCO3- exchange systems in Chinese hamster lung fibroblasts.

The PS120 variant of Chinese hamster lung fibroblasts which lacks Na+/H+ exchange activity was used to investigate bicarbonate transport systems and their role in intracellular pH (pHi) regulation. When pHi was decreased by acid load, bicarbonate caused pHi increase and stimulated 36Cl- efflux from the cells, both in a Na+-dependent manner. These results together with previous findings that bicarbonate stimulates 22Na+ uptake in PS120 cells (L'Allemain, G., Paris, S., and Pouyssegur, J. (1985) J. Biol. Chem. 260, 4877-4883) demonstrate the presence of a Na+-linked Cl-/HCO3- exchange system. In cells with normal initial pHi, bicarbonate caused Na+-independent pHi increase in Cl(-)-free solutions and stimulated Na+-independent 36Cl- efflux, indicating that a Na+-independent Cl-/HCO3- exchanger is also present in the cell. Na+-linked and Na+-independent Cl-/HCO3- exchange is apparently mediated by two distinct systems, since a [(tetrahydrofluorene-7-yl)oxy]acetic acid derivative selectively inhibits the Na+-independent exchanger. An additional distinctive feature is a 10-fold lower affinity for chloride of the Na+-linked exchanger. The Na+-linked and Na+-independent Cl-/HCO3- exchange systems are likely to protect the cell from acid and alkaline load, respectively.