Role of nonconserved charged residues of the AE2 transmembrane domain in regulation of anion exchange by pH

The ubiquitous AE2/SLC4A2 anion exchanger is acutely and independently regulated by intracellular (pH_i) and extracellular pH (pH_o), whereas the closely related AE1/SLC4A1 of the red cell and renal intercalated cell is relatively pH-insensitive. We have investigated the contribution of nonconserved charged residues within the C-terminal transmembrane domain (TMD) of AE2 to regulation by pH through mutation to the corresponding AE1 residues. AE2-mediated Cl⁻/Cl⁻ exchange was measured as 4,4′-di-isothiocyanatostilbene-2,2′-disulfonic acid-sensitive ³⁶Cl⁻ efflux from Xenopus oocytes by varying pH_i at constant pH_o, and by varying pH_o at near-constant pH_i. All mutations of nonconserved charged residues of the AE2 TMD yielded functional protein, but mutations of some conserved charged residues (R789E, R1056A, R1134C) reduced or abolished function. Individual mutation of AE2 TMD residues R921, F922, P1077, and R1107 exhibited reduced pH_i sensitivity compared to wt AE2, whereas TMD mutants K1153R, R1155K, R1202L displayed enhanced sensitivity to acidic pH_i. In addition, pH_o sensitivity was significantly acid- shifted when nonconserved AE2 TMD residues E981, K982, and D1075 were individually converted to the corresponding AE1 residues. These results demonstrate that multiple conserved charged residues are important for basal transport function of AE2 and that certain nonconserved charged residues of the AE2 TMD are essential for wild-type regulation of anion exchange by pH_i and pH_o. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. A. K. Stewart and C. E. Kurschat have contributed equally to this work.     

Effect of human defensins on the cytoplasmic Ca2+ content in platelets

The effect of the total fraction of human defensins (HNP-1, HNP-2, and HNP-3) on the cytoplasmic Ca²⁺ content ([Ca²⁺]_i) in the platelets of healthy donors was studied. At concentrations of 0.1–40 μg/ml and an incubation time of 10 min defensins have no effect on [Ca²⁺]_i in platelets labeled with Fura-2AM. However, at higher concentrations (100 μg/ml) they increased platelet [Ca²⁺]_i. In addition, defensins (40 μg/ml) inhibited the Ca²⁺ increase in platelets induced by thrombin, adenosine diphosphate, and the lipopolysaccharide ofS. typhimurium endotoxin. The most pronounced inhibitory effect was observed in a suspension of thrombin-stimulated platelets. It is shown that the effect of human defensins on the functional activity of platelets is due to the alterations in the intracellular Ca²⁺. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 12, pp. 600–603, December, 1994     

A study of the mechanism of action of befol on Ca2+ metabolism in cardiomyocytes using a FURA-2 fluorescent probe

The dynamics of the Ca-response of cardiomyocytes is studied and the efficiency of befol, verapamil, and amiodarone is compared using various experimental models of stimulation of [Ca²⁺]_i. Befol (1–5 μM) is shown to inhibit the caffeine-and strophanthin G-induced rise of [Ca²⁺]_i. Unlike verapamil and amiodarone, befol exhibits no Ca-blocking activity in modeled K-depolarization. It is concluded that the cardiotropic effect of befol is mediated through its primary action on Na⁺/Ca²⁺ exchange in cardiomyocytes, while the cardioplegic effect of verapamil and amiodarone is due to their ability to block the slow Ca²⁺ inward current. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, N ^o 3, pp. 288–291, March, 1996     

Intracellular pH and buffer power of type 1 and 2 fibres from skeletal muscle ofXenopus laevis

Intracellular pH (pH_i) and buffering power of type 1 and type 2 fibres from the iliofibularis muscle of the clawed frog,Xenopus laevis, have been measured using pH-sensitive microelectrodes. In phosphate buffered Ringer's solution (extracellular pH 7.25, 20–22°C), mean pH_i and its variance were similar in the two fibre types (6.86±SD 0.15±SEM 0.03,n=24, type 1, and 6.86±SD 0.12±SEM 0.03,n=15, type 2). On changing to Ringer's solution containing CO₂ and HCO ₃ ^– (extracellular pH 7.25, 20–22°C), pH_i became more acid in both fibre types. Although H⁺ ions were not at electrochemical equilibrium across the surface membrane, active transport did not return pH_i to its original value during exposure to CO₂. The buffering powers calculated from the changes in pH_i were not significantly different, 41.6 mmol·l^–1 per pH unit (±SEM 4.0,n=17) for type 1 and 49.3 mmol·l per pH unit (±SEM 7.2,n=11) for type 2 fibres. Thus differences in the mechanical properties of these fibre types are not due simply to a difference of the intracellular pH or buffering of resting fibres. Other possible explanations are discussed for the changes in some contractile properties that occur when pH_i is acidified.     

Modulation of the oxidative burst in mouse macrophages and human neutrophils by superlow concentrations of GM1 ganglioside

It is shown that ganglioside GM1 in picomolar concentrations stimulates the phorbol-12-myristate-13-acetate (PMA)-induced generation of active forms of oxygen by neutrophils and peritoneal macrophages. GM1 (10⁻¹¹ M) is found to enhance the luminol-dependent chemiluminescence induced by 10⁻⁸ M PMA in mouse macrophages in comparison with the effect of PMA alone. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 1, pp. 44–46, January, 1994 Presented by A. N. Klimov, Member of the Russian Academy of Medical Sciences     

Evidence for electrogenic sodium-bicarbonate cotransport in cultured rat cerebellar astrocytes

We have studied the regulation of intracellular pH (pH_i), and HCO ₃ ⁻ -dependent membrane currents in cultured astrocytes from neonatal rat cerebellum, using the fluorescent pH-sensitive dye 2,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and the whole-cell patch-clamp technique. The steady-state pH_i was 6.96 in both nominally CO₂/HCO ₃ ⁻ -free, HEPES-buffered saline (6.96 ±0.14;n=48) and in a saline containing 5% CO₂/24 mM HCO ₃ ⁻ (6.96±0.18;n=48) (at pH 7.4). Inhibition of the Na⁺/H⁺ exchange by amiloride (2 mM) caused a significant decrease of pH_i in nominally CO₂/HCO ₃ ⁻ -free saline. Addition of CO₂/HCO ₃ ⁻ in the continuous presence of amiloride induced a large and fast intracellular alkalinization. Removal of external Na⁺ also caused a fall of pH_i, and addition of CO₂/HCO ₃ ⁻ in Na⁺-free saline evoked a further fall of pH_i, while the outward current was reduced or even reversed. The stilbene 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS, 0.3 mM) reduced the pH_i recovery from the CO₂/HCO ₃ ⁻ -evoked acidification, and blocked the prominent intracellular acidification upon removal of CO₂/HCO ₃ ⁻ . Removal of external Cl⁻ had little effect on these pH_i changes. Lowering the external pH from 7.4 to 6.6 in CO₂/HCO ₃ ⁻ -containing saline produced a large and rapid intracellular acidification and inward current, which were both greatly reduced by DIDS and in the absence of CO₂/HCO ₃ ⁻ . The results suggest that the CO₂/HCO ₃ ⁻ -dependent current is partly due to a reversible bidirectional, electrogenic Na⁺-HCO ₃ ⁻ cotransporter, which helps to regulate pH_i in these cells. In addition, a prominent Na⁺/H⁺ exchanger contributes to extrude acid equivalents from these astrocytes to maintain the steadystate pH_i.     

Na+/H+ exchange regulates intracellular pH of rat gastric surface cells in vivo

Intracellular pH (pH_i) and viability of gastric surface cells of the rat stomach in response to luminal acidification, and the role of Na⁺/H⁺ exchange in maintaining pH_i homeostasis were studied in vivo using a fluorescent microscopic technique. pH_i was measured during superfusion with buffers of pH 1.2–7.4. When the pH of the superfusate was 7.4, baseline pH_i was unchanged. Superfusion with pH 3 buffer rapidly decreased pH_i to 6.7, with subsequent recovery to baseline pH_i within 15 min despite continuing acid exposure. Superfusion with buffers of pH 1.7 and 1.2 decreased pH_i continuously to below 6.2 with no recovery observed. Despite the relentless decline in pH_i during superfusion with pH-1.2 and –1.7 solutions, over 75% of the surface cells were still viable, as measured by exclusion of the vital dye propidium iodide. We then examined the role of Na⁺/H⁺ exchange in the regulation of pH_i. Superfusion with amiloride did not affect recovery of pH_i from intracellular acidification induced by a NH₄Cl prepulse. Exposure to the potent, lipophilic Na⁺/H⁺ exchange inhibitor 5-(N,N-hexaniethylene)-amiloride (HMA), either in the superfusate or by close arterial perfusion, decreased baseline pH_i from 7.1 to 6.8. Close arterial perfusion of HMA additionally attenuated the recovery of pH_i to baseline during superfusion with pH 3 buffer. We conclude that luminal protons permeate into the cytoplasm of gastric surface cells, where they are eliminated by an Na⁺/H⁺ exchanger, most probably localized to the basolateral membrane.     

The colon carcinoma cell line Caco-2 contains an H+/K+-ATPase that contributes to intracellular pH regulation

The presence of an H⁺/K⁺-ATPase and its contribution to the regulation of intracellular pH (pH_i) was investigated in Caco-2 cells. The H⁺/K⁺-ATPase was detected immunologically using the monoclonal antibody 5-B6, which was raised against hog gastric H⁺/K⁺-ATPase. Cell pH was determined using the pH-sensitive dye 2,7-bis(carboxyethyl)-carboxyfruorescein. Control pH_i, measured in HCO ₃ ^– -free medium, was 7.62±0.03 (n=27) when cells were cultured for 14 days and decreased to 7.40±0.03 (n=18) after 35 days in culture. Recovery of pH_i following a NH ₄ ⁺ /NH₃ pulse could be reduced by either 100 M SCH 28080 or 1 mM amiloride, or by removing extracellular Na⁺. The inhibitory effects of SCH 28080 and amiloride were additive, demonstrating the involvement of a gastric-like H⁺/K⁺-ATPase and a Na⁺/H⁺ exchanger in regulating pH_i. Recovery rates at pH_i 6.8 were not significantly different in cells cultured for up to 21 days, but were significantly lower in cells cultured for 28 and 35 days. This decrease in recovery rate was due to a decrease in the SCH-28080-insensitive recovery, indicating a reduction of the relative importance of Na⁺/H⁺ exchange to the recovery. Recovery of pH_i was also inhibited by 1 mM N-ethylmaleimide. However, it is unlikely that N-ethylmaleimide inhibited a vacuolar type of H⁺-ATPase, since bafilomycin A₁ had no effect on pH_i recovery. In conclusion, Caco-2 cells contain a SCH-28080-sensitive mechanism for regulating pH_i, which is most conveniently studied after 28 days in culture, when the relative contribution of a Na⁺/H⁺ exchanger to pH_i regulation is decreased.     

Electrophysiological investigation of microdissected gastric glands of bullfrog II. Basolateral membrane properties in the presence of histamine

Following the technical approach described in the preceding publication we have investigated if, and how, stimulation of gastric HCl secretion affects the basolateral ion transport properties of oxyntopeptic cells of Rana catesbeiana stomach. To this end microdissected gastric glands were punctured with conventional or H⁺-sensitive glass microelectrodes and the effects of changing bath ion concentrations on the cell membrane potential (V _b) and cell pH (pH_i) were determined. Except for a transient alkalinization, histamine (0.5 mmol/l) did not significantly affect V _b or pH_i. The latter averaged 7.18±0.03 (mean±SEM, n=5) under resting conditions (0.1 mmol/l cimetidine) and 7.21±0.07 (n=5) in the presence of histamine. In addition, neither the initial velocity nor the final steady-state value of the cell alkalinization following a 101 reduction of bath Cl^– concentration changed in the presence of histamine, and the same holds true for the cell acidification following a 101 reduction of bath HCO₃ ^– concentration. These observations indicate that the basolateral Cl^–/HCO₃ ^– exchanger was not stimulated by histamine, and that no other base transporters were activated. By contrast, the V _b response to elevation of bath K ⁺ concentration decreased, and so did the initial depolarizing V _b response to bath Cl^– substitution, while the secondary hyperpolarizing response increased. The latter observations are compatible with the notion that stimulation by histamine reduced a pH-insensitive part of the basolateral K⁺ conductance and reduced also the basolateral Cl^– conductance.     

Effect of cortisol and pertussis toxin on the cAMP concentration in human lymphocytes

It is demonstrated that pertussis toxin and hydrocortisone potentiate the adenosine-induced rise of the cAMP concentration in lymphocytes. Hydrocortisone elicits an immediate (for the simultaneous addition of adenosine and cortisol) and reversible effect. The effect of pertussis toxin has a latency and is irreversible. Added together, these agents exert no cumulative effect. It is assumed that hydrocortisone and pertussis toxin have the same target — The inhibiting regulatory protein G_i. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, N ^o 2, pp. 171–173, February, 1995 Presented by P. V. Sergeev, Member of the Russian Academy of Medical Sciences     

Induction of the monooxygenase system in rat blood neutrophils by methylcholanthrene and sovolin vivo and its effects on the properties of these cells

The xenobiotics methylcholanthrene and sovol (the latter being a mixture of polychlorinated biphenyls), which are monooxygenase system inducers, were tested for their effect on the respiratory burst in rat blood neutrophilsin vivo. The chemiluminescence accompanying this burst was more intensive in the neutrophils of rats treated with methylcholanthrene or sovol than in untreated rats. Observed changes in the 2A_max parameter of the electron paramagnetic resonance spectrum recorded for the spin probe 5-doxyl stearate in the presence of neutrophils indicated that methylcholanthrene and sovol can exert a direct effect on the viscous properties of neutrophil plasma membranesin vivo. These changes were similar in direction to those in the intensity of chemiluminescence during the respiratory burst in neutrophils. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 11, pp. 485–488, November, 1995 Presented by I. P. Ashmarin, Member of the Russian Academy of Medical Sciences     

Signaling pathways involved with the stimulatory effect of Angiotensin II on vacuolar H+-ATPase in proximal tubule cells

It has been documented that angiotensin II (ANG II) (10⁻⁹ M) stimulates proton extrusion via H⁺-adenosine triphosphatase (ATPase) in proximal tubule cells. In the present study, we investigated the signaling pathways involved in the effects of ANG II on H⁺-ATPase activity and on the cytosolic free calcium concentration in immortalized rat proximal tubule cells, a permanent cell line derived from rat proximal tubules. The effects of ANG on pH_i and [Ca⁺²]_i were assessed by the fluorescent probes, 2′,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxy-methyl ester and fluo-4-acetoxy-methyl ester, in the absence of Na⁺ to block the Na⁺/H⁺ exchanger. In the control situation, the pH recovery rate following intracellular acidification with NH₄Cl was 0.073±0.011 pH units/min (n=12). This recovery was significantly increased with ANG II (10⁻⁹ M), to 0.12±0.015 pH units/min, n=10. This last effect was also followed by a significant increase of Ca⁺² _i, from 99.72±1.704 nM (n=21) to 401.23±33.91 nM (n=39). The stimulatory effect of ANG II was blocked in the presence of losartan, an angiotensin II subtype 1 (AT₁) receptor antagonist. H89 [protein kinase A (PKA) inhibitor] plus ANG II had no effect on the pH recovery. Staurosporine [protein kinase C (PKC) inhibitor] impaired the effect of ANG II. Phorbol myristate acetate (PKC activator) mimicked in part the stimulatory effect of ANG II, but reduced Ca⁺² _i. 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (intracellular calcium chelator) alone reduced the pH_i recovery rate below control levels and impaired the effect of ANG II, in a way similar to that of trimethoxy benzoate (a blocker of Ca⁺² _i mobilization). We conclude that ANG II regulates rat proximal tubule vacuolar H⁺-ATPase by a PKA-independent mechanism and that PKC and intracellular calcium play a critical role in this regulation.     

Dependence of intracellular free calcium and tension on membrane potential and intracellular pH in single crayfish muscle fibres

The dependence of intracellular free calcium ([Ca²⁺]_i) and tension on membrane potential and intracellular pH (pH_i) was studied in single isolated fibres of the crayfish claw-opener muscle using ion-selective microelectrodes. Tension (T) was quantified as a percentage of the maximum force, or as force per cross-sectional area (N/cm²). In resting fibres, pH_i had a mean value of 7.06. Contractions evoked by an increase extracellular potassium ([K⁺]₀) produced a fall in pH_i of 0.01–0.05 units. The lowest measured levels of resting [Ca²⁺]_i corresponded to a pCa_i (= –log [Ca²⁺]_i) of 6.8. Intracellular Ca²⁺ transients recorded during K⁺-induced contractions did not reveal any distinct threshold for force development. Both the resting [Ca²⁺]_i and resting tension were decreased by an intracellular alkalosis and increased by an acidosis. The sensitivity of resting tension to a change in pH_i (quantified as –dT/ dpH_i) showed a progressive increase during a fall in pH_i within the range examined (pH_i 6.2–7.5). The pH_i/[Ca²⁺]_i and pH_i/tension relationships were monotonic throughout the multiphasic pH_i change caused by NH₄Cl. A fall of 0.5–0.6 units in pH_i did not produce a detectable shift in the pCa_i/tension relationship at low levels of force development. The results indicate that resting [Ca²⁺]_i is slightly higher than the level required for contractile activation. They also show that the dependence of tension on pH_i in crayfish muscle fibres is attributable to a direct H⁺ and Ca²⁺ interaction at the level of Ca²⁺ sequestration and/or transport. Finally, the results suggest that in situ, the effect of pH on the Ca²⁺ sensitivity of the myofibrillar system is not as large as could be expected on the basis of previous work on skinned crustacean muscle fibres.     

Induced activation of peripheral blood mononuclears and neutrophils in recipients of an allogeneic kidney

Peripheral blood mononuclears and neutrophils in recipients of an allogeneic kidney are better stimulated with zymosan and phorbol ester during the first days after transplantation and during allograft rejection. Antithymocytic globulin depresses chemiluminescence of both neutrophils and mononuclears. Antisera to human immunoglobulin suppress chemiluminescence of mononuclears but not of neutrophils. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, N ^o 5, pp. 523–525, May 1995     

Impact of plasmin and its complexes with α2-macroglobulin or α2-antiplasmin on the proliferative activity of human lymphocyteson the proliferative activity of human lymphocytes

It is shown that plasmin in doses of 0.1, 1, or 10 μg/ml did not influence significantly phytohemagglutinin-induced proliferation of mononuclear lymphocytes in a 3-day culture with these cells. Their proliferative response to pokeweed mitogen was stimulated by plasmin in the dose of 10 μg/ml only. Biogenic complexes of plasmin with α₂-macroglobulin or α₂-antiplasmin induced a moderate reduction of spontaneous proliferation after 3 days of culture, and so did plasmin after 5 days; α₂-macroglobulin induced a dose-dependent comitogenic effect with phytohemagglutinin and pokeweed mitogen, while α₂-antiplasmin induced a dose-independent comitogenic effect with pokeweed mitogen. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 9, pp. 304–305, September, 1994 Presented by V. A. Trufakin, Member of the Russian Academy of Medical Sciences     

Mechanisms of intracellular pH regulation in the hamster inner medullary collecting duct perfused in vitro

To examine the mechanisms of H⁺ transport in the mid-inner medullary collecting duct of hamsters, we measured the intracellular pH (pH_i) in the in vitro perfused tubules by microscopic fluorometry using 2,7-bis(carboxyethyl)-carboxyfluorescein (BCECF) as a fluorescent probe. In the basal condition, pH_i was 6.74±0.04 (n=45) in HCO ₃ ^– -free modified Ringer solution. Either elimination of Na⁺ from the bath or addition of amiloride (1 mM) to the bath produced a reversible fall in pH_i After acid loading with 25 mM NH₄Cl, pH_i spontaneously recovered with an initial recovery rate of 0.096±0.012 (n=23) pH unit/min. In the absence of ambient Na⁺, after removal of NH ₄ ⁺ , the pH_i remained low (5.95±0.10, n=8) and showed no signs of recovery. Subsequent restoration of Na⁺ only in the lumen had no effect on pH_i. However, when Na⁺ in the bath was returned to the control level, pH_i recovered completely. Amiloride (1 mM) in the bath completely inhibited the Na⁺-dependent pH_i recovery. Furthermore, elimination of Na⁺ from the bath, but not from the lumen, decreased pH_i from 6.97±0.07 to 6.44±0.05 (n=12) in the HCO ₃ ^– /Ringer solution or 6.70±0.03 to 6.02±0.05 (n=8) in the HCO ₃ ^– free solution. pH_i spontaneously returned to 6.76±0.08 with a recovery rate of 0.017±0.5 pH unit/min in the presence of CO₂/HCO ₃ ^– , whereas it did not recover in the absence of CO₂/HCO ₃ ^– . Although elimination of ambient Na⁺ depolarized the basolateral membrane voltage (V _B) from –78±1.2 to –72 ±0.6 mV (n=5, P<0.01), the level of V _B was not sufficient to explain the pH_i recovery solely by HCO ₃ ^– entry driven by the voltage. These results indicate that (a) pH_i of the inner medullary collecting duct is regulated mainly by a Na⁺/H⁺ exchanger in the basolateral membranes, (b) no apparent Na⁺-dependent H⁺ transport system exists in the luminal membranes and (c) Na⁺-independent H⁺ transport may also operate in the presence of CO₂/HCO ₃ ^– Preliminary data were reported at the Conference on Bicarbonate, Chloride, and Proton Transport Systems, New York, USA, in January 1989     

Video microscopy of intracellular pH in primary cultures of rabbit proximal and early distal tubules

The purpose of this study was to investigate intracytoplasmic pH (pH_i) regulation in primary cultures of proximal (PCT) and distal bright (DCTb) convoluted tubules. PCT and DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The cultured epithelia were grown on semi-transparent permeable supports. The pH_i was determined by video microscopy and digital image processing using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and measuring the ratio of BCECF fluorescence excited by two successive wavelengths (490 nm and 450 nm). Resting pH_i values, determined in bicarbonatefree medium (extracellular pH: 7.40), were 7.25±0.02 (n=23) and 7.17±0.04 (n=30) for cultured PCT and DCTb respecitively. After the acid-loading procedure, cultured proximal cells recovered their pH_i by means of the classic Na⁺/H⁺ antiporter, sensitive to amiloride and located in the apical membrane only. In cultured DCTb part of the pH_i recovery was mediated by a Na⁺/H⁺ exchange present in the basolateral side. Moreover, at physiological initial pH_i values, chloride removal from the apical solution caused the pH_i to increase in the presence of bicarbonate. In acidified cultured DCTb cells, a partial pH_i recovery was induced in sodium-free media by 15 mM HCO ₃ ^– in the presence of an outward chloride gradient. This pH_i change was completely abolished by 4,4-diisothiocyanostilbene 2,2-disulfonic acid (1 mM). These data suggest that DCTb cells possess in apical anion/base exchanger that resembles the Na⁺-independent Cl^–/HCO ₃ ^– exchanger.     

Action of ANG II and ANP on colon epithelial cells

The interaction of angiotensin II (ANG II) and atrial natriuretic peptide (ANP) on intracellular pH (pH_i) and calcium ([Ca²⁺]_i) was investigated in T84 cells (a permanent cell line derived from human colon epithelium) using the fluorescent stains BCECF/AM and Fluo 4/AM, respectively. pH_i recovery rate mediated by the Na⁺/H⁺ exchanger (NHE) was examined following an NH₄Cl pulse. Under control conditions pH_i recovered at 0.114±0.005 pH units/min (n=35). ANG II (10^–12 or 10^–9 M) increased this value, whilst ANG II (10^–7 M) decreased it. These effects of ANG II were impaired by simultaneous addition of 1 M or 25 M HOE-694, indicating that the stimulatory and inhibitory effects of ANG II on pH_i recovery are mediated in part via the NHE1 and NHE2 isoforms. ANG II increased [Ca²⁺]_i concentration-dependently. ANP (10^–6 M) or dimethyl-BAPTA/AM (50 M) blocked the effects of ANG II on [Ca²⁺]_i and on the rate of pH_i recovery. Thapsigargin (10^–5 M) enhanced the effect of ANG II on [Ca²⁺]_i and reversed its stimulatory effect on the rate of pH_i recovery to an inhibitory one. External Ca²⁺-free solution did not affect the effects of ANG II on these parameters. These data suggest that the [Ca²⁺]_i increase induced by ANG II is dependent on intracellular calcium stores. They are compatible with the demonstration of two sites on the C-terminal of the Na⁺/H⁺ exchanger, one stimulating Na⁺/H⁺ activity by increases of [Ca²⁺]_i in the lower range (at 10^–12 or 10^–9 M ANG II) and the other inhibiting this activity at high [Ca²⁺]_i levels (at 10^–7 M ANG II). ANP or dimethyl-BAPTA/AM, by impairing the pathway mediating the increase in [Ca²⁺]_i, block both the stimulatory and inhibitory effects of ANG II.     

Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells

Kinetic properties of the Na⁺-H⁺ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pH_i) and Na⁺ activity (aNa_i) with the aid of double-barreled H⁺- and Na⁺-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pH_i was 7.12±0.01 and aNa_i was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NH ₄ ⁺ solution followed by an Na⁺-free N-methyl-d-glucamine (NMDG⁺) solution. Intracellular Na⁺ and H⁺ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na⁺ solution rapidly increased pH_i. This Na⁺-induced increase in pH_i was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNa_i. The rate of pH_i recovery induced by the standard Na⁺ solution increased in a saturable manner as pH_i decreased, and was negligible at pH_i 7.2–7.3, indicating an inactivation of the Na⁺-H⁺ antiport. The apparent K _m for intracellular H⁺ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNa_i to less than 1 mmol/l by prolonged exposure to NMDG⁺ solution significantly increased the rate of Na⁺-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na⁺ concentration increased following Michaelis-Menten kinetics (V _max was 0.55 pH/min and the apparent K _m was 75 mmol/l at pH_i 6.88). The results clearly showed that the Na⁺-H⁺ antiport activity is dependent on the chemical potential gradient of both Na⁺ and H⁺ ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na⁺ and H⁺ concentrations.     

Changes in intracellular ion activities induced by adrenaline in human and rat skeletal muscle

To study the stimulating effect of adrenaline (ADR) on active Na⁺/K⁺ transport we used double-barrelled ion-sensitive micro-electrodes to measure the activities of extracellular K⁺ (aK_e) and intracellular Na⁺ (aNa_i) in isolated preparations of rat soleus muscle, normal human intercostal muscle and one case of hyperkalemic periodic paralysis (h.p.p.). In these preparations bath-application of ADR (10⁻⁶ M) resulted in a membrane hyperpolarization and transient decreasesaK_e andaNa_i which could be blocked by ouabain (3×10⁻⁴ M). In the h.p.p. muslce a continuous rise ofaNa_i induced by elevation ofaK_e to 5.2 mM could be stopped by ADR. In addition, the intracellular K⁺ activity (aK_i), the free intracellular Ca²⁺ concentration (pCa_i) and intracellular pH (pH_i) were monitored in rat soleus muscle. During ADRaK_i increased, pH_i remained constant and intracellular Ca²⁺ apparently decreased. In conclusion, our data show that ADR primarily stimulates the Na⁺/K⁺ pump in mammalian skeletal muscle. This stimulating action is not impaired in the h.p.p. muscle. Parts of the results have been presented to the German Physiological Society (Ballanyi and Grafe 1987)