A Mathematical Model of the Pancreatic Ductal Epithelium

A mathematical model of the HCO⁻ ₃-secreting pancreatic ductal epithelium was developed using network thermodynamics. With a minimal set of assumptions, the model accurately reproduced the experimentally measured membrane potentials, voltage divider ratio, transepithelial resistance and short-circuit current of nonstimulated ducts that were microperfused and bathed with a CO₂/HCO⁻ ₃-free, HEPES-buffered solution, and also the intracellular pH of duct cells bathed in a CO₂/HCO⁻ ₃-buffered solution. The model also accurately simulated: (i) the effect of step changes in basolateral K⁺ concentration, and the effect of K⁺ channel blockers on basolateral membrane potential; (ii) the intracellular acidification caused by a Na⁺-free extracellular solution and the effect of amiloride on this acidification; and (iii) the intracellular alkalinization caused by a Cl⁻-free extracellular solution and the effect of DIDS on this alkalinization. In addition, the model predicted that the luminal Cl⁻ conductance plays a key role in controlling both the HCO⁻ ₃ secretory rate and intracellular pH during HCO⁻ ₃ secretion. We believe that the model will be helpful in the analysis of experimental data and improve our understanding of HCO⁻ ₃-transporting mechanisms in pancreatic duct cells. Received: 18 October 1995/Revised: 5 July 1996     

H+ and Cl− secretion in the stomach of the teleost fish, Anguilla anguilla : stimulation by histamine and carbachol

The fundus of an eel stomach was mounted in an Ussing chamber and bathed with control Ringer on the serosal side and with unbuffered solution on the mucosal side. The gastric mucosa exhibited a mucosa negative transepithelial voltage (V _t), a “short circuit” current (I _SC) and a small spontaneous acid secretion rate (J _H). All these parameters were abolished by cimetidine treatment. Bilateral ion substitution experiments in tissues lacking spontaneous acid secretion suggested that a net Cl⁻ transport from serosa to mucosa was responsible for the genesis of the I _SC in the absence of H⁺ secretion. Serosal application of histamine (10⁻⁴ mol · l⁻¹) or carbachol (10⁻⁴ mol · l⁻¹) stimulated both I _SC and J _H. The action of carbachol was independent of histamine. The control as well as the histamine-stimulated I _SC was sensitive to both serosal bumetanide (10⁻⁵ mol · l⁻¹), inhibitor of the Na⁺-K⁺-2Cl⁻ cotransport, and 4,4-diisothiocyano-stilbene-2,2-disulphonic acid (DIDS, 5 · 10⁻⁴ mol · l⁻¹), inhibitor of the Cl⁻-HCO⁻ ₃ exchange, while the I _SC stimulated by carbachol was nullified by serosal DIDS. These data suggested that the non-acidic Cl⁻ uptake across the serosal membrane was linked to the activity of both Na⁺-K⁺-2Cl⁻ cotransport and Cl⁻-HCO⁻ ₃ antiporter; histamine stimulated both transporters while carbachol was limited to the anion exchanger. The finding that the acid secretion was strictly dependent on serosal Cl⁻ and was completely blocked by serosal DIDS suggested that the Cl⁻ accompanying H⁺ secretion entered the cell through the serosal membrane by the Cl⁻-HCO⁻ ₃ exchange. In addition, the acid secretion stimulated by carbachol was also dependent on serosal Na⁺ and sensitive to the application of 5-N-N-dimethyl-amiloride in the serosal bath, suggesting that the increased activity of the Cl⁻-HCO⁻ ₃ during carbachol treatment was linked to the activation of serosal Na⁺-H⁺ exchange. The inhibitory effect of luminal omeprazole (10⁻⁴ mol · l⁻¹) on acid secretion suggested the presence of the H⁺-K⁺ pump on the luminal membrane. Accepted: 18 September 1997     

Acid–base regulation in the plainfin midshipman (Porichthys notatus): an aglomerular marine teleost

The plainfin midshipman (Porichthys notatus) possesses an aglomerular kidney and like other marine teleosts, secretes base into the intestine to aid water absorption. Each of these features could potentially influence acid–base regulation during respiratory acidosis either by facilitating or constraining HCO₃ ⁻ accumulation, respectively. Thus, in the present study, we evaluated the capacity of P. notatus to regulate blood acid–base status during exposure to increasing levels of hypercapnia (nominally 1–5% CO₂). Fish exhibited a well-developed ability to increase plasma HCO₃ ⁻ levels with values of 39.8 ± 2.8 mmol l⁻¹ being achieved at the most severe stage of hypercapnic exposure (arterial blood PCO₂ = 21.9 ± 1.7 mmHg). Consequently, blood pH, while lowered by 0.15 units (pH = 7.63 ± 0.06) during the final step of hypercapnia, was regulated far above values predicted by chemical buffering (predicted pH = 7.0). The accumulation of plasma HCO₃ ⁻ during hypercapnia was associated with marked increases in branchial net acid excretion (J _NETH⁺) owing exclusively to increases in the titratable alkalinity component; total ammonia excretion was actually reduced during hypercapnia. The increase in J _NETH⁺ was accompanied by increases in branchial carbonic anhydrase (CA) enzymatic activity (2.8×) and CA protein levels (1.6×); branchial Na⁺/K⁺-ATPase activity was unaffected. Rectal fluids sampled from control fish contained on average HCO₃ ⁻ concentrations of 92.2 ± 4.8 mmol l⁻¹. At the highest level of hypercapnia, rectal fluid HCO₃ ⁻ levels were increased significantly to 141.8 ± 7.4 mmol l⁻¹ but returned to control levels during post-hypercapnia recovery (96.0 ± 13.2 mmol l⁻¹). Thus, the impressive accumulation of plasma HCO₃ ⁻ to compensate for hypercapnic acidosis occurred against a backdrop of increasing intestinal HCO₃ ⁻ excretion. Based on in vitro measurements of intestinal base secretion in Ussing chambers, it would appear that P. notatus did not respond by minimizing base loss during hypercapnia; the increases in base flux across the intestinal epithelium in response to alterations in serosal HCO₃ ⁻ concentration were similar in preparations obtained from control or hypercapnic fish. Fish returned to normocapnia developed profound metabolic alkalosis owing to unusually slow clearance of the accumulated plasma HCO₃ ⁻. The apparent inability of P. notatus to effectively excrete HCO₃ ⁻ following hypercapnia may reflect its aglomerular (i.e., non-filtering) kidney coupled with the normally low rates of urine production in marine teleosts.     

Protective effects of prostaglandins in the isolated gastric mucosa of the eel, Anguilla anguilla

The protective effect of endogenous prostaglandins on the fish gastric mucosa was evaluated by studying the effect of indomethacin and aspirin, known cyclooxigenase inhibitors, on the mucosal ulceration in the isolated gastric sacs of Anguilla anguilla. Gastric sacs devoid of muscle layers were incubated in the presence of indomethacin (10⁻⁴ mol · l⁻¹) or aspirin (10⁻⁴ mol · l⁻¹) in different experimental conditions. Both the anti-inflammatory drugs produced ulcers, but the effects were more severe in the presence of histamine and in the absence of HCO₃ ⁻ in the incubation bath. The effects of prostaglandin E₂ (PGE₂) on acid secretion rate (J_H) and on alkaline secretion rate (J_OH) were evaluated (with the aid of the pH stat method) in isolated gastric mucosa mounted in Ussing chambers. We found that PGE₂ (10⁻⁸–10⁻⁵ mol · l⁻¹) increased J_H in a dose-dependent manner. In tissues pretreated with luminal omeprazole (10⁻⁴ mol · l⁻¹), PGE₂ stimulated gastric alkaline secretion. It was nullified by serosal removal of HCO₃ ⁻ or Na⁺ and by serosal ouabain (10⁻⁴ mol · l⁻¹). These results suggested that prostaglandins also exert their protective effects in fish gastric mucosa. This protection seems partially due to a stimulation of exogenous HCO₃ ⁻ transport from the serosal to the mucosal side. It is likely that this transport is an active transcellular mechanism coupled to Na⁺ transport. Accepted: 14 April 2000     

HCO3 − Transport in a Mathematical Model of the Pancreatic Ductal Epithelium

We have used computer modeling to investigate how pancreatic duct cells can secrete a fluid containing near isotonic (∼140 mm) NaHCO₃. Experimental data suggest that NaHCO₃ secretion occurs in three steps: (i) accumulation of HCO⁻ ₃ across the basolateral membrane of the duct cell by Na(HCO₃) _n cotransporters, Na⁺/H⁺ exchangers and proton pumps; (ii) secretion of HCO⁻ ₃ across the luminal membrane on Cl⁻/HCO⁻ ₃ antiporters operating in parallel with Cl⁻ channels; and (iii) diffusion of Na⁺ through the paracellular pathway. Programming the currently available experimental data into our computer model shows that this mechanism for HCO⁻ ₃ secretion is deficient in one important respect. While it can produce a relatively large volume of a HCO⁻ ₃-rich fluid, it can only raise the luminal HCO⁻ ₃ concentration up to about 70 mm. To achieve secretion of 140 mm NaHCO₃ by the model it is necessary to: (i) reduce the conductive Cl⁻ permeability and increase the conductive HCO⁻ ₃ permeability of the luminal membrane of the duct cell, and (ii) reduce the activity of the luminal Cl⁻/HCO⁻ ₃ antiporters. Under these conditions most of the HCO⁻ ₃ is secreted via a conductive pathway. Based on our data, we propose that HCO⁻ ₃ secretion occurs mainly by the antiporter in duct segments near the acini (luminal HCO⁻ ₃ concentration up to ∼70 mm), but mainly via channels further down the ductal tree (raising luminal HCO⁻ ₃ to ∼140 mm). Received: 15 November 1999/Revised: 29 March 2000     

Oxygen consumption rate and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase activity in early developmental stages of the sea urchin <Emphasis Type="Italic">Paracentrotus lividus</Emphasis> Lam.

Changes in oxygen consumption rate and Na⁺/K⁺-ATPase activity during early development were studied in the sea urchin Paracentrotus lividus Lam. The oxygen consumption rate increased from 0.12 μmol O₂ mg protein⁻¹ h⁻¹ in unfertilized eggs to 0.38 μmol O₂ mg protein⁻¹ h⁻¹ 25 min after fertilization. Specific activity of the Na⁺/K⁺-ATPase was significantly stimulated after fertilization, ranging up to 1.07 μmol P_i h⁻¹ mg protein⁻¹ in the late blastula stage and slightly lower values in the early and late pluteus stages.     

Control of Cell pH in the T84 Colon Cell Line

Cell pH regulation was investigated in the T84 cell line derived from epithelial colon cancer. Cell pH was measured by ratiometric fluorescence microscopy using the fluorescent probe BCECF. Basal pH was 7.17 ± 0.023 (n= 48) in HEPES Ringer. After acidification by an ammonium pulse, cell pH recovered toward normal at a rate of 0.13 ± 0.011 pH units/min in the presence of Na⁺, but in the absence of this ion or after treatment with 0.1 mm hexamethylene amiloride (HMA) no significant recovery was observed, indicating absence of Na⁺ independent H⁺ transport mechanisms in HEPES Ringer. In CO₂/HCO⁻ ₃ Ringer, basal cell pH was 7.21 ± 0.020 (n= 35). Changing to HEPES Ringer, a marked alkalinization was observed due to loss of CO₂, followed by return to the initial pH at a rate of −0.14 ± 0.012 (n= 8) pH/min; this return was retarded or abolished in the absence of Cl⁻ or after addition of 0.2 mm DIDS, suggesting extrusion of bicarbonate by Cl⁻/HCO⁻ ₃ exchange. This exchange was not Na⁺ dependent. When Na⁺ was added to cells incubated in 0 Na⁺ Ringer while blocking Na⁺/H⁺ exchange by HMA, cell alkalinization by 0.19 ± 0.04 (n= 11) pH units was observed, suggesting the presence of Na⁺/HCO⁻ ₃ cotransport carrying HCO⁻ ₃ into these cells, which was abolished by DIDS. These experiments, thus, show that Na⁺/H⁺ and Cl⁻/HCO⁻ ₃ exchange and Na⁺/HCO⁻ ₃ cotransport participate in cell pH regulation in T84 cells. Received: 3 April 2000/Revised: 22 June 2000     

Gramicidin-Perforated Patch Analysis on HCO3 − Secretion Through a Forskolin-Activated Anion Channel in Rat Parotid Intralobular Duct Cells

Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO₃ ⁻ secretion in the intralobular duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K⁺, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 μm), a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel. The anion current was markedly suppressed by addition of 1 mm methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO₃ ⁻. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO₃ ⁻ significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl⁻ channel located in luminal membranes) and production of cytosolic HCO₃ ⁻ induce the inward anion current and resulting depolarization. Inhibition of the Na⁺-K⁺-2Cl⁻ cotransporter and the Cl⁻-HCO₃ ⁻ exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl⁻ via the Na⁺-K⁺-2Cl⁻ cotransporter or the Cl⁻-HCO₃ ⁻ exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO₃ ⁻ produced intracellularly, but not of Cl⁻ due to lack of active Cl⁻ transport in parotid duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport. Received: 17 June 2000/Revised: 14 November 2000     

Electrogenic Cl<Superscript>−</Superscript> secretion does not occur in the ileum of the Australian common brushtail possum, <Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>, due to low levels of expression of the NaK2Cl cotransporter,NKCC1

The colon of the brushtail possum does not have an electrogenic secretory response. Given the functional significance of electrogenic Cl⁻ secretion in the intestine of eutherian mammals, we have investigated the secretory response in the small intestine of this marsupial. In the Ussing chamber cAMP-dependent secretagogues stimulated a sustained increase in ileal short-circuit current (Isc), whereas Ca²⁺-dependent secretagogues induced a transient increase. Both the responses were inhibited by mucosal addition of the anion channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (100 μmol l⁻¹), consistent with an anion secretory response. However, the responses were not inhibited by serosal bumetanide (10 μmol l⁻¹) and were independent of bath Cl⁻, indicating that the stimulated ileal Isc does not involve electrogenic Cl⁻ secretion driven by the NaK2Cl cotransporter, NKCC1. Consistent with this, there were low levels of NKCC1 expression in the ileal epithelium. In particular, NKCC1 expression in the ileal crypt cells was comparable to that of the villous cells. This differs from eutherian mammals where high levels of NKCC1 expression in the ileal crypt cells are associated with their role in Cl⁻ secretion. The cAMP- and Ca²⁺-dependent secretory responses were inhibited by the removal of HCO₃ ⁻ suggesting that these responses were due to electrogenic HCO₃ ⁻ secretion. We conclude that the ileum of the possum does not secrete Cl⁻ due to low levels of NKCC1 expression. It does however appear to secrete HCO₃ ⁻. These results are further significant examples of differences in the transport function of the possum intestinal epithelium compared with eutherian mammals.     

Postprandial acid–base balance and ion regulation in freshwater and seawater-acclimated European flounder, <Emphasis Type="Italic">Platichthys flesus</Emphasis>

The effects of feeding on both acid–base and ion exchange with the environment, and internal acid–base and ion balance, in freshwater and seawater-acclimated flounder were investigated. Following voluntary feeding on a meal of 2.5–5% body mass and subsequent gastric acid secretion, no systemic alkaline tide or respiratory compensation was observed in either group. Ammonia efflux rates more than doubled from 489 ± 35 and 555 ± 64 μmol kg⁻¹ h⁻¹ under control conditions to 1,228 ± 127 and 1,300 ± 154 μmol kg⁻¹ h⁻¹ post-feeding in freshwater and seawater-acclimated fish, respectively. Based on predictions of gastric acid secreted during digestion, we calculated net postprandial internal base gains (i.e., HCO₃⁻ secreted from gastric parietal cells into the blood) of 3.4 mmol kg⁻¹ in seawater and 9.1 mmol kg⁻¹ in freshwater-acclimated flounder. However, net fluxes of ammonia, titratable alkalinity, Na⁺ and Cl⁻ indicated that branchial Cl⁻/HCO₃⁻ and Na⁺/H⁺ exchange played minimal roles in counteracting these predicted base gains and cannot explain the absence of alkaline tide. Instead, intestinal Cl⁻/HCO₃⁻ exchange appears to be enhanced after feeding in both freshwater and seawater flounder. This implicates the intestine rather than the gills as a potential route of postprandial base excretion in fish, to compensate for gastric acid secretion.     

Activity and Stoichiometry of Na+:HCO− 3 Cotransport in Immortalized Renal Proximal Tubule Cells

The proximal tubule Na⁺-HCO⁻ ₃ cotransporter is located in the basolateral plasma membrane and moves Na⁺, HCO⁻ ₃, and net negative charge together out of the cell. The presence of charge transport implies that at least two HCO⁻ ₃ anions are transported for each Na⁺ cation. The actual ratio is of physiological interest because it determines direction of net transport at a given membrane potential. To determine this ratio, a thermodynamic approach was employed that depends on measuring charge flux through the cotransporter under defined ion and electrical gradients across the basolateral plasma membrane. Cells from an immortalized rat proximal tubule line were grown as confluent monolayer on porous substrate and their luminal plasma membrane was permeabilized with amphotericin B. The electrical properties of these monolayers were measured in a Ussing chamber, and ion flux through the cotransporter was achieved by applying Na⁺ or HCO⁻ ₃ concentration gradients across the basolateral plasma membrane. Charge flux through the cotransporter was identified as difference current due to the reversible inhibitor dinitro-stilbene disulfonate. The cotransporter activity was Cl⁻ independent; its conductance ranged between 0.12 and 0.23 mS/cm² and was voltage independent between −60 and +40 mV. Reversal potentials obtained from current-voltage relations in the presence of Na⁺ gradients were fitted to the thermodynamic equivalent of the Nernst equation for coupled ion transport. The fit yielded a cotransport ratio of 3HCO⁻ ₃:1Na⁺. Received: 19 January 1996/Revised: 24 April 1996     

Concurrent and Independent HCO− 3 and Cl− Secretion in a Human Pancreatic Duct Cell Line (CAPAN-1)

The present study investigated both HCO⁻ ₃ and Cl⁻ secretions in a human pancreatic duct cell line, CAPAN-1, using the short-circuit current (I _sc ) technique. In Cl⁻/HCO⁻ ₃-containing solution, secretin (1 μm) or forskolin (10 μm) stimulated a biphasic rise in the I _sc which initially reached a peak level at about 3 min and then decayed to a plateau level after 7 min. Removal of external Cl⁻ abolished the initial transient phase in the forskolin-induced I _sc while the plateau remained. In HCO⁻ ₃/CO₂-free solution, on the contrary, only the initial transient increase in I _sc was prominent. Summation of the current magnitudes observed in Cl⁻-free and HCO⁻ ₃-free solutions over a time course of 10 min gave rise to a curve which was similar, both in magnitude and kinetics, to the current observed in Cl⁻/HCO⁻ ₃-containing solution. Removal of external Na⁺ greatly reduced the initial transient rise in the forskolin-induced I _sc response, and the plateau level observed under this condition was similar to that obtained in Cl⁻-free solution, suggesting that Cl⁻-dependent I _sc was also Na⁺-dependent. Bumetanide (50 μm), an inhibitor of the Na⁺-K⁺-2Cl⁻ cotransporter, and Ba²⁺ (1 mm), a K⁺ channel blocker, could reduce the forskolin-induced I _sc obtained in Cl⁻/HCO⁻ ₃-containing or HCO⁻ ₃-free solution. However, they were found to be ineffective when external Cl⁻ was removed, indicating the involvement of these mechanisms in Cl⁻ secretion. On the contrary, the HCO⁻ ₃-dependent (in the absence of external Cl⁻) forskolin-induced I _sc could be significantly reduced by carbonic anhydrase inhibitor, acetazolamide (45 μm). Basolateral application of amiloride (100 μm) inhibited the I _sc ; however, a specific Na⁺-H⁺ exchanger blocker, 5-N-methyl-N-isobutylamiloride (MIA, 5–10 μm) was found to be ineffective, excluding the involvement of the Na⁺-H⁺ exchanger. However, an inhibitor of H⁺-ATPase, N-ethylmaleimide did suppress the I _sc (IC₅₀= 22 μm). Immunohistochemical studies also confirmed the presence of a vacuolar type of H⁺-ATPase in these cells. H₂DIDS (100 μm), an inhibitor of Na⁺-HCO⁻ ₃ cotransporter, was without effect. Apical addition of Cl⁻ channel blocker, diphenylamine-2,2′-dicarboxylic acid (DPC, 1 mm), but not disulfonic acids, DIDS (100 μm) or SITS (100 μm), exerted an inhibitory effect on both Cl⁻ and HCO⁻ ₃-dependent forskolin-induced I _sc responses. Histochemical studies showed discrete stainings of carbonic anhydrase in the monolayer of CAPAN-1 cells, suggesting that HCO⁻ ₃ secretion may be specialized to a certain population of cells. The present results suggest that both HCO⁻ ₃ and Cl⁻ secretion by the human pancreatic duct cells may occur concurrently and independently. Received: 17 October 1997/Revised: 3 April 1998     

Intracellular pH regulation and buffer capacity in CO2/HCO− 3-buffered media in cultured epithelial cells from rainbow trout gills

The influence of a CO₂/HCO⁻ ₃-buffered medium on intracellular pH regulation of gill pavement cells from freshwater rainbow trout was examined in monolayers grown in primary culture on glass coverslips; intracellular pH (pH_i) was monitored by continuous spectrofluorometric recording from cells loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxy-fluoroscein. When cells in HEPES-buffered medium at normal pH=7.70 were transferred to normal CO₂/HCO⁻ ₃-buffered medium {P _CO2=3.71 mmHg, [HCO⁻ ₃]= 6.1 mmol l⁻¹, extracellular pH (pH_e)=7.70}, they exhibited a brief acidosis but subsequently regulated the same pHi (∼7.41) as in HEPES. Buffer capacity (β) increased by the expected amount (5.5–8.0 slykes) based on intracellular [HCO⁻ ₃], and was unaffected by most drugs and treatments. However, after transfer to high P _CO2=11.15 mmHg, [HCO⁻ ₃]= 18.2 mmol l⁻¹ at the same pH_e=7.70, the final regulated pHi was elevated (∼7.53). The rate of correction of alkalosis caused by washout of this high P _CO2, high-HCO⁻ ₃ medium was unaffected by removal of extracellular Cl⁻. Removal of extracellular Na⁺ lowered resting pH_i and greatly inhibited the rate of pH_i recovery from acidosis. Bafilomycin A₁ (3 μmol l⁻¹) had no effect on these responses. However amiloride (0.2 mmol l⁻¹) inhibited recovery from acidosis caused by washout of an ammonia prepulse, but did not affect resting pH_i, the latter differing from the response in HEPES where amiloride also lowered resting pH_i. Similarly 4-acetamido-4′- isothiocyanatostilbene-2,2′-disulfonic acid, sodium salt (0.1 mmol l⁻¹) did not affect resting pH_i but slowed the rate of recovery from acidosis, though to a lesser extent than amiloride. Removal of extracellular Cl⁻ also slowed the rate of recovery but greatly increased β by an unknown mechanism; when this was taken into account, H⁺ extrusion rate was unaffected. These results are consistent with the presence of Na⁺-(HCO⁻ ₃)_N co-transport and/or Na⁺-dependent HCO⁻ ₃/Cl⁻ exchange, in addition to Na⁺/H⁺ exchange, as mechanisms contributing to “housekeeping” pH_i regulation in gill cells in CO₂/HCO⁻ ₃ media, whereas only Na⁺/H⁺ exchange is seen in HEPES. Both Na⁺-independent Cl⁻/HCO⁻ ₃ exchange and V-type H⁺-ATPase mechanisms appear to be absent from these cells cultured in isotonic media. Accepted: 30 November 1999     

Renal Cortical Basolateral Na+/HCO− 3 Cotransporter: IV Characterization and Localization with Polyclonal Antibodies

We have previously partially purified the basolateral Na⁺/HCO⁻ ₃ cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na⁺/HCO⁻ ₃ cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity. Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against the Na⁺/HCO⁻ ₃ cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na⁺/HCO⁻ ₃ cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes. The purified antibody fraction caused a concentration-dependent inhibition of the Na⁺/HCO⁻ ₃ cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence of the substrates (NaHCO₃ or Na₂CO₃) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and small intestine but was not detected in red blood cell membranes. Localization of the Na⁺/HCO⁻ ₃ cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against the 56 kDa basolateral protein inhibit the activity of the Na⁺/HCO⁻ ₃ cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact at or near the substrate binding sites. The Na⁺/HCO cotransporter protein is expressed in different regions of the kidneys and in other tissues. Received: 27 January 1996/Revised: 23 July 1996     

Effect of 3-isobutyl-1-methylxanthine on HCO3− transport in turtle bladder: Evidence for electrogenic HCO3− secretion

Ouabain-treated turtle bladders bathed on both surfaces by identical HCO₃^?/CO₂-containing, Cl^?-free Na⁺ media exhibit a short-circuit current (I_sc) and transepithelial potential (p.d.) serosa electronegative to mucosa. Addition of 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic nucleotide phosphodiesterase, rapidly reverses the direction of the I_sc and p.d.. The IBMX-induced reversal of I_sc and p.d. is (1) dependent on the presence of HCO₃^? (and CO₂) in the serosal bathing fluid, (2) independent of Na⁺ and other ions in the bathing medium, (3) decreased by inhibitors of carbonic anhydrase or oxidative metabolism, (4) increased by the serosal addition of cyclic AMP or the disulfonic stilbene, SITS. The results constitute evidence that the reversed I_sc elicited by IBMX represents electrogenic secretion of HCO₃^?.     

Differential regulation of vacuolar H+-ATPase and Na+/H+ exchanger 3 in rat cholangiocytes after bile duct ligation

The cholangiocytes lining the intrahepatic bile ducts modify the primary secretion from the hepatocytes. The cholangiocytes secrete HCO₃⁻ into bile when stimulated with secretin in many species, including man. However, in rats, secretin stimulation neither affects biliary HCO₃⁻ concentration nor bile flow, whereas following bile duct ligation (BDL) it induces hypercholeresis with significant increase of NaHCO₃ concentration. We hypothesized that BDL might affect the expression of cholangiocyte H⁺ transporters and thereby choleresis, and determined the expression and localization of the 31 kDa vacuolar type H⁺-ATPase (V-ATPase) subunit and of Na⁺/H⁺ exchanger NHE3 in the livers of control and BDL rats by real-time PCR, in situ hybridization, immunoblotting, and immunohistochemistry. In controls, secretin had no effect on bile flow, whereas following BDL, secretin increased bile flow ∼threefold. V-ATPase and NHE3 were expressed in control cholangiocytes showing intracellular and apical distribution, respectively. BDL significantly up-regulated V-ATPase mRNA and protein expression and was associated with redistribution to the apical pole in ∼60% of the cholangiocytes lining the small bile ductules. In contrast, NHE3 expression was significantly down-regulated by BDL at the mRNA and protein level. The data demonstrate expression of V-ATPase in rat cholangiocytes. BDL-induced down-regulation of NHE3 may contribute to a reduction of Na⁺ and HCO₃⁻ reabsorption and thus to their net secretion into bile. Apical localization of V-ATPase in cholangiocytes may indicate its involvement in pH regulation and/or HCO₃⁻ salvage to compensate for NHE3 down-regulation in BDL.     

Chloride-bicarbonate exchange in the urinary bladder of the turtle. Independence from sodium ion

The rates of Cl^? absorption and HCO^?₃ secretion were not different in turtle urinary bladders bathed in Na⁺-containing and solutions.These results in turtle bladder are inconsistent with Na⁺-anion cotransport but can be accounted for by a Cl^?/HCO^?₃ exchange system.     

Tubular Fluid Secretion in the Seminiferous Epithelium: Ion Transporters and Aquaporins in Sertoli Cells

Sertoli cells play a key role in the establishment of an adequate luminal environment in the seminiferous tubules of the male reproductive tract. Secretion of the seminiferous tubular fluid (STF) is vital for the normal occurrence of spermatogenesis and for providing a means of transport to the developing spermatozoa. However, several studies on this subject have not completely clarified the origin and composition of this fluid. Electrolyte and water are central components of STF. Sertoli cells secrete an iso-osmotic fluid with a higher content of K⁺ than the blood and express various membrane and water transporters (Na⁺/K⁺-ATPase; Ca²⁺-ATPase; V-type ATPase; Cl⁻ channels; CFTR Cl⁻ channels; K⁺ channels; L-, T- and N-type Ca²⁺ channels; Na⁺/H⁺ exchangers; Na⁺-driven HCO₃ ⁻/Cl⁻ exchangers (NDCBEs); Na⁺/HCO₃ ⁻ cotransporters (NBCes); Na⁺–K⁺–2Cl⁻ cotransporter; Na⁺/Ca²⁺ exchanger; and aquaporins 0 and 8) involved in cellular and secretory functions. Studies with knockout mice for some of these transporters showed tubular fluid accumulation and associated infertility, revealing the relevance of these processes for the normal occurrence of spermatogenesis. Nevertheless, the role of the several membrane transporters in the establishment of STF electrolyte composition needs to be further elucidated. This review summarizes the available data on the ionic composition of STF and on the Sertoli cell membrane mechanisms responsible for ion and water movement. Deepening the knowledge on the mechanisms involved in the secretion, composition and regulation of SFT is essential and will be a major step in understanding the infertility associated with some pathological conditions.     

Effects of different ionic compositions on survival and growth of <Emphasis Type="Italic">Physa acuta</Emphasis>

Most inland saline waters in southern Australia predominantly contain Na⁺ and Cl⁻ as major ions. The proportions of Ca²⁺, Mg²⁺, SO₄ ²⁻, HCO₃ ⁻ and CO₃ ²⁻ in these waters somewhat vary and might influence salinity tolerance of freshwater organisms. Here the salinity stress of five ionic compositions to the freshwater snail Physa acuta Draparnaud (Gastropoda: Physidae) was compared: commercial sea salt Ocean Nature (ON), synthetic Ocean Nature (ONS) and three saline water types that are common in southern Australia (ONS but without [1]: SO₄ ²⁻, HCO₃ ⁻ and CO₃ ²⁻, [2]: Ca²⁺, HCO₃ ⁻ and CO₃ ²⁻, [3]: Ca²⁺, Mg²⁺), Milli-Q water was used as a negative control. The 96-h LC₅₀ values for all treatments did not differ. However in prolonged sub-lethal exposures, results varied depending on the ionic composition. Growth was negative and shell strength reduced in treatments lacking Ca. Though the content of major cationic elements (Ca, Mg, Na and K) did not differ per unit dry weight of snail across the treatments, the total load of these elements per individual snail varied among treatments. Furthermore, at the sub-lethal salinities tested, 1 and 5 mS cm⁻¹, ionic compositions had more effect on the snail’s growth than salinity. The long-term effects on freshwater animals, especially taxa with calcium-based exoskeletons, from exposure to common saline water types with low calcium concentrations will likely be greater than from exposure to saline waters with an ionic composition similar to seawater.