Basolateral pH Research Articles

Revisiting the pKa-Flux method for determining intrinsic membrane permeability

Intrinsic membrane permeability is one of several factors that critically determine the intestinal absorption of a chemical. The intrinsic membrane permeability of a chemical is usually extracted from transwell experiments with Caco-2 or MDCK cells, preferably by the pKa-Flux method, which is considered the method of choice when aqueous boundary layer effects need to be excluded. The pKa-Flux method has two variants, the iso-pH method, where apical and basolateral pH are equal, and the gradient-pH method, where apical and basolateral pH are different. The most commonly used method is the gradient-pH method, as it is intended to reflect the pH-conditions in the gastrointestinal tract. However, concentration-shift effects caused by the applied pH-difference between apical and basolateral compartment in the gradient-pH method have not been considered in the evaluation of the experimental data in the past. Consequently, incorrect intrinsic membrane permeabilities have been determined. In this work, we present a revised method for extracting the intrinsic membrane permeability from gradient-pH data that considers concentration-shift effects in the basolateral aqueous boundary layer and filter as well as in the cytosol. Furthermore, we propose the use of the iso-pH method, where only concentration-shift effects in the cytosol need to be considered, as an alternative to the gradient-pH method. We use the five lipophilic bases amantadine, chloroquine, propranolol, venlafaxine and verapamil as examples to compare gradient-pH method and iso-pH method with regard to the extractability of the intrinsic membrane permeability. For lipophilic bases, the iso-pH method proves to be advantageous. All intrinsic membrane permeabilities determined in this work were substantially higher than the intrinsic membrane permeabilities reported in literature.

Extracellular pH affects phosphorylation and intracellular trafficking of AQP2 in inner medullary collecting duct cells.

Kidney collecting duct cells are continuously exposed to the changes of extracellular pH (pHe). We aimed to study the effects of altered pHe on desmopressin (dDAVP)-induced phosphorylation (Ser(256), Ser(261), Ser(264), and Ser(269)) and apical targeting of aquaporin-2 (AQP2) in rat kidney inner medullary collecting duct (IMCD) cells. When freshly prepared IMCD tubule suspensions exposed to HEPES buffer with pH 5.4, 6.4, 7.4, or 8.4 for 1 h were stimulated with dDAVP (10(-10) M, 3 min), AQP2 phosphorylation at Ser(256), Ser(264), and Ser(269) was significantly attenuated under acidic conditions. Next, IMCD cells primary cultured in transwell chambers were exposed to a transepithelial pH gradient for 1 h (apical pH 6.4, 7.4, or 8.4 vs. basolateral pH 7.4 and vice versa). Immunocytochemistry and cell surface biotinylation assay revealed that exposure to either apical pH 6.4 or basolateral pH 6.4 for 1 h was associated with decreased dDAVP (10(-9) M, 15 min, basolateral)-induced apical targeting of AQP2 and surface expression of AQP2. Fluorescence resonance energy transfer analysis revealed that the dDAVP (10(-9) M)-induced increase of PKA activity was significantly attenuated when LLC-PK1 cells were exposed to pHe 6.4 compared with pHe 7.4 and 8.4. In contrast, forskolin (10(-7) M)-induced PKA activation and dDAVP (10(-9) M)-induced increases of intracellular Ca(2+) were not affected. Taken together, dDAVP-induced phosphorylation and apical targeting of AQP2 are attenuated in IMCD cells under acidic pHe, likely via an inhibition of vasopressin V2 receptor-G protein-cAMP-PKA actions.

Intestinal Permeability Study of Minoxidil: Assessment of Minoxidil as a High Permeability Reference Drug for Biopharmaceutics Classification

The purpose of this study was to evaluate minoxidil as a high permeability reference drug for Biopharmaceutics Classification System (BCS). The permeability of minoxidil was determined in in situ intestinal perfusion studies in rodents and permeability studies across Caco-2 cell monolayers. The permeability of minoxidil was compared with that of metoprolol, an FDA reference drug for BCS classification. In rat perfusion studies, the permeability of minoxidil was somewhat higher than that of metoprolol in the jejunum, while minoxidil showed lower permeability than metoprolol in the ileum. The permeability of minoxidil was independent of intestinal segment, while the permeability of metoprolol was region-dependent. Similarly, in mouse perfusion study, the jejunal permeability of minoxidil was 2.5-fold higher than that of metoprolol. Minoxidil and metoprolol showed similar permeability in Caco-2 study at apical pH of 6.5 and basolateral pH of 7.4. The permeability of minoxidil was independent of pH, while metoprolol showed pH-dependent transport in Caco-2 study. Minoxidil exhibited similar permeability in the absorptive direction (AP-BL) in comparison with secretory direction (BL-AP), while metoprolol had higher efflux ratio (ER > 2) at apical pH of 6.5 and basolateral pH of 7.4. No concentration-dependent transport was observed for either minoxidil or metoprolol transport in Caco-2 study. Verapamil did not alter the transport of either compounds across Caco-2 cell monolayers. The permeability of minoxidil was independent of both pH and intestinal segment in intestinal perfusion studies and Caco-2 studies. Caco-2 studies also showed no involvement of carrier mediated transport in the absorption process of minoxidil. These results suggest that minoxidil may be an acceptable reference drug for BCS high permeability classification. However, minoxidil exhibited higher jejunal permeability than metoprolol and thus to use minoxidil as a reference drug would raise the permeability criteria for BCS high permeability classification.

Basolateral glycylsarcosine (Gly-Sar) transport in Caco-2 cell monolayers is pH dependent

Transepithelial di/tripeptide transport in enterocytes occurs via the apical proton-coupled peptide transporter, hPEPT1 (SLC15A1) and a basolateral peptide transporter, which has only been characterized functionally. In this study we examined the pH dependency, substrate uptake kinetics and substrate specificity of the transporter. We studied the uptake of [(14) C]Gly-Sar from basolateral solution into Caco-2 cell monolayers grown for 17-22 days on permeable supports, at a range of basolateral pH values. Basolateral Gly-Sar uptake was pH dependent, with a maximal uptake rate at a basolateral pH of 5.5. Uptake of Gly-Sar decreased in the presence of the protonophore nigericin, indicating that the uptake was proton-coupled. The uptake was saturable, with a maximal flux (Vmax ) of 408 ± 71, 307 ± 25 and 188 ± 19 pmol/cm(2) /min (mean ± S.E., n = 3) at basolateral pH 5.0, 6.0 and 7.4, respectively. The compounds Gly-Asp, Glu-Phe-Tyr, Gly-Glu-Gly, Gly-Phe-Gly, lidocaine and, to a smaller degree, para-aminohippuric acid were all shown to inhibit the basolateral uptake of Gly-Sar. The study showed that basolateral Gly-Sar transport in the intestinal cell line Caco-2 is proton-coupled. The inhibitor profile indicated that the transporter has broad substrate specificity.

Biopharmaceutics Classification System: Validation and Learnings of an in Vitro Permeability Assay

The Biopharmaceutics Classification System (BCS) is the scientific basis for classifying drugs based on their aqueous solubility and intestinal permeability that supports in vivo bioavailability and bioequivalence waivers for immediate-release solid dosage form drugs. One requirement of the BCS is that the permeability method must be validated. In order to accommodate the variety of in vitro/in situ permeability models, the BCS Guidance gives a general framework for the validation requirements, necessitating implemented experimental details to be selected by the applicant laboratory. The objective of this work was to define the parameters for a cell based in vitro permeability method (e.g., cell type, pH, transport direction, time, and concentration) and validate the method to support formal BCS classification of drugs. Twenty reference drugs were selected and permeability values determined using the Madin-Darby canine kidney type II cell line heterologously expressing the human P-glycoprotein transporter (MDCKII-MDR1). A rank order relationship was established between the in vitro permeability value and human intestinal absorption values. This relationship was as predicted and validates the MDCKII-MDR1 permeability method as defined by the BCS Guidance. The final validated in vitro permeability method employs the MDCKII-MDR1 cell line incubated with the Pgp inhibitor GF120918. It is a unidirectional apical-to-basolateral transport assay performed at apical pH values of 5.5 and 7.4 and a basolateral pH of 7.4. Four reference standards (metoprolol, pindolol, labetalol and ranitidine) dosed and analyzed as a single cassette are included in each experiment. A strategy on selection of drug concentrations and on how to deal with problematic compounds (i.e., those suffering from poor mass balance) is discussed.

Peptide transporter substrate identification during permeability screening in drug discovery: Comparison of transfected MDCK-hPepT1 cells to caco-2 cells

The purpose of this study was to investigate the utility of stably transfected MDCK-hPepT1 cells for identifying peptide transporter substrates in early drug discovery and compare the characteristics of this cell line with Caco-2 cells. MDCK-hPepT1, MDCK-mock, and Caco-2 cells grown to confluence on 24-well Transwell were used for this study. Expression levels of different transporter proteins (PepT1, PepT2, P-gp) in these cell lines were assessed by qRT-PCR. Permeability studies were conducted in parallel in all the cells with a diverse set of peptide substrates using the optimized experimental condition: 100 microM, apical pH 6.0, basolateral pH 7.4, 2 hr incubation at 37 degrees C. Permeability studies were also conducted with classical P-gp substrates (tested in bi-directional mode) and paracellularly absorbed probes to investigate the differences between the cell lines. As expected, MDCK-hPepT1 cells express significantly higher level of PepT1 mRNA compared to both Caco-2 and MDCK-mock cells. Efflux transporter, P-gp, was expressed adequately in all the cell lines. Permeability studies demonstrated that classical peptide substrates had significantly higher permeability in stably transfected MDCK-hPepT1 cells compared to MDCK-mock and Caco-2 cells. The transfected MDCK-hPepT1 cells were qualitatively similar to Caco-2 cells with respect to functional P-gp efflux activity and paracellular pore activity. Stably transfected MDCK-hPepT1 cells have been demonstrated as a viable alternative to Caco-2 cells for estimating the human absorption potential of peptide transporter substrates. These cells behave similar to Caco-2 cells with regards to P-gp efflux and paracellular pore activity but demonstrate greater predictability of absorption values for classical peptide substrates (for which Caco-2 cells under-estimate oral absorption).

PH-Dependent passive and active transport of acidic drugs across Caco-2 cell monolayers

The aim of this study was to investigate pH-dependent passive and active transport of acidic drugs across Caco-2 cells. Therefore, the bidirectional pH-dependent transport of two acidic drugs, indomethacin and salicylic acid, across Caco-2 cells was studied in the physiological pH range of the gastrointestinal tract. The transport of both drugs decreased with increased pH, as expected from the pH-partition hypothesis. Net absorption occurred when the basolateral pH exceeded the apical pH. Concentration dependence and transporter inhibition studies indicated passive transport for indomethacin and a mixture of pH-dependent passive and active influx for salicylic acid. Unexpectedly, active and passive drug transport results were indistinguishable in temperature dependency studies. The transport of salicylic acid (apical pH 5.0; basolateral pH 7.4) was partly blocked by inhibitors of the proton-dependent transporters MCT1 (SLC16A1) and OATP-B (SLC21A9, SLCO2B1). This study shows that the asymmetry in bidirectional transport of acidic drugs is affected by both passive and active components in the presence of pH gradients across Caco-2 cells. Thus, combined studies of concentration-dependency and transport-inhibition are preferred when acidic drug transport is studied in a pH gradient. The findings of this in vitro study can be extrapolated to in vivo situations involving an acidic microclimate.

Effect of isolated removal of either basolateral HCO3-or basolateral CO2on HCO3-reabsorption by rabbit S2 proximal tubule

The equilibrium CO2+H2O right arrow over left arrow H++HCO3- had made it impossible to determine how isolated changes in basolateral CO2 ([CO2]) or HCO3- concentration ([HCO3-]), at a fixed basolateral pH, modulate renal HCO3- or reabsorption. In the present study, we have begun to address this issue by measuring HCO3- reabsorption (JHCO3) and intracellular pH (pHi) in isolated perfused rabbit S2 proximal tubules exposed to three different basolateral (bath) solutions: 1) equilibrated 5% CO2/22 mM HCO3-/pH 7.40, 2) an out-of-equilibrium (OOE) solution containing 5% CO2/pH 7.40 but minimal HCO3- ("pure CO2"), and 3) an OOE solution containing 22 mM HCO3-/pH 7.40 but minimal CO2 ("pure HCO3-"). Tubule lumens were constantly perfused with equilibrated 5% CO2/22 mM HCO3-. Compared with the equilibrated bath solution (JHCO3 = 76.5 +/- 7.7 pmol.min-1.mm-1, pHi = 7.09 +/- 0.04), the pure CO2 bath solution increased JHCO3 by approximately 25% but decreased pHi by 0.19. In contrast, the pure HCO3- bath solution decreased JHCO3 by 37% but increased pHi by 0.24. Our data are consistent with two competing hypotheses: 1) the isolated removal of basolateral HCO3- (or CO2) causes a pHi decrease (increase) that in turn raises (lowers) JHCO3; and 2) HCO3- removal raises JHCO3 by reducing inhibition of basolateral Na/HCO3 cotransport and/or reducing HCO3- backleak, whereas CO2 removal lowers JHCO3 by reducing stimulation of a CO2 sensor.

Interaction of ochratoxin A with human intestinal Caco-2 cells: possible implication of a multidrug resistance-associated protein (MRP2)

Ochratoxin A (OTA), a nephrotoxic mycotoxin, is absorbed from small intestine and, in plasma, binds to serum albumin. Prolonged half-live results from reabsorption by proximal tubules and enterohepatic circulation. The mechanism whereby OTA crosses intestine was investigated by means of a cell culture system consisting of Caco-2 cells, as in vitro model of human intestinal epithelium. Cytotoxicity assays on proliferating Caco-2 cells showed that 0.4 μM OTA inhibits MTT reduction by 50%. Transepithelial transport and intracellular accumulation of OTA were studied in Caco-2 cells, differentiated in bicameral inserts. At pH 7.4, OTA is transported preferentially in basolateral (BL) to apical (AP) direction, suggesting a net secretion. Conditions closer to in vivo situation in duodenum (AP pH 6.0, BL pH 7.4) increase intracellular accumulation and transepithelial transport. AP to BL transport becomes higher than BL to AP transport, suggesting OTA absorption. Addition of serum albumin in BL compartment further increases OTA absorption across Caco-2 cells and suggests that in vivo OTA transport from serosal to luminal side of enterocytes is prevented, due to its binding to plasma proteins. Competition experiments showed that carrier systems for large neutral amino acids, H +/dipeptides cotransporter, organic anion ( p-aminohippurate) carrier and organic anion transporter (oatp) are not implicated in OTA transport across Caco-2 cells, in contrast to what was reported in kidney and liver. AP and BL transport and intracellular accumulation of OTA are increased in the presence of non specific inhibitors of MRPs (indomethacin, genistein and probenecid) and of 1-chloro-2,4-dinitrobenzene (biotransformed into 2,4-dinitrophenyl-gluthatione, a specific inhibitor of MRPs), but are affected by verapamil, an inhibitor of P-gp. This suggests that the multidrug resistance-associated protein (MRP2) could be implicated in transepithelial transport. Therefore, absorption of OTA across the intestinal mucosa would be limited thanks to its excretion through MRP2 at the apical pole of enterocytes.

PH-dependent bidirectional transport of weakly basic drugs across Caco-2 monolayers: implications for drug-drug interactions.

The purpose of this study was to investigate the pH-dependent passive and active transport of weakly basic drugs across the human intestinal epithelium. The bidirectional pH-dependent transport of weak bases was studied in Caco-2 cell monolayers in the physiologic pH range of the gastrointestinal tract. A net secretion of atenolol and metoprolol was observed when a pH gradient was applied. However, the bidirectional transport of both compounds was equal in the nongradient system. Hence, at lower apical than basolateral pH a change in passive transport caused by an imbalance in the concentration of the uncharged drug species resulted in a "false" asymmetry (efflux ratio). Furthermore, a mixture of pH-dependent passive and active efflux was found for the P-glycoprotein (P-gp, MDR1, ABCB1) substrates, talinolol and quinidine, but not for the neutral drug, digoxin. However, the clinically important digoxin-quinidine interaction depended on the presence of a pH gradient. Hence, the degree of interaction depends on the amount of quinidine available at the binding site of the P-gp. Active efflux of weak bases can only be accounted for when the fraction of unionized drug species is equal in all compartments because the transport is biased by a pH-dependent passive component. However, this component may take part in vivo and contribute to drug-drug interactions involving P-gp.

The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers

We determined the extent of Na(+)-independent, proton-driven amino acid transport in human intestinal epithelia (Caco-2). In Na(+)-free conditions, acidification of the apical medium (apical pH 6.0, basolateral pH 7.4) is associated with a saturable net absorption of glycine. With Na(+)-free media and apical pH set at 6.0, (basolateral pH 7.4), competition studies with glycine indicate that proline, hydroxyproline, sarcosine, betaine, taurine, beta-alanine, alpha-aminoisobutyric acid (AIB), alpha-methylaminoisobutyric acid (MeAIB), tau-amino-n-butyric acid and L-alanine are likely substrates for pH-dependent transport in the brush border of Caco-2 cells. Both D-serine and D-alanine were also substrates. In contrast leucine, isoleucine, valine, phenylalanine, methionine, threonine, cysteine, asparagine, glutamine, histidine, arginine, lysine, glutamate and D-aspartate were not effective substrates. Perfusion of those amino acids capable of inhibition of acid-stimulated net glycine transport at the brush-border surface of Caco-2 cell monolayers loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl-5(6)-carboxyfluorescein) (BCECF) caused cytosolic acidification consistent with proton/amino acid symport. In addition, these amino acids stimulate an inward short-circuit current (Isc) in voltage-clamped Caco-2 cell monolayers in Na(+)-free media (pH 6.0). Other amino acids such as leucine, isoleucine, phenylalanine, tryptophan, methionine, valine, serine, glutamine, asparagine, D-aspartic acid, glutamic acid, cysteine, lysine, arginine and histidine were without effect on both pHi and inward Isc. In conclusion, Caco-2 cells express a Na(+)-independent, H(+)-coupled, rheogenic amino acid transporter at the apical brush-border membrane which plays an important role in the transepithelial transport of a range of amino acids across this human intestinal epithelium.

H +-coupled α-methylaminoisobutyric acid transport in human intestinal Caco-2 cells

Transepithelial apical-to-basal transport and cellular uptake of the non-metabolisable amino acid α-methylaminoisobutyric acid (MeAIB) across confluent monolayers of the human intestinal epithelial cell line Caco-2 are enhanced by a transepithelial pH gradient (apical pH 6.0, basolateral pH 7.4). In Na +-free conditions (apical pH 7.4, basolateral pH 7.4), net absorption (120 ± 58 pmol/cm 2per h, n = 13) and uptake across the apical membrane (cell/medium ratio 0.56 ± 0.06, n = 13) are low. However, in Na +-free conditions with apical pH 6.0, net absorption (685 ± 95 pmol/cm 2 per h, n = 15) and intracellular accumulation (cell/medium ratio 3.63 ± 0.29, n = 14) were marked. Continuous monitoring of intracellular pH (pH i) in BCECF (2′,7′-bis(2-carbozyethyl)-5(6)-carboxyfluorescein)-loaded Caco-2 cell monolayers indicated that apical addition of MeAIB (20 mM) was associated with H +-flow across the apical membrane in both Na + and Na +-free conditions. This transport process is rheogenic in Na +-free media, stimulating an inward short-circuit current in voltage-clamped Caco-2 cell monolayers. On the basis of competition for MeAIB accumulation and pH i experiments, l-proline, glycine, l-alanine and β-alanine are also substrates for H +-linked transport at the apical membrane of Caco-2 cells but l-valine, l-leucine and l-phenylalanine are not. These data are consistent with the expression, in the apical brush-border membrane of Caco-2 cells, of a H +-coupled, Na +-independent MeAIB carrier.

Luminal perfusion of isolated gastric glands

We have extended to rabbit gastric glands the technique for perfusing single isolated renal tubules. We isolated glands by hand dissection and used concentric glass pipettes to hold them and perfuse their lumina. Parietal cells (PCs), which tended to be located toward the gland opening, were identified by their pyramidal shape, large size, and autofluorescence. Chief cells (CCs) were identified by their round shape and smaller size. In some experiments, we perfused the lumen with hydroxypyrenetrisulfonate, a pH-sensitive fluorophore, at pH 7.4 and used digital image processing to monitor luminal pH (pH1). Solutions were buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid to pH 7.4 at 37 degrees C. With fast perfusion, we found no evidence of decreased pH1, even with stimulation by 10 microM carbachol. With slow perfusion, pH1 often fell below the dye's sensitive range (pH < 5), especially at low perfusate buffering power. In other experiments, we loaded cells with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and monitored intracellular pH (pHi) in multiple individual PCs and CCs in a single gland. Mean pHi was 7.21 +/- 0.02 (n = 136 cells) for PCs and 7.27 +/- 0.03 (n = 103) for CCs. To examine the response to decreased pH1 and basolateral pH (pHb), we lowered pHb to 6.4 or lowered pH1 to 3.4 or 1.4. Lowering pHb to 6.4 for approximately 1 min caused pHi to fall reversibly by 0.39 +/- 0.05 (n = 53) in PCs and 0.58 +/- 0.03 (n = 50) in CCs. Lowering pH1 to 3.4 or 1.4 caused no significant pHi changes in PCs (n = 38 and 82) or in CCs (n = 44 and 77). Carbachol did not affect the response to changes in pH1 or pHb. We conclude that the apical surfaces of PCs and CCs are unusually resistant to extreme pH gradients.

Uptake and transepithelial transport of the orally absorbed cephalosporin cephalexin, in the human intestinal cell line, Caco-2

Cephalexin (CPX) uptake in cultures of the human colon adenocarcinoma cell lines, Caco-2 and HT-29 has been shown to involve the di-/tripeptide transporter (DPT). However, little is known about the mechanism mediating the transepithelial (TE) transport of CPX either in vivo or in cultured cells. In this study, uptake and TE transport of CPX were investigated in Caco-2 monolayers grown on microporous membranes. Caco-2 cells did not show net TE transport of CPX when the pH of both apical (AP) and basolateral (BL) bathing solutions was 7.4. When the pH of the AP bathing solution was decreased from 7.4 to 6.0, while maintaining the pH of the BL bathing solution at 7.4, AP-BL transport of CPX (0.1 mM) increased from 0.1 to 0.23% h −1 cm −2. Reversal of the pH gradient across the monolayer (AP, pH 7.4; BL, pH 6.0) did not alter the BL-AP flux of CPX. Manipulation of the AP or BL pH between 5.5 and 7.4 affected neither the AP-BL nor the BL-AP flux of mannitol (both ~0.1% h −1 cm −2), an internal marker of passive, paracellular diffusion. The pH-dependent AP uptake and AP-BL flux of CPX were time-, concentration- and temperature-dependent. Apparent half-maximal transport concentration ( K t) and maximal transport velocity ( V t) were 2.9 mM and 1.0 nmol min −1 mg protein −1 for AP uptake, and 4.7 mM and 0.13 nmol min −1 cm −2 (0.30 nmol min −1 mg protein −1) for AP-BL transport. The carrier-mediated AP uptake and AP-BL transport of CPX were inhibited by Gly-Pro, Pro-Gly, cephradine, cefadroxil, benzylpenicillin and ampicillin, but not by proline, glycine, valine, lysine or aspartic acid. In addition, CPX uptake was not inhibited by the nucleoside, adenosine, or the bile acid, taurocholic acid, suggesting that the uptake and TE transport of CPX involves the DPT but not other carriers present in the intestinal mucosa. We confirmed that the AP uptake of CPX involves mainly the DPT and conclude the following: (a) AP-BL transport of CPX is predominantly transcellular and involves a carrier, probably the DPT; (b) the rate-limiting step in AP-BL transport of cephalexin appears to be BL efflux and not AP uptake; (c) interaction with the DPT alone may be a poor predictor of substrate transport via this carrier; and (c) the Caco-2 culture system is a good model for studying mucosal uptake and TE transport of small peptides and peptidomimetic drugs.

Effects of pH on apical calcium entry and active calcium transport in rabbit cortical collecting system.

Rabbit connecting tubules and cortical collecting ducts were isolated by immunodissection and cultured on permeable supports. The monolayers actively transported Ca2+ with a net transcellular rate of 92 +/- 3 nmol.h-1.cm-2. Methoxyverapamil, felodipine, diltiazem, omega-conotoxin GVIA, and omega-agatoxin IVA when added to the apical side had no effect on Ca2+ absorption. Neither hyperpolarization nor depolarization of the apical membrane affected Ca2+ transport rates significantly. Stepwise lowering of the apical pH (pHa) from 8.0 to 5.6 gradually inhibited Ca2+ transport from 88 +/- 5 to 7 +/- 2 nmol.h-1.cm-2. Measuring the intracellular pH (pHi) with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein revealed that lowering the pHa from 8.0 to 5.6 decreased pHi from 7.8 to 6.7. To determine whether inhibition of Ca2+ absorption results from intracellular acidification, pHi was lowered using an NH4Cl pulse while extracellular pH was kept constant. Intracellular acidification from 7.4 +/- 0.2 to 6.9 +/- 0.1 reduced Ca2+ absorption by 26 +/- 6% only. In addition, lowering of the basolateral pH to 6.2 resulted in a pHi of 6.8 +/- 0.1, without affecting Ca2+ absorption rates. In conclusion, the basal Ca2+ influx mechanism in the apical membrane is most likely a voltage-independent Ca2+ transporter, insensitive to Ca2+ channel blockers, but strongly inhibited by apical acidification.

H +-coupled dipeptide (glycylsarcosine) transport across apical and basal borders of human intestinal Caco-2 cell monolayers display distinctive characteristics

Transepithelial transport and intracellular accumulation of the dipeptide glycylsarcosine (Gly-Sar) were studied using intact monolayers of the human intestinal epithelial cell line, Caco-2. Gly-Sar transport was demonstrated in both absorptive (apical-to-basal) and secretory (basal-to-apical) directions. In both directions, transport and accumulation were enhanced in the presence of a pH gradient ( pH o < pH i ). Under conditions similar to those found at the intestinal membrane in vivo (apical pH 6.0, basolateral pH 7.4), net absorption (145.2 pmol/cm 2 per h) was observed, although experimental conditions could also be manipulated (apical pH 7.4, basolateral pH 6.0) so that net secretion was observed. Transport and accumulation (in both directions) were inhibited in the presence of either 20 mM (unlabelled) Gly-Sar or 20 mM cephalexin (an aminocephalosporin antibiotic). When added to either the apical or basolateral surface of BCECF (2′,7′,-bis(2-carboxyethyl)-5(6)-carboxyfluorescein)-loaded Caco-2 cell monolayers Gly-Sar (20 mM), at pH 6.0, caused a marked intracellular acidification, demonstrating that dipeptide absorption is accompanied by H +-flow into the cells. Cephalexin (20 mM) had similar effects (as Gly-Sar) when presented at the apical surface but also caused a marked intracellular acidification when perfused into the basolateral chamber at pH 7.4. In contrast, addition of Gly-Sar (20 mM) to the basolateral chamber (at pH 7.4) had no effect. Transepithelial absorption of dipeptides (Gly-Sar) and β-lactam antibiotics (cephalexin) at low concentrations is predominately via a transcellular route mediated by carrier mechanisms located at both apical and basolateral membranes. Interestingly, Gly-Sar and cephalexin transport across the basolateral membrane (and, therefore, exit from the cell) display both common and distinct characteristics suggesting that more than one mechanism may be responsible for exit into the basolateral space.

Oxygen uptake of isolated toad skin epithelium: micromeasurement and effect of ionic acclimation

Oxidative metabolism of isolated toad skin epithelium (Bufo viridis) was investigated in vitro under open-circuit conditions using the spectrophotometric oxyhemoglobin micromethod. This highly sensitive technique has been adapted for studying several epithelia in parallel and for detecting possible regional variations of oxygen uptake in individual epithelium. Changes in the proportion of mitochondria-rich cells (MRC) by ionic acclimation affected oxidative metabolism under nontransporting condition. After acclimation of animals to either NaNO3 or NaCl solutions (100 mmol/l, for greater than 2 wk), the number of MRC per square millimeter in epithelia from nonacclimated and NaNO3- and NaCl-acclimated animals was 350 +/- 113, 460 +/- 196, and 107 +/- 52, respectively. O2 uptake of nonacclimated and NaNO3-acclimated epithelia was significantly higher than that of NaCl-acclimated epithelia (i.e., 0.89 and 0.90 vs. 0.57 nmol O2.h-1.mm-2, respectively). The correlation established between O2 uptake and number of MRC allowed evaluation of the respiration rate of one single MRC, i.e., approximately 1 pmol O2/h. The lowest mitochondrial oxidative activity was found in the epithelia from NaCl-acclimated toads where the uncoupler 2,4-dinitrophenol (50 mumols/l) had the highest relative stimulatory effect (+114%). Acetazolamide (50 mumols/l), a potent inhibitor of carbonic anhydrase mainly present in the MRC, reduced selectively by 31% O2 uptake of the MRC-rich epithelia (NaNO3 acclimated). O2 uptake increased significantly by approximately 80% when basolateral pH increased from 5.8 to 7.8, but did not depend on apical pH. These findings indicate that under nontransporting (open-circuit) conditions, aerobic metabolism of the isolated toad skin epithelium is related to the density and/or characteristics of the MRC.(ABSTRACT TRUNCATED AT 250 WORDS)

Transepithelial acidification by cultures of rabbit proximal tubules grown on filters

Transepithelial acidification in the proximal tubule occurs by the simultaneous actions of the Na(+)-H+ exchanger in the brush border and the basolateral Na(+)-HCO3- cotransporter. The presence of these systems has been demonstrated for cultured cells; however, their contributions to the transepithelial movement of acid equivalents has not been confirmed in monolayers. To examine transepithelial acidification by intact cells, tubules were grown on membrane filters. Confluent cultures developed a transepithelial pH gradient within 6 h by decreasing the pH of medium in the apical chamber (6.66 +/- 0.03) while raising the basolateral pH to 7.40 +/- 0.02. Cells maintained on plastic did not acidify the medium during this time. Amiloride (10-100 microM) inhibited development of the gradient only when placed in the top chamber. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 10-100 microM), which inhibits basolateral Na(+)-HCO3- cotransport, decreased the gradient only when added to the bottom. These results demonstrate that cultured proximal tubule cells can develop a transepithelial pH gradient and that the polarized distribution of the transport systems is maintained in vitro.

Electrogenic Na/HCO3 cotransport across basolateral membrane of isolated perfused Necturus proximal tubule.

This study was undertaken to determine whether the proximal tubule of the mud puppy Necturus maculosus possesses a basolateral Na/HCO3 cotransporter. We examined the effects on basolateral membrane potential (Vbl) and intracellular pH (pHi) of 1) lowering basolateral [HCO3-] at constant PCO2, and 2) replacing Na+ with N-methyl-D-glucamine. Vbl and pHi were measured with Ling-Gerard and liquid-membrane pH microelectrodes, respectively, in isolated tubules perfused in vitro. We found that decreasing basolateral [HCO3-] from 10 mM (pH 7.5) to 2 mM (pH 6.8) resulted in an immediate depolarization of 14.9 mV, and a pHi decrease of 0.35. SITS (4-acetamido-4'-isothiocyanostibene-2,2'-disulfonic acid, 0.5 mM) inhibited the HCO3-induced depolarization by 87% and inhibited the initial rate of the pHi decrease by 79%. Replacement of basolateral Na+ with N-methyl-D-glucamine resulted in an immediate depolarization of 11.3 mV, and a pHi decrease of 0.36. SITS inhibited the zero Na-induced depolarization by 86% and the initial rate of the pHi decrease by 81%. Nominal removal of basolateral HCO3- (replaced with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) inhibited the zero Na-induced depolarization by 64%, whereas nominal removal of Na+ inhibited the 2 mM HCO3-induced depolarization by 67%. Replacement of all basolateral Cl- with glucuronate did not inhibit the changes in Vbl induced by changing [HCO3-] or [Na+]. Observations similar to those described above have been made previously on Ambystoma proximal tubules, and attributed to an electrogenic Na/HCO3 cotransport mechanism that carries HCO3-, Na+, and net negative charge in the same direction. We conclude that Necturus proximal tubules possess a similar, if not identical, electrogenic Na/HCO3 cotransport mechanism.

Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCO3- transport.

We have used pH-, Na-, and Cl-sensitive microelectrodes to study basolateral HCO3- transport in isolated, perfused proximal tubules of the tiger salamander Ambystoma tigrinum. In one series of experiments, we lowered basolateral pH (pHb) from 7.5 to 6.8 by reducing [HCO3-]b from 10 to 2 mM at a constant pCO2. This reduction of pHb and [HCO3-]b causes a large (approximately 0.35), rapid fall in pHi as well as a transient depolarization of the basolateral membrane. Returning pHb and [HCO3-]b to normal has the opposite effects. Similar reductions of luminal pH (pHl) and [HCO3-]l have only minor effects. The reduction of [HCO3-]b and pHb also produces a reversible fall in aiNa. In a second series of experiments, we reduced [Na+]b at constant [HCO3-]b and pHb, and also observed a rapid fall in pHi and a transient basolateral depolarization. These changes are reversed by returning [Na+]b to normal. The effects of altering [Na+]l in the presence of HCO3-, or of altering [Na+]b in the nominal absence of HCO3-, are substantially less. Although the effects on pHi and basolateral membrane potential of altering either [HCO3-]b or [Na+]b are largely blocked by 4-acetamido-4-isothiocyanostilbene-2,2'-disulfonate (SITS), they are not affected by removal of Cl-, nor are there accompanying changes in aiCl consistent with a tight linkage between Cl- fluxes and those of Na+ and HCO3-. The aforementioned changes are apparently mediated by a single transport system, not involving Cl-. We conclude that HCO3- transport is restricted to the basolateral membrane, and that HCO3- fluxes are linked to those of Na+. The data are compatible with an electrogenic Na/HCO3 transporter that carries Na+, HCO3-, and net negative charge in the same direction.