Expression of OCTN2 and OCTN3 in the apical membrane of rat renal cortex and medulla.

Immunological assays and transport measurements in apical membrane vesicles revealed that the apical membrane of rat kidney cortex and medulla presents OCTN2 and OCTN3 proteins and transports L-[(3)H]-carnitine in a Na(+)-dependent and -independent manner. OCTN2 mediates the Na(+)/L-carnitine transport activity measured in medulla because (i) the transport showed the same characteristics as the cortical Na(+)/L-carnitine transporter and (ii) the medulla expressed OCTN2 mRNA and protein. The Na(+)-independent L-carnitine transport activity appears to be mediated by both OCTN2 and OCTN3 since: (i) Na(+)-independent L-carnitine uptake was inhibited by both, anti-OCTN2 and anti-OCTN3 antibodies, (ii) kinetics studies revealed the involvement of a high- and a low-affinity transport systems, and (iii) Western and immunohistochemistry studies revealed that OCTN3 protein is located at the apical membrane of the kidney epithelia. The Na(+)-independent L-carnitine uptake exhibited trans-stimulation by intravesicular L-carnitine or betaine. This trans-stimulation was inhibited by anti-OCTN3 antibody, but not by anti-OCTN2 antibody, indicating that OCTN3 can function as an L-carnitine/organic compound exchanger. This is the first report showing a functional apical OCTN2 in the renal medulla and a functional apical OCTN3 in both renal cortex and medulla.

Ontogeny regulates creatine metabolism in rat small and large intestine.

The ontogeny of intestinal CRT, AGAT and GAMT was investigated in foetuses, newborn, suckling, weaning and adult rats. In the colon, CRT mediates creatine transport because it was Na(+)- and Cl(-) dependent and inhibited by creatine and GPA. In addition, Northern assays showed two CRT transcripts (2.7-kb and 4.2-kb) and the in situ hybridisation revealed that CRT mRNA is restricted to the colon epithelial cells. The immunohistochemistry revealed that CRT protein was at the apical membrane of colon epithelia. Maturation decreased colonic CRT activity to undetectable levels and increased CRT mRNA abundance. Western assays revealed 57-, 65-, 80- and 116-kDa polypeptides at the intestinal apical membrane. The abundance of the 65-, 80- and 116-kDa polypeptides decreased with age, and that of 57-kDa was only observed in adult rats. The small and large intestine express AGAT and GAMT mRNAs. Maturation decreased AGAT mRNA abundance without affecting that of GAMT. For comparison, renal AGAT mRNA levels were measured and they were increased with age. The study reports for the first time that: i) the apical membrane of rat colon have an active CRT, ii) development down-regulates CRT activity via post-transcriptional mechanism(s), iii) the intestine might synthesize creatine and iv) intestinal and renal creatine synthesis is ontogenically regulated at the level of AGAT gene expression.

Ontogeny up-regulates renal Na(+)/Cl(-)/creatine transporter in rat.

Creatine plays a role in energy storage and transport/shuttle of high-energy phosphate in heart, brain, retina, testis and skeletal muscle. These tissues take creatine from the plasma via a 2Na(+)/1Cl(-)/1creatine cotransporter (CRT). We have previously demonstrated that renal apical membrane presents a 2Na(+)/1Cl(-)/1creatine cotransport activity. The goal of this study was to determine whether this transporter is ontogenically regulated. Na(+)/Cl(-)/creatine transport activity was evaluated by measuring [(14)C]-creatine uptake into renal brush-border membrane vesicles. CRT mRNA expression was measured by Northern and real-time PCR assays. E20 foetuses, newborn, suckling, weaning and adult (2- and 8-month-old) Wistar rats were used. The results revealed that neither the vesicular volume, the binding of creatine to the brush-border membrane vesicles, nor the purity of the brush-border membrane vesicle preparations was affected by maturation. Fetal and neonatal kidneys contained a creatine transporter that was qualitatively indistinguishable from that in the adult: it was concentrative, Na(+)- and Cl(-)-dependent, electrogenic and inhibited by guanidinopropionic acid. Maturation increased this transport activity by increasing the maximal rate of transport (V(max)) without significantly changing the apparent K(m). Northern analysis revealed two transcripts for CRT of 2.7 kb and 4.2 kb in all the ages tested. Northern and real-time PCR assays showed that, as seen with NaCl-dependent creatine transport activity, maturation increased CRT mRNA expression. This study reports for the first time that: (i) an apical renal Na(+)/Cl(-)/creatine cotransporter is already active in rat foetuses and (ii) development regulates Na(+)/Cl(-)/creatine cotransport activity by increasing the density and/or turnover of the transporters.

OCTN3: A Na+-independent L-carnitine transporter in enterocytes basolateral membrane.

L-carnitine transport has been measured in enterocytes and basolateral membrane vesicles (BLMV) isolated from chicken intestinal epithelia. In the nominally Na+-free conditions chicken enterocytes take up L-carnitine until the cell to medium L-carnitine ratio is 1. This uptake was inhibited by L-carnitine, D-carnitine, gamma-butyrobetaine, acetylcarnitine, tetraethylammonium (TEA), and betaine. L-3H-carnitine uptake into BLMV showed no overshoot, and it was (i) Na+-independent, (ii) trans-stimulated by intravesicular L-carnitine, and (iii) cis-inhibited by TEA and cold L-carnitine. L-3H-carnitine efflux from L-3H-carnitine preloaded enterocytes was also Na+-independent, and trans-stimulated by L-carnitine, D-carnitine, gamma-butyrobetaine, acetylcarnitine, TEA, and betaine. Both, uptake and efflux of L-carnitine were inhibited by verapamil and unaffected by either extracellular pH or palmitoyl-L-carnitine. RT-PCR with specific primers for the mouse OCTN3 transporter revealed the existence of OCTN3 mRNA in mouse intestine, which was confirmed by in situ hybridization studies. Immunohystochemical analysis showed that OCTN3 protein was mainly associated with the basolateral membrane of rat and chicken enterocytes, whereas OCTN2 was detected at the apical membrane. In conclusion, the results demonstrate for the first time that (i) mammalian small intestine expresses OCTN3 mRNA along the villus and (ii) that OCTN3 protein is located in the basolateral membrane. They also suggest that OCTN3 could mediate the passive, Na+ and pH-independent L-carnitine transport activity measured in the three experimental conditions.

Human, rat and chicken small intestinal Na+ - Cl- -creatine transporter: functional, molecular characterization and localization.

In spite of all the fascinating properties of oral creatine supplementation, the mechanism(s) mediating its intestinal absorption has(have) not been investigated. The purpose of this study was to characterize intestinal creatine transport. [(14)C] creatine uptake was measured in chicken enterocytes and rat ileum, and expression of the creatine transporter CRT was examined in human, rat and chicken small intestine by reverse transcription-polymerase chain reaction, Northern blot, in situ hybridization, immunoblotting and immunohistochemistry. Results show that enterocytes accumulate creatine against its concentration gradient. This accumulation was electrogenic, Na(+)- and Cl(-)-dependent, with a probable stoichiometry of 2 Na(+): 1 Cl(-): 1 creatine, and inhibited by ouabain and iodoacetic acid. The kinetic study revealed a K(m) for creatine of 29 microM. [(14)C] creatine uptake was efficiently antagonized by non-labelled creatine, guanidinopropionic acid and cyclocreatine. More distant structural analogues of creatine, such as GABA, choline, glycine, beta-alanine, taurine and betaine, had no effect on intestinal creatine uptake, indicating a high substrate specificity of the creatine transporter. Consistent with these functional data, messenger RNA for CRT was detected only in the cells lining the intestinal villus. The sequences of partial clones, and of the full-length cDNA clone, isolated from human and rat small intestine were identical to previously cloned CRT cDNAs. Immunological analysis revealed that CRT protein was mainly associated with the apical membrane of the enterocytes. This study reports for the first time that mammalian and avian enterocytes express CRT along the villus, where it mediates high-affinity, Na(+)- and Cl(-)-dependent, apical creatine uptake.

Functional characterization of intestinal L-carnitine transport.

The carnitine transporter OCTN2 is responsible for the renal reabsorption of filtered L-carnitine. However, there is controversy regarding the intestinal L-carnitine transport mechanism(s). In this study, the characteristics of L-carnitine transport in both, isolated chicken enterocytes and brush-border membrane vesicles (BBMV) were studied. In situ hybridization was also performed in chicken small intestine. Chicken enterocytes maintain a steady-state L-carnitine gradient of 5 to 1 and 90% of the transported L-carnitine remains in a readily diffusive form. After 5 min, L-Carnitine uptake into BBMV overshot the equilibrium value by a factor of 2.5. Concentrative L-carnitine transport is Na+-, membrane voltage-and pH-dependent, has a high affinity for L-carnitine (Km 26 - 31 microM ) and a 1:1 Na+: L-carnitine stoichiometry. L-Carnitine uptake into either enterocytes or BBMV was inhibited by excess amount of cold L-carnitine > D-carnitine = acetyl-L-carnitine = gamma-butyrobetaine > palmitoyl-L-carnitine > betaine > TEA, whereas alanine, histidine, GABA or choline were without significant effect. In situ hybridization studies revealed that only the cells lining the intestinal villus expressed OCTN2 mRNA. This is the first demonstration of the operation of a Na+/L-carnitine cotransport system in the apical membrane of enterocytes. This transporter has properties similar to those of OCTN2.

Na(+)-dependent D-mannose transport at the apical membrane of rat small intestine and kidney cortex.

The presence of a Na(+)/D-mannose cotransport activity in brush-border membrane vesicles (BBMV), isolated from either rat small intestine or rat kidney cortex, is examined. In the presence of an electrochemical Na(+) gradient, but not in its absence, D-mannose was transiently accumulated by the BBMV. D-Mannose uptake into the BBMV was energized by both the electrical membrane potential and the Na(+) chemical gradient. D-Mannose transport vs. external D-mannose concentration can be described by an equation that represents a superposition of a saturable component and another component that cannot be saturated up to 50 microM D-mannose. D-Mannose uptake was inhibited by D-mannose >> D-glucose>phlorizin, whereas for alpha-methyl glucopyranoside the order was D-glucose=phlorizin >> D-mannose. The initial rate of D-mannose uptake increased as the extravesicular Na(+) concentration increased, with a Hill coefficient of 1, suggesting that the Na(+):D-mannose cotransport stoichiometry is 1:1. It is concluded that both rat intestinal and renal apical membrane have a concentrative, saturable, electrogenic and Na(+)-dependent D-mannose transport mechanism, which is different from SGLT1.

A Na+-dependent D-mannose transporter in the apical membrane of chicken small intestine epithelial cells.

The presence of a Na+/D-mannose cotransporter in brush-border membrane vesicles (BBMV) isolated from chicken small intestine was examined. In the presence of an electrochemical gradient for Na+, but not in its absence, D-mannose was accumulated transiently by the BBMV. D-Mannose uptake into the BBMV was energized by both the membrane potential and the chemical gradient for Na+. The relationship between D-mannose transport and external D-mannose concentration was described by an equation that represented the superposition of a saturable component (Michaelis-Menten constant Km 12.5 microM) and another component unsaturatable up to 80 microM D-mannose. D-Mannose uptake was inhibited by various substances in the following order of potency: D-mannose>>D-glucose>phlorizin>phloretin>D-fructose. For the uptake of alpha-methyl-glucopyranoside the order was D-glucose=phlorizin>>phloretin=D-fructose=D-mannose. The initial rate of D-mannose uptake increased as the extravesicular [Na+] increased, with a Hill coefficient of 1, suggesting that the Na+:D-mannose cotransport stoichiometry is 1:1. It is concluded that the intestinal apical membrane has a saturable, electrogenic and concentration- and Na+-dependent mannose transport mechanism that differs from the sodium-dependent glucose transporter SGLT1.

Hormonal regulation of chicken intestinal NHE and SGLT-1 activities.

The effects of aldosterone and arginine vasotocin (AVT) on intestinal Na(+)/H(+) exchange (NHE) and Na(+)-sugar cotransport (SGLT-1) activities have been investigated using brush-border membrane vesicles isolated from Hubbard chicken small and large intestines, and they were compared with those induced by either Na(+) depletion or dehydration. Na(+) depletion was induced by feeding the chickens with either a low- or a high-Na(+) diet for either 0.5, 1, 2, 4, or 8 days. Ileal and colonic NHE2 activity increased with the duration of the Na(+) depletion, whereas that of intestinal SGLT-1 decreased, reaching a plateau after 2 days of treatment. Three-hour incubation of the intestine with aldosterone produced the same effects on NHE activity as does Na(+) depletion, without altering SGLT-1 activity. However, 3-h incubation of the intestine with AVT increased intestinal SGLT-1 activity, without affecting intestinal NHE activity. It is concluded that aldosterone regulates apical ileal and colonic NHE2 activity, whereas that of SGLT-1 is regulated by AVT.

K+ transport in colonocytes isolated from the chick: effect of anisosmotic buffers.

Potassium transport was measured in isolated chicken colonocytes using 85Rb+ as a tracer for K+. Rb+ was determined by atomic absorption spectrometry. The results revealed that net K+ uptake occurred via at least four mechanisms: (i) Na+,K(+)-ATPase, (ii) K(+)-ATPase, (iii) Na(+)-K(+)-2Cl- cotransport system and (iv) a mechanism(s) which is resistant to both ouabain and bumetanide. The rate of K+(Rb+) efflux is stimulated by the calcium ionophore A23187, inhibited by either quinine, verapamil or Ba2+, and unaffected by either apamin, 3,4-diaminopyridine (3,4-DAP), H2-DIDS or bumetanide. The A23187-induced increase in K+(Rb+) efflux was abolished by apamin. These findings suggest that K+(Rb+) efflux from chicken colonocytes occurs at least in part through Ca(2+)-activated K+ channels. The present results also show that all these K+ transport systems are involved in cell volume regulation. Thus, external hyposmolarity decreased net K+(Rb+) uptake mediated by Na+,K(+)-ATPase, K(+)-ATPase and the Na(+)-K(+)-2Cl- cotransporter and increased K+(Rb+) efflux rate. The opposite was observed under hyperosmotic conditions.

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.

HCO3(-)-dependent ion transport systems and intracellular pH regulation in colonocytes from the chick.

The current study examines the presence of the Na+/HCO3- cotransporter and of the Cl-/HCO3- exchanger in chicken colonocytes and their role in cytosolic pH (pHi) homeostasis. pHi was measured with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) at 25 degreesC. Basal pHi was 7.16 in HEPES-buffered solutions and 7.06 in those buffered with HCO3-. Removal of external Cl- increased pHi and Cl- reinstatement brought the pHi towards resting values. These Cl--induced pHi changes were Na+-independent, inhibited by H2-DIDS and faster in the presence than in the absence of HCO3-. Cells recovered from alkaline loads by a mechanism that was Cl--dependent, Na+-independent and inhibited by H2-DIDS. This rate of Cl--dependent cell acidification decreased as the pHi decreased, with a Hill coefficient value close to 4. Removal of external Na+ decreased pHi and readdition of Na+ brought pHi towards the control values. The rate of the Na+-induced changes was not modified by the presence of HCO3- and was prevented by EIPA and unaffected by H2-DIDS. In the presence of EIPA cells partially recovered from a moderate acid load only when both Na+ and HCO3- were present. The EIPA resistant Na+- and bicarbonate-dependent pHi recovery was inhibited by H2-DIDS and occurred at equal rates in both Cl--containing and Cl--free solutions. It is concluded that in chicken colonocytes bathed in HCO3--buffered solutions, both the Na+/H+ exchanger and the Cl-/HCO3- exchanger participate in setting the resting pHi value. The latter transporter helps the cells to recover from alkaline loads and the first transporter, together with the Na+/HCO3- cotransporter, is involved in pHi recovery from an acid load.

In birds, NHE2 is major brush-border Na+/H+ exchanger in colon and is increased by a low-NaCl diet.

We previously reported that mammalian small intestinal and colonic brush borders (BBs) contained both epithelial Na+/H+ exchangers NHE2 and NHE3. We now show that, in the avian (chicken) colon, NHE2 is the major functional isoform under basal conditions and when stimulated by a low-NaCl diet. Hubbard chickens were maintained for 2 wk on a high- or low-NaCl diet. After the chickens were killed, the ileum and colon were removed, and BBs were prepared by Mg2+ precipitation and 22Na and D-[14C]glucose uptake determined in the BB vesicles. NHE2 and NHE3 were separated by differential sensitivity to HOE-694 (NHE2 defined as Na+/H+ exchange inhibited by 50 microM HOE-694). Chickens on a low-Na+ diet have increased plasma aldosterone (10 vs. 207 pg/ml). On the high-NaCl diet, both NHE2 and NHE3 contributed to ileal and colonic apical Na+/H+ exchange, contributing equally in ileum, but NHE2 being the major component in colon (86%). Low-NaCl diet significantly increased ileal and colonic BB Na+/H+ exchange; the increase in BB Na+/H+ exchange in both ileum and colon was entirely due to an increase in NHE2 with no change in NHE3 activity. In contrast, low-NaCl diet decreased ileal and colonic Na+-dependent D-glucose uptake. Western analysis showed that low-Na+ diet increased the amount of NHE2 in the ileal and colonic BB and decreased the amount of ileal Na+-dependent glucose transporter SGLT1. Both NHE2 and NHE3 were present in the apical but not basolateral membranes (BLM) of ileal and colonic epithelial cells. In summary, 1) NHE2 and NHE3 are both present in the BB and not BLM of chicken ileum and colon; 2) NHE2 is the major physiological colonic BB Na+/H+ exchanger under basal conditions; 3) low-NaCl diet, which increases plasma aldosterone, increases ileal and colonic BB Na+/H+ exchange and decreases Na+-dependent D-glucose uptake; 4) the stimulation of colonic BB Na+/H+ exchange is due to increased activity and amount of NHE2; and 5) the inhibition of ileal D-glucose uptake is associated with a decrease in SGLT1 amount. NHE2 is the major chicken colonic BB Na+/H+ exchanger.

Na+-H+ exchange and intracellular pH regulation in colonocytes from the chick.

The involvement of Na(+)-H+ exchange in chicken colonocyte homeostasis was investigated. Colonocyte pH (pH(i)) was measured with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The proton ionophore FCCP reduced basal pH(i), indicating that cytosolic [H+] is not at electrochemical equilibrium across the membrane. External Na+ removal decreased pH(i) and subsequent addition of Na+ returns pH(i) towards its control value. The rate of pH(i) recovery from an acid load was Na(+)-dependent (K(m) for Na+, 24 mM) and inhibited by EIPA (IC50, 0.18 microM). The initial rate of Na(+)-dependent cell alkalization increased as the pH(i) decreased from 7.2 to 6.6 (Hill coefficient, 1.88). Radioisotope flux studies revealed that an outwardly directed proton gradient transiently stimulated Na+ uptake into BBMV isolated from the chick colon. EIPA and amiloride inhibited pH gradient-driven Na+ uptake (IC50 of 4 microM and 32 microM, respectively). The K(m) for Na+ of pH gradient-driven Na+ uptake was 6.8 mM. The Hill coefficient of the relationship between the initial rate of pH-driven Na+ uptake and the intravesicular pH was 0.70. It is concluded that a Na(+)-H+ exchanger is involved in pH(i) homeostasis in chicken colonocytes and that these cells possess at least two types of Na(+)-H+ antiporters with different sensitivity to EIPA and different kinetic parameters.

Apical ouabain-sensitive K+-activated-ATPase activity in colon and caecum of the chick.

This study sought to investigate the presence and characteristics of K+-ATPase activity in chicken intestinal epithelia. A cytochemical method revealed Na+-independent, ouabain-sensitive, K+-ATPase activity in the apical, but not in the basolateral, membrane of chicken colonic and caecal epithelial cells. K+-ATPase activity was not observed in the small intestine. The measurement of K+-activated pNPPase activity was used to characterize the K+-ATPase activity evidenced by the cytochemical method. In addition, K+ and NH4+, but neither Na+ nor Li+, could activate pNPPase activity in chicken intestinal epithelia. Vanadate abolished ouabain-sensitive, K+-activated pNPPase activity in the three membrane preparations tested, whereas oligomycin and SCH 28080 were without effect. The Km for K+ and the ouabain IC50 values for the apical colonic and caecal K+-activated pNPPase activity were higher than those measured for K+-activated pNPPase activity measured in the basolateral membrane of chicken jejunal enterocytes. The results indicate that the apical membranes of chicken colon and caecum possess Na+-independent, ouabain-sensitive K+-activated-ATPase activity.

PKC activators stimulate H+ conductance in chicken enterocytes.

Chicken enterocytes present a H+-conducting pathway involved in the recovery of intracellular pH (pHi) from an acid load. In the current study we have tested the effect of protein kinase C (PKC) activators on the rate of proton efflux through the H+-conducting pathway. The rate of proton efflux was increased by the addition of 1,2-dioctanoyl-rac-glycerol (DOG) or phorbol 12-myristate 13-acetate (PMA), but it was not affected by the addition of the inactive phorbol ester analogue, 4alpha-phorbol 12, 13-didecanoate. DOG stimulated the process in a dose-dependent manner with a half-maximal effect at 45 microM. Staurosporine and Zn2+ prevented the DOG-dependent increase in the rate of proton efflux. The rate of proton efflux was affected by the pH, and DOG shifted this relationship upward and to the right. These results suggest that the proton-conducting pathway is regulated by PKC.

Intracellular pH regulation in chicken enterocytes: the importance of extracellular pH.

The present work reports the effect of pHo on pHi and Na(+)-H+ exchanger activity. Intracellular pH tended to follow pHo, but the proton distribution across the cell membrane is not at electrochemical equilibrium. Removal of external Na+ acidified the cells by both reversing the direction of the Na(+)-H+ exchanger and hyperpolarizing the cell membrane potential. The relationship between pHo and the rate of Na(+)-dependent proton efflux following an acid load suggests that external protons interact with the Na(+)-H+ exchanger at a single site with an apparent pK (-log of the dissociation constant) of 7.22. The results demonstrate that maintenance of pHo in the physiological range is essential for maintenance of normal cell pH and that the activity of the Na(+)-H+ exchanger involved in pHi regulation is affected by external protons. The results also suggest that, at least at low pHo, some intracellular mechanism is involved in pHi regulation.

Na+-HCO3(-) cotransporter and intracellular pH regulation in chicken enterocytes.

The current studies examine the presence of the Na+-HCO3(-) cotransporter in chicken enterocytes and its role in cytosolic pH (pHi) regulation. The pH-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF) was used to monitor pHi. Under resting conditions, pHi was 7.25 in solutions buffered with bis(2-hydroxyethyl)-1-piperazine ethanesulphonic acid (HEPES) and 7.17 in those buffered with HCO3(-). Removal of external Na+ decreased pHi and readdition of Na+ rapidly increased pHi towards the control values. These Na+-dependent changes were greater in HCO3(-)- than in HEPES-buffered solutions. In HCO3- - free solutions the Na+-dependent changes in pHi were prevented by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and unaffected by 4,4'-diisothiocyanatostilbene disulphonic acid (H2-DIDS). In the presence of HCO3-, the Na+-induced changes in pHi were sensitive to both EIPA and H2-DIDS. In the presence of EIPA, cells partially recovered from a moderate acid load only when both Na+ and HCO3- were present. This pHi recovery, which was EIPA resistant, and dependent on Na+ and HCO3-, was inhibited by H2-DIDS and occurred at equal rates in both Cl--containing and Cl--free solutions. Kinetic analysis of the rate of HCO3- and Na+-dependent pHi recovery from an acid load as a function of the Na+ concentration revealed first-order kinetics with a Michaelis constant, Km, of 11 mmol/l Na+. It is concluded that in HCO3(-) buffered solutions both the Na+/H+ exchanger and the Na+-HCO3(-) cotransporter participate in setting the resting pHi in isolated chicken enterocytes and help the recovery from acid loads.

Cytosolic pH regulation in chicken enterocytes: Na(+)-independent regulatory cell alkalinization.

The mechanisms involved in intracellular pH (pHi) recovery from an acid load have been investigated in enterocytes isolated from chicken. Following an intracellular acidification, by abrupt withdrawal of NH4Cl, pHi alkalinized in the nominally absence of Na+ and bicarbonate. This Na(+)- and bicarbonate-independent (NBI) regulatory cell alkalinization became negligible when the pHi has reached a value of approx. 6.85. Addition of Na+ induced a rapid pHi recovery to control values. Rotenone, DCCD, vanadate, NBD-Cl, SCH 28080 and EIPA inhibited the NBI cell alkalinization, whereas bafilomycin A1, ouabain and H2-DIDS were without effect. Na(+)-dependent pHi recovery from an acid load was inhibited by EIPA and unaffected by SCH 28080 or DCCD. The rate of NBI cell alkalinization was a linear function of the electrochemical proton gradient. In high external K+ buffer plus valinomycin the line goes through the origin. Gramicidin accelerated the rate of NBI cell alkalinization, whereas it was slightly reduced by low external potassium. The results demonstrate that in intestinal epithelial cells exist at least two mechanisms for proton secretion: a Na(+)-H+ exchanger and a Na(+)- and bicarbonate-independent proton transport system. This latter mechanism appears to be a proton conductance pathway.

Intracellular pH regulation in cecal epithelial cells from the chick.

Intracellular pH (pHi) regulation has been investigated in cells isolated from the proximal ceca of the chicken. pHi was measured with the pH-sensitive dye, 2',7'-bis(carboxyethyl)-5 (6)-carboxyfluorescein in nominally HCO(3-)-free solutions. Under resting conditions the pHi was 7.08. Removal of extracellular Na+ decreased pHi by approx. 0.24 pH units and the subsequent addition of Na+ increased pHi towards the control value. This Na(+)-dependent pHi recovery was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Following an intracellular acidification, by abrupt withdrawal of NH4Cl, pHi alkalinized in the nominally absence of Na+. Rotenone, N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, iodoacetic acid and SCH 28080 inhibited the Na(+)-independent pHi recovery rate by 82, 82, 67, 74, 77 and 50% respectively. Bafilomycin A1 was without effect. Na(+)-independent cell alkalization was stimulated by external K+. In the presence of N-ethylmaleimide addition of Na+ induced a rapid pHi recovery. The initial rate of this recovery exhibited first-order dependence on Na+ concentration and it was inhibited by EIPA. The initial rate of Na(+)-dependent cell alkalization increased with a Hill coefficient greater than one when pHi was reduced from 7.2 to 6.2. The 'set point' for the exchanger is approx. 7.5. These studies demonstrate that in cecal epithelial cells exist at least two mechanisms for proton secretion: a Na(+)-H+ exchanger and a Na(+)-independent proton transport system.