Effect of chloride on pH microclimate and electrogenic Na+ absorption across the rumen epithelium of goat and sheep

Active Na+ absorption across rumen epithelium comprises Na+/H+ exchange and a nonselective cation conductance (NSCC). Luminal chloride is able to stimulate Na+ absorption, which has been attributed to an interaction between Cl-/HCO3- and Na+/H+ exchangers. However, isolated rumen epithelial cells also express a Cl- conductance. We investigated whether Cl- has an additional effect on electrogenic Na+ absorption via NSCC. NSCC was estimated from short-circuit current (Isc) across epithelia of goat and sheep rumen in Ussing chambers. Epithelial surface pH (pHs) was measured with 5-N-hexadecanoyl-aminofluorescence. Membrane potentials were measured with microelelectrodes. Luminal, but not serosal, Cl- stimulated the Ca2+ and Mg2+ sensitive Isc. This effect was independent of the replacing anion (gluconate or acetate) and of the presence of bicarbonate. The mean pHs of rumen epithelium amounted to 7.47 +/- 0.03 in a low-Cl- solution. It was increased by 0.21 pH units when luminal Cl- was increased from 10 to 68 mM. Increasing mucosal pH from 7.5 to 8.0 also increased the Ca2+ and Mg2+ sensitive Isc and transepithelial conductance and reduced the fractional resistance of the apical membrane. Luminal Cl- depolarized the apical membrane of rumen epithelium. 5-Nitro-2-(3-phenylpropylamino)-benzoate reduced the divalent cation sensitive Isc, but only in low-Cl- solutions. The results show that luminal Cl- can increase the microclimate pH via apical Cl-/HCO3- or Cl-/OH- exchangers. Electrogenic Na+ absorption via NSCC increases with pH, explaining part of the Cl- effects on Na+ absorption. The data further show that the Cl- conductance of rumen epithelium must be located at the basolateral membrane.     

Identification of uterine ion transporters for mineralisation precursors of the avian eggshell

ABSTRACT: BACKGROUND: In Gallus gallus, eggshell formation takes place daily in the hen uterus and requires large amounts of the ionic precursors for calcium carbonate (CaCO3). Both elements (Ca2+, HCO3-) are supplied by the blood via trans-epithelial transport. Our aims were to identify genes coding for ion transporters that are upregulated in the uterine portion of the oviduct during eggshell calcification, compared to other tissues and other physiological states, and incorporate these proteins into a general model for mineral transfer across the tubular gland cells during eggshell formation. RESULTS: A total of 37 candidate ion transport genes were selected from our database of overexpressed uterine genes associated with eggshell calcification, and by analogy with mammalian transporters. Their uterine expression was compared by qRTPCR in the presence and absence of eggshell formation, and with relative expression levels in magnum (low Ca2+/HCO3- movement) and duodenum (high rates of Ca2+/HCO3- trans-epithelial transfer). We identified overexpression of eleven genes related to calcium movement: the TRPV6 Ca2+ channel (basolateral uptake of Ca2+), 28 kDa calbindin (intracellular Ca2+ buffering), the endoplasmic reticulum type 2 and 3 Ca2+ pumps (ER uptake), and the inositol trisphosphate receptors type 1, 2 and 3 (ER release). Ca2+ movement across the apical membrane likely involves membrane Ca2+ pumps and Ca2+/Na+ exchangers. Our data suggests that Na+ transport involved the SCNN1 channel and the Na+/Ca2+ exchangers SLC8A1, 3 for cell uptake, the Na+/K+ ATPase for cell output. K+ uptake resulted from the Na+/K+ ATPase, and its output from the K+ channels (KCNJ2, 15, 16 and KCNMA1).We propose that the HCO3- is mainly produced from CO2 by the carbonic anhydrase 2 (CA2) and that HCO3- is secreted through the HCO3-/Cl- exchanger SLC26A9. HCO3- synthesis and precipitation with Ca2+ produce two H+. Protons are absorbed via the membrane's Ca2+ pumps ATP2B1, 2 in the apical membrane and the vacuolar (H+)-atpases at the basolateral level. Our model incorporate Cl- ions which are absorbed by the HCO3-/Cl- exchanger SLC26A9 and by Cl- channels (CLCN2, CFTR) and might be extruded by Cl-/H+ exchanger (CLCN5), but also by Na+ K+ 2 Cl- and K+ Cl- cotransporters. CONCLUSIONS: Our Gallus gallus uterine model proposes a large list of ion transfer proteins supplying Ca2+ and HCO3- and maintaining cellular ionic homeostasis. This avian model should contribute towards understanding the mechanisms and regulation for ionic precursors of CaCO3, and provide insight in other species where epithelia transport large amount of calcium or bicarbonate.     

Evidence for NHE3-mediated Na transport in sheep and bovine forestomach

Na absorption across the cornified, multilayered, and squamous rumen epithelium is mediated by electrogenic amiloride-insensitive transport and by electroneutral Na transport. High concentrations of amiloride (>100 μM) inhibit Na transport, indicating Na(+)/H(+) exchange (NHE) activity. The underlying NHE isoform for transepithelial Na absorption was characterized by mucosal application of the specific inhibitor HOE642 for NHE1 and S3226 for NHE3 in Ussing chamber studies with isolated epithelia from bovine and sheep forestomach. S3226 (1 μM; NHE3 inhibitor) abolished electroneutral Na transport under control conditions and also the short-chain fatty acid-induced increase of Na transport via NHE. However, HOE642 (30 μM; NHE1 inhibitor) did not change Na transport rates. NHE3 was immunohistochemically localized in membranes of the upper layers toward the lumen. Expression of NHE1 and NHE3 has been previously demonstrated by RT-PCR, and earlier experiments with isolated rumen epithelial cells have shown the activity of both NHE1 and NHE3. Obviously, both isoforms are involved in the regulation of intracellular pH, pH(i). However, transepithelial Na transport is only mediated by apical uptake via NHE3 in connection with extrusion of Na by the basolaterally located Na-K-ATPase. The missing involvement of NHE1 in transepithelial Na transport suggests that the proposed "job sharing" in epithelia between these two isoforms probably also applies to forestomach epithelia: NHE3 for transepithelial transport and NHE1 for, among others, pH(i) and volume regulation.     

Divalent cations reduce the electrogenic transport of monovalent cations across rumen epithelium

The rumen epithelium of sheep and goats showed an increase in short circuit current ( Isc) and transepithelial conductance (gt) upon mucosal removal of divalent cations. A divalent-sensitive Isc and gt were present in Na+, K+ or Rb+ buffer, but nearly abolished in mucosal NMDG+ (N-methyl-D-glucamine) buffer. High K buffer, addition of BaCl2 or of ouabain on the serosal side also reduced or abolished the divalent-sensitive Isc. Mucosal Ca2+ was more potent in blocking Isc, but had the same potency as Mg2+ in blocking gt. A prolonged mucosal deprivation of Mg2+ ions increased gt, potential difference and basal as well as the Ca2+-sensitive Isc. Mucosal addition of Mg2+ had a smaller effect on gt after serosal preincubation with Ba. The data suggest that rumen epithelial cells exhibit an apical non-selective cation conductance, which permits the passage of monovalents in the mucosal absence of divalents. The development of a divalent-sensitive Isc in Na buffer requires Na+/K+ pumps and K+ recycling through Ba2+-sensitive K+ conductances on the basolateral side. This Isc is blocked by extracellular Ca2+ and both extracellular and intracellular Mg2+ ions. A prolonged deprivation of mucosal Mg2+ alone seems to affect intracellular Mg2+ in this Mg2+-absorbing tissue.     

Modulation of electroneutral Na transport in sheep rumen epithelium by luminal ammonia

Ammonia is an abundant fermentation product in the forestomachs of ruminants and the intestine of other species. Uptake as NH3 or NH4+ should modulate cytosolic pH and sodium-proton exchange via Na+/H+ exchanger (NHE). Transport rates of Na+, NH4+, and NH3 across the isolated rumen epithelium were studied at various luminal ammonia concentrations and pH values using the Ussing chamber method. The patch-clamp technique was used to identify an uptake route for NH4+. The data show that luminal ammonia inhibits electroneutral Na transport at pH 7.4 and abolishes it at 30 mM (P < 0.05). In contrast, at pH 6.4, ammonia stimulates Na transport (P < 0.05). Flux data reveal that at pH 6.4, approximately 70% of ammonia is absorbed in the form of NH4+, whereas at pH 7.4, uptake of NH3 exceeds that of NH4+ by a factor of approximately four. The patch-clamp data show a quinidine-sensitive permeability for NH4+ and K+ but not Na+. Conductance was 135 +/- 12 pS in symmetrical NH(4)Cl solution (130 mM). Permeability was modulated by the concentration of permeant ions, with P(K) > P(NH4) at high and P(NH4) > P(K) at lower external concentrations. Joint application of both ions led to anomalous mole fraction effects. In conclusion, the luminal pH determines the predominant form of ammonia absorption from the rumen and the effect of ammonia on electroneutral Na transport. Protons that enter the cytosol through potassium channels in the form of NH4+ stimulate and nonionic diffusion of NH3 blocks NHE, thus contributing to sodium transport and regulation of pH.     

Common channels for water and protons at apical and basolateral cell membranes of frog skin and urinary bladder epithelia. Effects of oxytocin, heavy metals, and inhibitors of H(+)-adenosine triphosphatase

We have compared the response of proton and water transport to oxytocin treatment in isolated frog skin and urinary bladder epithelia to provide further insights into the nature of water flow and H+ flux across individual apical and basolateral cell membranes. In isolated spontaneous sodium-transporting frog skin epithelia, lowering the pH of the apical solution from 7.4 to 6.4, 5.5, or 4.5 produced a fall in pHi in principal cells which was completely blocked by amiloride (50 microM), indicating that apical Na+ channels are permeable to protons. When sodium transport was blocked by amiloride, the H+ permeability of the apical membranes of principal cells was negligible but increased dramatically after treatment with antidiuretic hormone (ADH). In the latter condition, lowering the pH of the apical solution caused a voltage-dependent intracellular acidification, accompanied by membrane depolarization, and an increase in membrane conductance and transepithelial current. These effects were inhibited by adding Hg2+ (100 microM) or dicyclohexylcarbodiimide (DCCD, 10(-5) M) to the apical bath. Net titratable H+ flux across frog skin was increased from 30 +/- 8 to 115 +/- 18 neq.h-1.cm-2 (n = 8) after oxytocin treatment (at apical pH 5.5 and serosal pH 7.4) and was completely inhibited by DCCD (10(-5) M). The basolateral membranes of the principal cells in frog skin epithelium were found to be spontaneously permeable to H+ and passive electrogenic H+ transport across this membrane was not affected by oxytocin. Lowering the pH of the basolateral bathing solution (pHb) produced an intracellular acidification and membrane depolarization (and an increase in conductance when the normal dominant K+ conductance of this membrane was abolished by Ba2+ 1 mM). These effects of low pHb were blocked by micromolar concentrations of heavy metals (Zn2+, Ni2+, Co2+, Cd2+, and Hg2+). Lowering pHb in the presence of oxytocin (50 mU/ml) produced a transepithelial current (3 microA.cm-2 at pHb 5.5) which was blocked by 100 microM of Hg2+, Zn2+, or Ni2+ at the basolateral side, and by DCCD (10(-5) M) or Hg2+ (100 microM) from the apical side. The net hydroosmotic water flux (JH2O) induced by oxytocin in frog bladder sacs was blocked by inhibitors of H(+)-adenosine triphosphatase (ATPase). Diethylstilbestrol (DES 10(-5) M), oligomycin (10(-8) M), and DCCD (10(-5) M) prevented JH2O when present in the lumen. These effects cannot be attributed to inhibition of metabolism since cyanide (10(-4) M), or 2-deoxyglucose (10(-3) M) had no effect on JH2O.(ABSTRACT TRUNCATED AT 400 WORDS)     

Anion transport in the colon

The principal anions transported by colonic epithelium are Cl-, HCO3- and organic anions (OA-), particularly acetate, butyrate and pyruvate, these last being formed by microbial degradation of carbohydrate. In the normal absorptive rat colon, Cl- is transported from lumen to plasma both by the transcellular and paracellular pathways. The transcellular route appears to depend on amiloride-insensitive coupling of Na+-Cl- at the mucosal (apical) membrane, the Na+ electrochemical gradient energizing Cl- uptake. Intraluminal [HCO3-] rises as Cl- as absorbed, and a mucosal Cl- -HCO3- exchange carrier has been postulated. In some species (and in distal colon of the rat when sodium-depleted), the putative Na+-Cl- carrier is absent so that Cl- absorption then depends largely on the paracellular electrochemical gradient. Absorption of OA- is independent of the transepithelial p.d., is associated with HCO3- secretion and is considerably reduced by acetazolamide. In the absence of Cl-, OA- supports Na+ absorption but does not depend on it continuing unchanged when the latter is blocked. Colonic epithelium can become secretory and an example of this state is congenital chloridorrhoea in which an elevated transepithelial p.d. is associated with excessive Cl- secretion. Here, it appears that the Na+-Cl- and Cl- -HCO3- carriers are lost and Cl- conductance of the mucosal membrane substantially increased. The transepithelial uphill movements of Cl- or HCO3- in the absorptive and secretory colon appear to depend on coupling to other ionic flows, and there seems to be no need to postulate active transport of these ions.     

Electrolyte transport in the epithelium of pulmonary segments of normal and cystic fibrosis lung.

The epithelium of pulmonary segments from trachea to aveoli actively transports electrolytes and allows osmotic movement of water to maintain the ionic environment in the airway lumen. Models of airway absorption and secretion depict the operation of transporters localized to apical or basolateral membrane. In many epithelia, a variety of electrolyte transporters operate in different combinations to produce absorption or secretion. This also applies to pulmonary epithelium of the large airways (trachea, main-stem bronchi), bronchioles, and alveoli. Na+ absorption occurs in all three pulmonary segments but by different transporters: apical Na+ channels in large airways and bronchioles; Na+/H+ exchange and Na+ channels in adult alveoli. The Na+ channels in each pulmonary segment share a sensitivity to amiloride, a potent inhibitory of epithelial Na+ channels. Fetal alveoli display spontaneous Cl- secretion, as do the large airways of some mammals, such as dog and bovine trachea. Cl- channels differ in conductance properties and in regulation by intracellular second messengers, osmolarity, and voltage mediate stimulated Cl- secretion. Electroneutral carriers, such as NaCl(K) cotransport, Cl-/HCO3- exchange, and Na+/HCO3- exchange, operate in large airways and alveoli during absorption and secretion. Abnormal ion transport in airways of cystic fibrosis (CF) patients is manifest as a reduced Cl- conductance and increased Na+ conductance. Isolation of the CF gene and identification of its product CFTR now allow investigations into the basic defect. Intrinsic to these investigations is the development of systems to study the function of CFTR and its relation to electrolyte transporters and their regulation.     

Ambroxol-induced modification of ion transport in human airway Calu-3 epithelia

Ambroxol is often used as a mucolytic agent in various lung diseases. However, it is unclear how ambroxol acts on bronchial epithelial cells. To clarify the action of ambroxol, we studied the effects of ambroxol on the ion transport in human Calu-3 cells, a human submucosal serous cell line, measuring the transepithelial short-circuit current and conductance across monolayers of Calu-3 cells. Ambroxol of 100 microM diminished the terbutaline (a beta2-adrenergic agonist)-stimulated Cl-/HCO3(-)-dependent secretion without any decreases in the conductance of cystic fibrosis transmembrane conductance regulator (CFTR) channel locating on the apical membrane. On the other hand, under the basal (unstimulated) condition ambroxol increased the Cl(-)-dependent secretion with no significant change in the apical CFTR channel conductance and decreased the HCO3- secretion associated with a decrease in the apical CFTR channel conductance. Ambroxol had no major action on the epithelial Na+ channel (ENaC) or the ENaC-mediated Na+ absorption. These results indicate that in Calu-3 cells: (1) under the basal (unstimulated) condition ambroxol increases Cl- secretion by stimulating the entry step of Cl- and decreases HCO3- secretion by diminishing the activity of the CFTR channel and/or the Na+/HCO3(-)-dependent cotransporter, (2) under the adrenergic agonist-stimulated condition, ambroxol decreases Cl- secretion by acting on the Cl-/HCO3- exchanger, and (3) ambroxol has a more powerful action than the adrenergic agonist on the Cl-/HCO3- exchanger, leading fluid secretion to a moderately stimulated level from a hyper-stimulated level.     

K(+)- and HCO3(-)-dependent acid-base transport in squid giant axons. I. Base efflux

We used microelectrodes to monitor the recovery (i.e., decrease) of intracellular pH (pHi) after using internal dialysis to load squid giant axons with alkali to pHi values of 7.7, 8.0, or 8.3. The dialysis fluid (DF) contained 400 mM K+ but was free of Na+ and Cl-. The artificial seawater (ASW) lacked Na+, K+, and Cl-, thereby eliminating effects of known acid-base transporters on pHi. Under these conditions, halting dialysis unmasked a slow pHi decrease caused at least in part by acid-base transport we refer to as "base efflux." Replacing K+ in the DF with either NMDG+ or TEA+ significantly reduced base efflux and made membrane voltage (Vm) more positive. Base efflux in K(+)-dialyzed axons was stimulated by decreasing the pH of the ASW (pHo) from 8 to 7, implicating transport of acid or base. Although postdialysis acidifications also occurred in axons in which we replaced the K+ in the DF with Li+, Na+, Rb+, or Cs+, only with Rb+ was base efflux stimulated by low pHo. Thus, the base effluxes supported by K+ and Rb+ appear to be unrelated mechanistically to those observed with Li+, Na+, or Cs+. The combination of 437 mM K+ and 12 mM HCO3- in the ASW, which eliminates the gradient favoring a hypothetical K+/HCO3- efflux, blocked pHi recovery in K(+)-dialyzed axons. However, the pHi recovery was not blocked by the combination of 437 mM Na+, veratridine, and CO2/HCO3- in the ASW, a treatment that inverts electrochemical gradients for H+ and HCO3- and would favor passive H+ and HCO3- fluxes that would have alkalinized the axon. Similarly, the recovery was not blocked by K+ alone or HCO3- alone in the ASW, nor was it inhibited by the K-H pump blocker Sch28080 nor by the Na-H exchange inhibitors amiloride and hexamethyleneamiloride. Our data suggest that a major component of base efflux in alkali-loaded axons cannot be explained by metabolism, a H+ or HCO3- conductance, or by a K-H exchanger. However, this component could be mediated by a novel K/HCO3- cotransporter.     

Interaction between the basolateral K+ and apical Na+ conductances in Necturus urinary bladder

Experimental modulation of the apical membrane Na+ conductance or basolateral membrane Na+-K+ pump activity has been shown to result in parallel changes in the basolateral K+ conductance in a number of epithelia. To determine whether modulation of the basolateral K+ conductance would result in parallel changes in apical Na+ conductance and basolateral pump activity, Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that allowed rapid serosal solution changes. Exposure of the basolateral membrane to the K+ channel blockers Ba2+ (0.5 mM/liter), Cs+ (10 mM/liter), or Rb+ (10 mM/liter) increased the basolateral resistance (Rb) by greater than 75% in each case. The increases in Rb were accompanied simultaneously by significant increases in apical resistance (Ra) of greater than 20% and decreases in transepithelial Na+ transport. The increases in Ra, measured as slope resistances, cannot be attributed to nonlinearity of the I-V relationship of the apical membrane, since the measured cell membrane potentials with the K+ channel blockers present were not significantly different from those resulting from increasing serosal K+, a maneuver that did not affect Ra. Thus, blocking the K+ conductance causes a reduction in net Na+ transport by reducing K+ exit from the cell and simultaneously reducing Na+ entry into the cell. Close correlations between the calculated short-circuit current and the apical and basolateral conductances were preserved after the basolateral K+ conductance pathways had been blocked. Thus, the interaction between the basolateral and apical conductances revealed by blocking the basolateral K+ channels is part of a network of feedback relationships that normally serves to maintain cellular homeostasis during changes in the rate of transepithelial Na+ transport.     

Sodium-phosphate symport by Aplysia californica gut

Phosphate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Phosphate transport across apical membranes of vertebrate gastrointestinal epithelia requires sodium; whereas, its transport across the basolateral membrane requires antiport processes involving primarily chloride or bicarbonate. To decipher the phosphate transport mechanism in the foregut apical membrane of the mollusc, Aplysia californica, in vitro short-circuited Aplysia californica gut was used. Bidirectional transepithelial fluxes of both sodium and phosphate were measured to see whether there was interaction between the fluxes. The net mucosal-to-serosal flux of Na+ was enhanced by the presence of phosphate and it was abolished by the presence of serosal ouabain. Similarly, the net mucosal-to-serosal flux of phosphate was dependent upon the presence of Na+ and was abolished by the presence of serosal ouabain. Theophylline, DIDS and bumetande, added to either side, had no effect on transepithelial difference or short-circuit current in the Aplysia gut bathed in a Na2HPO4 seawater medium. However, mucosal arsenate inhibited the net mucosal-to-serosal fluxes of both phosphate and Na+ and the arsenate-sensitive Na+ flux to that of phosphate was 2:1. These results suggest the presence of a Na-PO4 symporter in the mucosal membrane of the Aplysia californica foregut absorptive cell.     

Kinetic transport model for cellular regulation of pH and solute concentration in the renal proximal tubule.

An open circuit kinetic model was developed to calculate the time course of proximal tubule cell pH, solute concentrations, and volume in response to induced perturbations in luminal or peritubular fluid composition. Solute fluxes were calculated from electrokinetic equations containing terms for known carrier saturabilities, allosteric dependences, and ion coupling ratios. Apical and basolateral membrane potentials were determined iteratively from the requirements of cell electroneutrality and equal opposing transcellular and paracellular currents. The model converged to membrane potentials accurate to 0.05% in one to four iterations. Model variables included cell concentrations of Na, K, HCO3, glucose, pH (uniform CO2), volume, and apical and basolateral membrane potentials. The basic model contained passive apical membrane transport of Na/H, Na/glucose, H and K, basolateral transport of Na/3HCO3, K, H, and glucose, and paracellular transport of Na, K, Cl, and HCO3; apical H and basolateral 3Na/2K-ATPases were present. Apical Na/H and basolateral K transport were regulated allosterically by pH. Apical Na/H transport, basolateral Na/3HCO3 transport, and the 3Na/2K-ATPase were saturable. Model parameters were chosen from data in the rat proximal tubule. Model predictions for the magnitude and time course of cell pH, Na, and membrane potential in response to rapid changes in apical and peritubular Na and HCO3 were in excellent agreement with experiment. In addition, the model requires that there exist an apical H-ATPase, basolateral Na/3HCO3 transport saturable with HCO3, and electroneutral basolateral K transport.     

Phenylalkylamine-sensitive calcium channels in osteoblast-like osteosarcoma cells. Characterization by ligand binding and single channel recordings

(-)-[3H]Desmethoxyverapamil ((-)-DMV) binds saturably to homogenates of the osteoblast-like cell lines UMR 106 and ROS 17/2.8 with KD values of 45 and 61 nM and Bmax values of 6.0 and 5 pmol/mg protein, respectively. Binding is stereoselective with (-)-DMV 8-10 times more potent than (+)-DMV. None of the dihydropyridine or benzothiazepine Ca2+ antagonists examined affect (-)-[3H]DMV binding. Monovalent cations such as Li+, Na+, and K+ inhibit (-)[3H]DMV binding in the 100-400 mM range. Divalent cations such as Ba2+, Sr2+, Ca2+, and Mg2+ are effective binding inhibitors in the 2-5 mM range. ROS 17/2.8 cells express a channel on the apical plasma membrane which conducts Ba2+ and Ca2+. With 110 mM BaCl2 or CaCl2 as charge carriers the single channel conductance is 3-5 picosiemens. In cell-excised patches the channel selects for Ba2+ over Na+ 3.3:1. In the absence of divalent ions the channel conducts Na+ ions with a single channel conductance of 13 picosiemens. This Na+ conductance decreases with physiological levels of Ca2+. The channel appears related to the (-)-[3H]DMV binding site, since its conductance is blocked by verapamil in a dose-dependent manner. Moreover, DMV blocks the channel stereoselectively with relative potencies of the isomers corresponding to their affinities for the binding site. The dihydropyridine drugs BAY K 8644 or (+)-202-791 do not affect channel opening. These binding and biophysical data indicate that osteoblast cells have a phenylalkylamine receptor associated with a Ca2+ channel.     

The action of epinephrine on Madin-Darby canine kidney cells

We have used cultured monolayers of Madin-Darby canine kidney (MDCK) cells, which form epithelial layers of high transepithelial resistance, grown on Millipore filters, for transport studies. In the absence of hormones net ion transport is of small magnitude and is consistent with a net absorptive flow (apical to basal) of Na+. Epinephrine, effective only from the basolateral cell surface, stimulates a net secretion (basal to apical) of Cl-. A substantial portion of net Cl- secretion is inhibited by loop diuretics such as furosemide applied to the basolateral cell aspects. The participation of a diuretic-sensitive cotransport system for Na+, K+, and Cl-, similar to that found in other cells, in transepithelial Cl- flux is postulated. The action of catecholamines on MDCK cell adenylate cyclase and on a Ca2+-activated K+ conductance is described.     

Monensin Inhibition of Na+-Dependent HCO3- Transport Distinguishes It from Na+-Independent HCO3- Transport and Provides Evidence for Na+/HCO3- Symport in the Cyanobacterium Synechococcus UTEX 625

The effect of monensin, an ionophore that mediates Na+/H+ exchange, on the activity of the inorganic carbon transport systems of the cyanobacterium Synechococcus UTEX 625 was investigated using transport assays based on the measurement of chlorophyll a fluorescence emission or 14C uptake. In Synechococcus cells grown in standing culture at about 20 [mu]M CO2 + HCO3-, 50 [mu]M monensin transiently inhibited active CO2 and Na+-independent HCO3- transport, intracellular CO2 and HCO3- accumulation, and photosynthesis in the presence but not in the absence of 25 mM Na+. These activities returned to near-normal levels within 15 min. Transient inhibition was attributed to monensin-mediated intracellular alkalinization, whereas recovery may have been facilitated by cellular mechanisms involved in pH homeostasis or by monensin-mediated H+ uptake with concomitant K+ efflux. In air-grown cells grown at 200 [mu]M CO2 + HCO3- and standing culture cells, Na+-dependent HCO3- transport, intracellular HCO3- accumulation, and photosynthesis were also inhibited by monensin, but there was little recovery in activity over time. However, normal photosynthetic activity could be restored to air-grown cells by the addition of carbonic anhydrase, which increased the rate of CO2 supply to the cells. This observation indicated that of all the processes required to support photosynthesis only Na+-dependent HCO3- transport was significantly inhibited by monensin. Monensin-mediated dissipation of the Na+ chemical gradient between the medium and the cells largely accounted for the decline in the HCO3- accumulation ratio from 751 to 55. The two HCO3- transport systems were further distinguished in that Na+-dependent HCO3- transport was inhibited by Li+, whereas Na+-independent HCO3- transport was not. It is suggested that Na+-dependent HCO3- transport involves an Na+/HCO3- symport mechanism that is energized by the Na+ electrochemical potential.     

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

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

Cation-selective channels in amphibian epithelia: electrophysiological properties and activation

1. Na+ as well as Li+ move across the apical membrane through amiloride-sensitive ionic channels. 2. K+ movements across the apical membrane occur through Ba2+- and Cs+-sensitive channels which do not allow the passage of Na+ or Li+. 3. A third pathway in the apical membrane is permeable for Na+, K+, Cs+, Rb+, NH+4 and Ti+. The currents carried by these monovalent cations are blocked by Ca2+ and divalent cations as well as La3+. 4. In the urinary bladder, the Ca2+-sensitive currents are stimulated by oxytocin, activators of cytosolic cAMP and cAMP analogues. Also the oxytocin activated currents are blocked by divalent cations and La3+. 5. Nanomolar concentrations of mucosal Ag+ activate the third channel and open the pathway for movements of Ca2+, Ba2+ and Mg2+, which are known to permeate through Ca2+ channels in excitable tissues.     

Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium

Inward-rectifier K channel: using macroscopic voltage clamp and single- channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current- voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell- attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. Conclusions: under physiological ionic gradients, a 15-pS inward- rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.     

SLC26A9 is expressed in gastric surface epithelial cells, mediates Cl-/HCO3- exchange, and is inhibited by NH4+

HCO3- secretion by gastric mucous cells is essential for protection against acidic injury and peptic ulcer. Herein we report the identification of an apical HCO3- transporter in gastric surface epithelial cells. Northern hybridization and RT-PCR demonstrate the expression of this transporter, also known as SLC26A9, in mouse and rat stomach and trachea (but not kidney). In situ hybridization in mouse stomach showed abundant expression of SLC26A9 in surface epithelial cells with apical localization on immunofluorescence labeling. Functional studies in HEK-293 cells demonstrated that SLC26A9 mediates Cl-/HCO3- exchange and is also capable of Cl--independent HCO3- extrusion. Unlike other anion exchangers or transport proteins reported to date, SLC26A9 activity is inhibited by ammonium (NH4+). The inhibitory effect of NH4+ on gastric HCO3- secretion was also indicated by reduced gastric juxtamucosal pH (pHjm) in rat stomach in vivo. This report is the first to describe the inhibition of HCO3- transport in vitro and the reduction of pHjm in stomach in vivo by NH4+. Given its critical localization on the apical membrane of surface epithelial cells, its ability to transport HCO3-, and its inhibition by NH4+, we propose that SLC26A9 mediates HCO3- secretion in surface epithelial cells and is essential for protection against acidic injury in the stomach. Disease states that are associated with increased ammonia (NH3)/NH4+ generation (e.g., Helicobacter pylori) may impair gastric HCO3- secretion and therefore predispose patients to peptic ulcer by inhibiting SLC26A9.