Metabolic acidosis and parathyroidectomy increase Na+-H+ exchange in brush border vesicles

Na+-H+ exchange across the brush border membrane of the renal proximal tubular cell is a mechanism for Na+ reabsorption and H+ secretion. An electroneutral Na+-H+ exchange activity has been identified in isolated renal brush border membrane vesicles from rat and dog kidney, and increased Na+-H+ exchange has been measured in brush border membrane vesicles from remnant kidneys of dogs with chronic renal failure. To ascertain whether changes in H+ secretion by the kidney observed in chronic metabolic acidosis and in states of altered parathyroid function might result from altered Na+-H+ exchange across the renal cortical cellular brush border membrane, we measured Na+-H+ exchange in brush border membrane vesicles from kidneys of dogs with chronic metabolic acidosis and from kidneys of thyroparathyroidectomized dogs. Increased amiloride-sensitive Na+-H+ exchange was demonstrated in brush border membrane vesicles from kidneys of both groups of dogs, suggesting that adaptations in H+ excretion in chronic metabolic acidosis and hypoparathyroidism might be explained by increased activity of a renal brush border membrane Na+-H+ exchanger.     

Effect of dehydration on apical Na+-H+ exchange activity and Na+-dependent sugar transport in brush-border membrane vesicles isolated from chick intestine

 The current work examines the effect of 4 days of water deprivation on Na⁺-H⁺ exchange and Na⁺-sugar cotransport systems in brush-border membrane vesicles isolated from either the jejunum, ileum or the colon of the chick. Apical Na⁺-H⁺ exchange activity was evaluated by measuring the pH-gradient-dependent Na⁺ uptake. The contribution of the Na⁺-H⁺ exchangers NHE2 and NHE3 to total Na⁺-H⁺ exchange activity was evaluated from their sensitivity to the amiloride-related drug HOE694. Dehydration increased plasma aldosterone levels from 12 to 70 pg/ml and also the activities of both Na⁺-H⁺ exchange and Na⁺-dependent sugar transport in the three intestinal regions tested. Na⁺-independent sugar transport was not modified by 4 days of water deprivation. In the ileum and colon the increase in Na⁺-H⁺ exchange activity was due to an increase in NHE2 activity, whereas in the jejunum it was due to an increase in both NHE2 and NHE3. Since we have previously reported that chronic Na⁺ depletion increases plasma aldosterone levels and NHE2 activity in ileum and colon, decreased small and large intestine Na⁺-sugar cotransport activity and had no effect on jejunal apical Na⁺-H⁺ exchange activity, it can be concluded that: (1) aldosterone does not regulate intestinal Na⁺-dependent sugar transport, and (2) the regulation of jejunal Na⁺-H⁺ exchange activity differs from that of either the ileum or the colon. Received: 31 October 1997 / Received after revision: 17 December 1997 / Accepted: 8 January 1998     

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.     

Na+-H+ exchange is not important for pancreatic HCO-3 secretion in the pig

Veel , T., Villanger , O., Holthe , M.R., Cragoe jr ., E.J. & Reder , M.G. 1992. Na⁺-H exchange is not important for pancreatic HCO^-₃ secretion in the pig. Acta Physiol Scand 144 , 239–246. Received 13 September 1991, accepted 14 November 1991. ISSN 0001–6772. University of Oslo, Institute for Experimental Medical Research and Surgical Department, Ullevaal Hospital, Oslo, Norway. Pancreatic inter- and intralobular duct cells extrude H⁺-ions to interstitial fluid when they secrete HCO^-₃ to pancreatic juice. This study assesses the potential importance of Na⁺-H⁺-ion exchange for H⁺-ion extrusion and secretion of HCO^-₃ using the Na⁺-H⁺ exchange blockers amiloride and hexamethylene-amiloride. Intracellular pH (pH,) in inter- and intralobular pancreatic duct epithelium was measured using BCECF fluorescence. H⁺-ion efflux was measured using a NH₄Cl prepulse, acid-loading technique. In HCO^-₃-free media, pH₁ recovery following acid loading was blocked by amiloride (10^-4 m) and hexamethylene-amiloride (10^-6 m) , demonstrating amiloride-and hexamethylene-amiloride-sensitive Na⁺-H⁺ exchange. However, 5 × 10^-6 M hexamethylene-amiloride did not reduce secretin-dependent pancreatic HCO, secretion in vivo. Maximal H⁺-efflux through Na⁺-H⁺ exchange was 1.5 ± 0.2μmol min^-1 ml cell volume^-l, i.e. less than 1 % of estimated net H⁺-ion efflux during HCO^-₃ secretion. Conclusion: amiloride- and hexamethylene amiloride sensitive Na⁺-H⁺ exchange is not important for secretin-dependent pancreatic HCO^-₃ secretion in the pig. Other mechanisms for H⁺ extrusion dominate.     

Stimulation of Na+/H+ exchange activity by endothelin in opossum kidney cells

Endothelin-1 (ET-1) controls multiple aspects of kidney function. In this study we have analysed the effects of ET-1 on apical Na⁺/H⁺ exchange activity in opossum kidney (OK) cells. ET-1 (at 10^–10 M and 10^–8 M) activated Na⁺/H⁺ exchange activity within 5 min of exposure. ET-1 (10^–8 M) prevented PTH-induced (parathyroid hormone; 10^–8 M) inhibition of Na⁺/H⁺ exchange activity; it also abolished transport inhibition in response to 10^–3 M IBMX (isobutylmethylxanthine) and 3×10^–7 M TPA (phorbol 12-myristate 13-acetate), but had no effect on the 8-bromo-cAMP-induced (10^–4 M) decrease of transport rate. Basal cAMP content, IBMX- and PTH-stimulated cAMP production were unaffected by ET-1 (10^–8 M). The stimulatory action of ET-1 (10^–8 M) on Na⁺/H⁺ exchange activity was prevented by calphostin C (10^–8 M). These data document that OK cells might serve as a useful in vitro model for analysis of cellular mechanisms involved in endothelin action; proteine kinase C activation seems to participate in the observed endothelin effects.     

Two different receptor sites for Ca2+ and Na+ in frog taste responses

Distilled water, 1 mM CaCl2 and 500 mM NaCl (pH 4.5) are effective stimuli which excite chemoreceptors of the frog tongue. To learn whether or not these taste stimuli react with different taste receptor sites, a proteolytic enzyme was topically applied to the tongue dorsum. Responses were recorded from the frog glossopharyngeal nerve during taste stimulation. After application of 0.1% pronase E to the dorsal tongue surface, the magnitude of the NaCl response remained unchanged, but the magnitude of the water and CaCl2 responses was markedly decreased. The selective suppression by the pronase E treatment indicates that there are two different receptor sites for Ca2+ and Na+ in the frog taste receptor cell and the receptor sites responsible for the generation of the water and the Ca2+ response may be composed of a protein.     

Cell shrinkage evoked by Ca2+-free solution in rat alveolar type II cells: Ca2+ regulation of Na+-H+ exchange

The effects of intracellular Ca2+ concentration, [Ca2+]i, on the volume of rat alveolar type II cells (AT-II cells) were examined. Perfusion with a Ca2+-free solution induced shrinkage of the AT-II cell volume in the absence or presence of amiloride (1 microm, an inhibitor of Na+ channels); however, it did not in the presence of 5-(N-methyl-N-isobutyl)-amiloride (MIA, an inhibitor of Na+-H+ exchange). MIA decreased the volume of AT-II cells. Inhibitors of Cl(-)-HCO3- exchange, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) also decreased the volume of AT-II cells. This indicates that the cell shrinkage induced by a Ca2+-free solution is caused by a decrease in NaCl influx via Na+-H+ exchange and Cl(-)-HCO3- exchange. Addition of ionomycin (1 microm), in contrast, induced cell swelling when AT-II cells were pretreated with quinine and amiloride. This swelling of the AT-II cells is not detected in the presence of MIA. Intracellular pH (pHi) measurements demonstrated that the Ca2+-free solution or MIA decreases pHi, and that ionomycin increases it. Ionomycin stimulated the pHi recovery after an acid loading (NH4+ pulse method), which was not noted in MIA-treated AT-II cells. Ionomycin increased [Ca2+]i in fura-2-loaded AT-II cells. In conclusion, the Na+-H+ exchange activities of AT-II cells, which maintain the volume and pHi, are regulated by [Ca2+]i.     

Volume regulation by Necturus gallbladder: apical Na+-H+ and Cl(-)-HCO-3 exchange

Necturus gallbladder epithelial cells exhibited volume regulatory swelling when exposed to a hypertonic mucosal bathing solution. The initial, osmotically induced shrinkage was followed by a rapid increase in cell volume back to the control value despite continuing hypertonicity of the mucosal perfusate. This volume regulatory increase occurred by osmotic water flow accompanying the transient cellular uptake of NaCl from the mucosal bathing solution. Volume regulatory increase required Na+ and Cl- in the mucosal bath; it was inhibited by amiloride or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid but not by bumetanide or ouabain. The K1/2 for Na+ was 2.8 mM, the K1/2 for Cl- was 1.9 mM, and maximum velocity of fluid flow into the cell for both ions was greater than 10 x 10(-6) cm/s. Both volume regulatory increase and transepithelial fluid absorption involve NaCl flux across the apical membrane into the cells, but the nature of the NaCl fluxes differ in the two processes. During volume regulatory increase NaCl enters the cells by parallel Na+-H+ and Cl(-)-HCO-3 exchanges, whereas during transepithelial fluid absorption NaCl enters the cell by the coupled flux of NaCl.     

Na(+)-H+ exchange is not important for pancreatic HCO3- secretion in the pig.

Pancreatic inter- and intralobular duct cells extrude H(+)-ions to interstitial fluid when they secrete HCO3- to pancreatic juice. This study assesses the potential importance of Na(+)-H(+)-ion exchange for H(+)-ion extrusion and secretion of HCO3-, using the Na(+)-H+ exchange blockers amiloride and hexamethylene-amiloride. Intracellular pH (pHi) in inter- and intralobular pancreatic duct epithelium was measured using BCECF fluorescence. H(+)-ion efflux was measured using a NH4Cl prepulse, acid-loading technique. In HCO3(-)-free media, pHi recovery following acid loading was blocked by amiloride (10(-4) M) and hexamethylene-amiloride (10(-6) M), demonstrating amiloride- and hexamethylene-amiloride-sensitive Na(+)-H+ exchange. However, 5 x 10(-6) M hexamethylene-amiloride did not reduce secretin-dependent pancreatic HCO3- secretion in vivo. Maximal H(+)-efflux through Na(+)-H+ exchange was 1.5 +/- 0.2 mumol min-1 ml cell volume-1, i.e. less than 1% of estimated net H(+)-ion efflux during HCO3- secretion. Conclusion: amiloride- and hexamethylene amiloride sensitive Na(+)-H+ exchange is not important for secretin-dependent pancreatic HCO3- secretion in the pig. Other mechanisms for H+ extrusion dominate.     

Acceleration of H+ extrusion via Na(+)-H+ exchange in guinea-pig ventricular papillary muscle under intracellular acidic condition.

We investigated mechanisms by which intracellular pH was regulated under intracellular acidic condition in resting guinea-pig ventricular papillary muscles in vitro. Intracellular sodium ion activity (aiNa), intracellular and surface pH (pHi and pHs) were measured with Na(+)- and H(+)-selective microelectrodes and resting tension was measured. By exposure to 0 mM K solution aiNa and resting tension increased progressively while pHi decreased but reached the steady level of pH 6.95. pHs which was lower than external bulk pH (pHo) decreased progressively by exposure to 0 mM K solution. In 4 mM K solution, amiloride (1 mM), an inhibitor of Na(+)-H+ exchange, induced a reversible decrease in both aiNa and pHi, and an increase in pHs. Changes in pHi and pHs induced by application of amiloride in 0 mM K solution were larger than those in 4 mM K solution. The rate of decrease in pHi induced by amiloride became larger at longer exposure to 0 mM K solution. Lowering pHo from 7.4 to 6.4 induced a larger decrease in pHi in 0 mM K solution than that in 4 mM K solution. Lowering pHo from 7.4 to 5.4 reversed the difference between pHs and pHo. These results suggest that in guinea-pig papillary muscle, Na(+)-H+ exchange is active to regulate intracellular H+ under resting condition and under intracellular acidic condition, H+ extrusion via the Na(+)-H+ exchange would be accelerated not only by the net thermodynamic driving force for Na+ and H+ but also by other factors.     

Na+/H+ exchange in glial cells of Necturus optic nerve

Single and double-barreled pH-sensitive electrodes were used to study intracellular pH (pHi) regulation in glial cells of Necturus optic nerve in the nominal absence of HCO3-/CO2. After the cells were acidified by the addition and withdrawal of NH4+, the pHi recovered toward the original steady-state pHi. The recovery from acidification was Na+-dependent and inhibited by 1 mM amiloride. These results suggest the existence in intact vertebrate glial cells of a Na+/H+ exchanger which functions in acid extrusion.     

Inhibition of taste responses to Na+ salts by epithelial Na+ channel blockers in gerbil

The Na+ transport inhibitor amiloride blocks taste responses to NaCl by 60-70%. The purpose of the present study was to determine if greater inhibition could be achieved with three potent amiloride analogs that are specific for the epithelial Na+ channel: phenamil, 2',4'-dimethylbenzamil, and 3',4'-dichlorobenzamil. Application of phenamil (100 microM) to the anterior tongue blocked integrated responses to NaCl from the chorda tympani nerve by 98.04%, but had no significant effect on sucrose or NH4Cl. This finding suggests that the epithelial Na+ channel alone transduces the taste of NaCl in gerbil. The residual 30-40% of the response that is not blocked by amiloride can simply be explained by the fact that amiloride is less potent than phenamil. On average, 100 microM phenamil blocked responses to Na+ salts with a variety of anions by 94.2%; 100 microM 2',4'-dimethylbenzamil, by 89.83%; and 100 microM 3',4'-dichlorobenzamil, by 72.56%. Small residual responses to salts of glutamate and phosphate were not eliminated by the amiloride analogs; this suggests that other transduction mechanisms may account for a small portion of taste responses for these salts in the gerbil.     

Assessment of evidence for K+-H+ exchange in isolated type-1 cells of neonatal rat carotid body

 Intracellular pH (pH_i) was measured in enzymically isolated, neonatal rat carotid body type-1 cells, using the fluorophore carboxy-SNARF-1 (AM-loaded), and using the nigericin technique for in situ fluorescence calibration (nigericin is a membrane-soluble K⁺-H⁺ exchanger). In CO₂/HCO₃ ^–-free media, inhibiting Na⁺-H⁺ exchange produced a prompt fall of pH_i (background acid-loading), the rate of which was reduced by raising the extracellular K⁺ concentration, [K⁺]_o. pH_i recovery from an intracellular acid or alkali load was also sensitive to changes of [K⁺]_o. These results are similar to those of Wilding et al. (J Gen Physiol 100:593–608, 1992), who proposed the existence of an acid-loading, K⁺-H⁺ exchanger (KHE) in the type-1 cell. However, when nigericin was not used for post-experimental calibration, and the superfusion system was flushed exhaustively with strong detergent, alcohol and distilled water, then background acid-loading was attenuated, and the K⁺ _o sensitivity of pH_i insignificant. Background loading was increased again, and K⁺ _o sensitivity restored, when cells were monitored in a superfusion system which had previously been exposed to a single nigericin-calibration protocol (followed by a short system wash with strong detergent and distilled water). We conclude that the previously reported expression of KHE in carotid body type-1 cells is an artefact caused by nigericin contamination. We have therefore quantified the pH_i dependence of background loading in uncontaminated type-1 cells. We consider the possible implications of our work for reports of KHE in other cell types. Received: 3 March 1997 / Received after revision: 1 April 1997 / Accepted: 2 April 1997     

Aldosterone actions on basolateral Na+/H+ exchange in Madin-Darby canine kidney cells

In recent studies, there has been a re-evaluation of the polarity of Na⁺/H⁺ exchange in Madin-Darby canine kidney (MDCK) cells. This study was designed to examine aldosterone actions on basolaterally located Na⁺/H⁺ exchange of MDCK cell monolayers grown on permeant filter supports; pH_i was analysed in the absence of bicarbonate by using the pH-sensitive fluorescent probe 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein. Pre-exposure of MDCK cells to aldosterone led within 10–20 min to an alkalization of pH_i ( 0.3 pH unit); this effect is prevented by an addition of dimethylamiloride to the basolateral superfusate. Addition of aldosterone led to stimulation of the basolaterally located Na⁺/H⁺ exchange activity (Na⁺-dependent recovery from an acid load); this effect required preincubation (more then 3 min) and was observed at 0.1 nM aldosterone. Preexposure (15 min) of MDCK monolayers to phorbol 12-myristate 13-acetate also led to an activation of Na⁺/H⁺ exchange; pre-exposure to 8-bromo-cAMP led to inhibition of Na⁺/H⁺ exchange activity. An inhibitory effect of aldosterone was observed if Na⁺/H⁺ exchange activity was analysed in the presence of aldosterone; the highest inhibitory effects (20%–30%) occurred at concentrations of 5 nM and higher. Aldosterone-dependent inhibition does not require preincubation and is fully reversible; it was only observed at low (20 mM) but not at high Na⁺ concentrations (130 mM). The data suggest that aldosterone has an instantaneous inhibitory effect on basolaterally located Na⁺/H⁺ exchange activity under conditions of low Na⁺, but stimulates the rate of transport activity upon preincubation under conditions of physiological Na⁺ concentrations.     

A transient receptor potential channel expressed in taste receptor cells

We used differential screening of cDNAs from individual taste receptor cells to identify candidate taste transduction elements in mice. Among the differentially expressed clones, one encoded Trpm5, a member of the mammalian family of transient receptor potential (TRP) channels. We found Trpm5 to be expressed in a restricted manner, with particularly high levels in taste tissue. In taste cells, Trpm5 was coexpressed with taste-signaling molecules such as alpha-gustducin, Ggamma13, phospholipase C-beta2 (PLC-beta2) and inositol 1,4,5-trisphosphate receptor type III (IP3R3). Our heterologous expression studies of Trpm5 indicate that it functions as a cationic channel that is gated when internal calcium stores are depleted. Trpm5 may be responsible for capacitative calcium entry in taste receptor cells that respond to bitter and/or sweet compounds.     

Polarized expression of Na+/H+ exchange activity in LLC-PK1/PKE20 cells: II. Hormonal regulation

LLC-PK₁/PKE₂₀ cells (a continuous epithelial cell line) has two different Na/H exchange activities: Na/H-1 located in the basolateral membrane and Na/H-2 located in the apical membrane [Casavola et al. (1989) Biochem Biophys Res Commun 165:833–837; Haggerty et al. (1988) Proc Natl Acad Sci USA 86:6797–6801]. In the present report we have studied hormone regulation of these exchange activities by measuring Na-dependent recovery of pH_i from an acid load (by using microspectrofluorometry and 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein) in response to activation of regulatory cascades by either pharmacological agents or by vasopressin or calcitonin. Agents leading to activation of protein kinase A (cAMP-dependent), such as forskolin (10 M), 8-Br-cAMP (0.25 mM), and isobutylmethylxanthine (0.5 mM), inhibited Na/H-2 and Na/H-1 by an average of 49%. Stimulation of protein kinase C by a phorbol ester (phorbol 12-myristate 13-acetate, TPA, 100 nM) inhibited Na/H-2 (by an average of 48%) and stimulated Na/H-1 (by an average of 38%); these effects of TPA were also observed in the presence of forskolin (100 M). Addition of either vasopressin (2 M) or calcitonin (0.3 M) onto both sides of the monolayer decreased the activity of Na/H-2 by an average of 26.3% and 27.7% respectively, and stimulated the activity of Na/H-1 by an average of 17.4% and 38.7% respectively; exposure of cells to either hormone stimulated production of cAMP and inositol trisphosphate, respectively. Separate hormone additions to either the apical or basolateral cell surface led to effects similar to those produced by simultaneous hormone additions onto both cell surfaces, although the relative response of Na/H exchangers to either agonist is variable. In summary, these results suggest that in LLC-PK ₁/PKE₂₀ cells, vasopressin and calcitonin can act via receptor systems coupled either to adenylate cyclase or to phospholipase C. Activation of these receptor systems can lead to inhibition of Na/H-2 and stimulation of Na/H-1.     

Extracellular protons activate K+ current in a subpopulation of frog taste receptor cells.

Based on morphological and electrophysiological criteria, two distinct subpopulations (group A and group B) taste receptor cells (TRCs) isolated from frog (Rana temporaria) taste disks were identified. TRCs from the group A were depolarized by acid stimuli which, in contrast, caused hyperpolarizing responses in group B TRCs. By using patch clamp technique we explored ion mechanisms mediating the pH dependent TRC hyperpolarization, and found that K+ conductance of TRCs from the group B increased as bath solution pH reduced. Our findings provide rationale to hypothesize that TRC from the group B express H+ gated K+ channels.