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Background and Purpose: Oral drug bioavailability is limited by intestinal expression of P-glycoprotein (MDR1, Pgp, ABCB1) whose capacity is regulated via nuclear receptors e.g. the pregnane X receptor (PXR, SXR, NR1I2). In order to study dynamic regulation of MDR1 transport capacity we have identified the T84 epithelial cell-line as a model for human intestine co-expressing MDR1 with PXR. The ability of rifampin, a known PXR agonist and digoxin, a model MDR1 substrate, to regulate MDR1 expression and transport activity has been tested, in these T84 cells., Experimental Approach: Transport was assayed by bi-directional [(3)H]-digoxin transepithelial fluxes across epithelial layers of T84 cells seeded onto permeable filter supports following pre-exposure to rifampin and digoxin. Quantitative real-time PCR, Western blotting and immunocytochemistry were used to correlate induction of MDR1 transcript and protein levels with transport activity., Key Results: Rifampin exposure (10 microM, 72 hours) increased MDR1 transcript levels (3.4 fold), MDR1 total protein levels (4.4 fold), apical MDR1 protein (2.7 fold) and functional activity of MDR1 (1.2 fold). Pre-incubation with digoxin (1 microM, 72 hours) potently induced MDR1 transcript levels (92 fold), total protein (7 fold), apical MDR1 protein (4.7 fold) and functional activity (1.75 fold). Whereas PXR expression was increased by rifampin incubation (2 fold), digoxin reduced PXR expression (0.3 fold)., Conclusions and Implications: Chronic digoxin pre-treatment markedly upregulates MDR1 expression and secretory capacity of T84 epithelia. Digoxin-induced changes in MDR1 levels are distinct from PXR-mediated changes resulting from rifampin exposure.

1. Human intestinal epithelial Caco-2 cells, T84 cells, and MDCKII cells transfected with human MDR1, were used to investigate the mechanistic basis of transintestinal fluoroquinolone secretion. 2. The fluoroquinolone grepafloxacin was secreted across Caco-2 monolayers by a saturable process (V(max)=16.9 +/- 3.4 nmol.cm(-2).h(-1)). Net secretion was reduced by 2-deoxyglucose/azide treatment to reduce intracellular ATP. 3. Grepafloxacin inhibited [(14)C]-ciprofloxacin (100 microM) secretion across Caco-2 monolayers (K(0.5)=0.8 mM), and concurrently increased the cellular accumulation of ciprofloxacin from the basal medium, indicating inhibition of export across the apical membrane. 4. The unconjugated bile acid, cholic acid, was secreted across Caco-2 monolayers, and this secretion was sensitive to inhibition by the MRP-selective inhibitor MK-571, suggesting MRP2 involvement. Secretion of cholic acid (10 microM) across the apical membrane was also inhibited by grepafloxacin (K(0.5)=0.3 mM), but not by ciprofloxacin. 5. In MDCKII-MDR1 monolayers, net secretion of grepafloxacin was increased by 3.5 fold compared with untransfected controls. Neither ciprofloxacin nor cholic acid showed net secretion in either MDCKII or MDCKII-MDR1 monolayers, showing that in contrast to grepafloxacin, neither are substrates for MDR1. 6. In T84 monolayers, which express MDR1 but not MRP2, neither ciprofloxacin nor cholic acid was secreted, whilst the V(max) for grepafloxacin secretion was lower than in Caco-2 cells, which express both MDR1 and MRP2. 7. In conclusion, the transepithelial secretion of grepafloxacin is mediated by both MRP2 and MDR1, whereas ciprofloxacin is a substrate for neither. Grepafloxacin also competes for the ciprofloxacin-sensitive pathway, which remains to be elucidated.

The mechanism by which bradykinin regulates renal epithelial salt transport has been investigated using a mouse inner medullary renal collecting duct cell-line mIMCD-K2. Using fura-2 loaded mIMCD-K2 cells bradykinin (100 nM) has been shown to induce a transient increase in intracellular Ca(2+) via activation of bradykinin B2 receptors localized to both the apical and basolateral epithelial cell surfaces. In mIMCD-K2 epithelial cell-layers clamped in Ussing chambers, 100 nM bradykinin via apical and basolateral bradykinin B2 receptors stimulated a transient increase in inward short-circuit current (I:(sc)) of similar duration to the increase in intracellular Ca(2+). Replacements of the bathing solution Na(+) by the impermeant cation, N-methyl-D-glucamine and of Cl(-) and HCO(3)(-) by the impermeant anion gluconate at either the apical (no reduction) or basal bathing solutions (abolition of the response) are consistent with the bradykinin-stimulated increase in inward I:(sc) resulting from basal to apical Cl(-) (anion) secretion. Using the slow whole cell configuration of the patch-clamp technique, bradykinin was shown to activate a transient Cl(-) selective whole cell current which showed time-dependent activation at positive membrane potentials and time-dependent inactivation at negative membrane potentials. These currents were distinct from those activated by forskolin (CFTR), but identical to those activated by exogenous ATP and are therefore consistent with bradykinin activation of a Ca(2+)-dependent Cl(-) conductance. The molecular identity of the Ca(2+)-dependent Cl(-) conductance has been investigated by an RT - PCR approach. Expression of an mRNA transcript with 96% identity to mCLCA1/2 was confirmed, however an additional but distinct mRNA transcript with only 81% of the identity to mCLCA1/2 was identified.

1. Transintestinal absorption of gamma-aminobutyric acid (GABA) via a pH-dependent mechanism is demonstrated in the model human intestinal epithelial cell line Caco-2. 2. Experiments with BCECF [2',7',-bis(2-carboxyethyl)-5(6)- carboxyfluorescein]-loaded Caco-2 cells demonstrate that GABA transport across the apical membrane is coupled to proton flow into the cell. 3. Short-circuit current (ISC) measurements using Caco-2 cell monolayers under voltage-clamped conditions demonstrate that pH-dependent GABA transport is a rheogenic process even in the absence of extracellular Na+, consistent with H+/GABA symport. 4. A range of GABA analogues were tested for their abilities to: (a) inhibit pH-dependent [3H]GABA uptake across the apical membrane; (b) stimulate H+ flow across the apical surface of BCECF-loaded Caco-2 cell monolayers; (c) increase inward ISC across voltage-clamped Caco-2 cell monolayers. 5. Nipecotic acid, isonipecotic acid, D,L-beta-aminobutyric acid, and 3-amino-1-propanesulphonic acid each caused a marked acidification of intracellular pH and an increase in ISC when superfused at the apical surface of Caco-2 cell monolayers. In contrast L-alpha-amino-n-butyric acid failed to induce proton flow or ISC. The ability of these compounds to induce proton or current flow across the apical surface of this intestinal epithelium was closely related to the relative inhibitory effects on [3H]GABA uptake. 6. These observations demonstrate H+/GABA symport and suggest that this transport mechanism may be accessible as a route for oral absorption of therapeutically-useful GABA analogues.

1. Human intestinal epithelial Caco-2 cells were used to investigate the mechanistic basis of transepithelial secretion of the fluoroquinolone antibiotic ciprofloxacin. 2. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to competitive inhibition by sulphate, thiosulphate, oxalate, succinate and para-amino hippurate, probenecid (10 mM), taurocholate (100 microM) or bromosulphophthalein (100 microM). Similarly tetraethylammonium and N-'methylnicotinamide (10 mM) were without effect. 3. Net secretion of ciprofloxacin was inhibited by the organic exchange inhibitor 4,4'-diisothiocyanostilbene-2-2'-disulphonic acid (DIDS, 400 microM). 4. Net secretion of ciprofloxacin was partially inhibited by 100 microM verapamil, whilst net secretion of the P-glycoprotein substrate vinblastine was totally abolished under these conditions. Ciprofloxacin secretion was unaltered after preincubation of cells with two anti-P-glycoprotein antibodies (UIC2 and MRK16), which both significantly reduced secretory vinblastine flux (measured in the same cell batch). Ciprofloxacin (3 mM) failed to inhibit vinblastine net secretin in Caco-2 epithelia, and was not itself secreted by the P-glycoprotein expressing and vinblastine secreting dog kidney cell line, MDCK. 5. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to alterations of either cytosolic or medium pH, or dependent on the presence of medium Na+, Cl- or K+ in the bathing media. 6. The substrate specificity of the ciprofloxacin secretory transport in Caco-2 epithelia is distinct from both the renal organic anion and cation transport. A role for P-glycoprotein in ciprofloxacin secretion may also be excluded. A novel transport mechanism, sensitive to both DIDS and verapamil mediates secretion of ciprofloxacin by human intestinal Caco-2 epithelia.

1. Human intestinal epithelial Caco-2 cells have been used to investigate the transepithelial permeation of the cardiac glycoside, digoxin. 2. Transepithelial basal to apical [3H]-digoxin flux exceeds apical to basal flux, a net secretion of [3H]-digoxin being observed. At 200 microM digoxin, net secretory flux (Jnet) was 10.8 +/- 0.6 nmol cm-2 h-1. Maximal secretory flux (Jmax) of vinblastine was 1.3 +/- 0.1 nmol cm-2 h-1. Cellular uptake of digoxin was different across apical and basal cell boundaries. It was greatest across the basal surface at 1 microM, whereas at 200 microM, apical uptake exceeded basal uptake. 3. Net secretion of [3H]-digoxin was subject to inhibition by digitoxin and bufalin but was not inhibited by ouabain, convallatoxin, and strophanthidin (all 100 microM). Inhibition was due to both a decrease in Jb-a and an increase in Ja-b. Uptake of [3H]-digoxin at the apical surface was increased by digitoxin and bufalin. All cardiac glycosides decreased [3H]-digoxin uptake at the basal cell surface (except for 100 microM digitoxin). 4. The competitive P-glycoprotein inhibitors, verapamil (100 microM), nifedipine (50 microM) and vinblastine (50 microM) all abolished net secretion of [3H]-digoxin due to both a decrease in Jb-a and an increase in Ja-b. Cellular accumulation of [3H]-digoxin was also increased across both the apical and basal cell surfaces. I-Chloro-2,4,-dinitrobenzene (10 microM), a substrate for glutathione-S-transferase and subsequent ATP-dependent glutathione-S-conjugate secretion, failed to inhibit net secretion of [3H]-digoxin. The increase in absorptive permeability Pa-b (= Ja-b/Ca) and cellular [3H]-digoxin uptake upon P-glycoprotein inhibition, showed that the intestinal epithelium was rendered effectively impermeable by ATP-dependent extrusion at the apical surface. 5. A model for [3H]-digoxin secretion by the intestinal epithelium is likely to involve both diffusional uptake and Na(+)-K+ pump-mediated endocytosis, followed by active extrusion at the apical membrane.

1. The ability of D-cycloserine to act as a substrate for H+/amino acid symport has been tested in epithelial layers of Caco-2 human intestinal cells. 2. In Na(+)-free media with the apical bathing media held at pH 6.0, D-cycloserine (20 mM) is an effective inhibitor of net transepithelial transport (Jnet) of L-alanine (100 microM) and its accumulation (across the apical membrane) in a similar manner to amino acid substrates (L-alanine, beta-alanine, L-proline and glycine). In contrast L-valine was ineffective as an inhibitor for H+/amino acid symport. Both inhibition of L-alanine Jnet and its accumulation by D-cycloserine were dose-dependent, maximal inhibition being achieved by 5-10 mM. 3. Both D-cycloserine and known substrates for H+/amino acid symport stimulated an inward short circuit current (Isc) when voltage-clamped monolayers of Caco-2 epithelia, mounted in Ussing chambers, were exposed to apical substrate in Na(+)-free media, with apical pH held at 6.0. The D-cycloserine dependent increase in Isc was dose-dependent with an apparent Km = 15.8 +/- 2.0 (mean +/- s.e. mean) mM, and Vmax = 373 +/- 21 nmol cm-2h-1. 4. D-Cycloserine (20 mM) induced a prompt acidification of Caco-2 cell cytosol when superfused at the apical surface in both Na+ and Na(+)-free conditions. Cytosolic acidification in response to D-cycloserine was dependent upon superfusate pH, being attenuated at pH 8 and enhanced in acidic media. 5. The increment in Isc with 20 mM D-cycloserine was non-additive with other amino acid substrates for H+/amino acid symport.

1. The role of proton-linked solute transport in the absorption of the angiotensin-converting enzyme (ACE) inhibitors captopril, enalapril maleate and lisinopril has been investigated in human intestinal epithelial (Caco-2) cell monolayers. 2. In Caco-2 cell monolayers the transepithelial apical-to-basal transport and intracellular accumulation (across the apical membrane) of the hydrolysis-resistant dipeptide, glycylsarcosine (Gly-Sar), were stimulated by acidification (pH 6.0) of the apical environment. In contrast, transport and intracellular accumulation of the angiotensin-converting enzyme (ACE) inhibitor, lisinopril, were low (lower than the paracellular marker mannitol) and were not stimulated by apical acidification. Furthermore, [14C]-lisinopril transport showed little reduction when excess unlabelled lisinopril (20 mM) was added. 3. pH-dependent [14C]-Gly-Sar transport was inhibited by the orally-active ACE inhibitors, enalapril maleate and captopril (both at 20 mM). Lisinopril (20 mM) had a relatively small inhibitory effect on [14C]-Gly-Sar transport. pH-dependent [3H]-proline transport was not inhibited by captopril, enalapril maleate or lisinopril. 4. Experiments with BCECF[2',7',-bis(2-carboxyethyl)-5(6)-carboxyfluorescein]-loaded Caco-2 cells demonstrate that dipeptide transport across the apical membrane is associated with proton flow into the cell. The dipeptide, carnosine (beta-alanyl-L-histidine) and the ACE inhibitors enalapril maleate and captopril, all lowered intracellular pH when perfused at the apical surface of Caco-2 cell monolayers. However, lisinopril was without effect. 5. The effects of enalapril maleate and captopril on [14C]-Gly-Sar transport and pHi suggest that these two ACE inhibitors share the H(+)-coupled mechanism involved in dipeptide transport. The absence of pH-dependent [14C]-lisinopril transport, the relatively small inhibitory effect on [14C]-Gly-Sar transport,and the absence of lisinopril-induced pHi changes, all suggest that lisinopril is a poor substrate for thedi/tripeptide carrier in Caco-2 cells. These observations are consistent with the greater oral availability and time-dependent absorption profile of enalapril maleate and captopril, compared to lisinopril.

1. pH-dependent transepithelial transport and intracellular accumulation of the hydrolysis-resistant dipeptide glycylsarcosine (Gly-Sar) have been demonstrated in the model human intestinal epithelial cell line, Caco-2. 2. Experiments with BCECF (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein)-loaded Caco-2 cells demonstrated that dipeptide (Gly-Sar) transport across the apical membrane is coupled to proton flow into the cell. 3. A range of postulated substrates for the intestinal di/tripeptide carrier were tested for their abilities to: (a) inhibit pH-dependent [14C]Gly-Sar apical-to-basal transport and intracellular accumulation and (b) stimulate H+ flow across the apical surface of BCECF-loaded Caco-2 cell monolayers. 4. A range of compounds (including Gly-Gly, Leu-Leu, Gly-Gly-Gly, cefadroxil and cephalexin) caused marked acidification of intracellular pH when perfused at the apical surface of Caco-2 cell monolayers. In contrast leucine and D-Leu-D-Leu failed to induce proton flow. The ability to induce proton-flow across the apical surface by these compounds, in this intestinal epithelium, was directly correlated to the relative inhibitory effects on [14C]-Gly-Sar transport and accumulation. 5. The determination of substrate-induced intracellular pH change in the Caco-2 cell system may provide a useful rapid screen for candidate substrates for absorption via H(+)-coupled transport mechanisms such as the intestinal di/tripeptide carrier in an appropriate physiological context.

The bidirectional transepithelial fluxes of ciprofloxacin, an antibacterial fluoroquinolone, across the human intestinal epithelial Caco-2 cell-line show marked asymmetry. Basal-to-apical flux of ciprofloxacin (10 microM) exceeds apical-to-basal flux indicating net secretion. Net ciprofloxacin secretion is abolished by azide/2-deoxy-D-glucose treatment, displays saturation kinetics (Km = 0.89 +/- 0.23 mM, Vmax 44.3 +/- 4.9 nmol cm-2.h) and competition by other fluoroquinolones. A specific, active secretion in Caco-2 epithelia may explain the transintestinal elimination of ciprofloxacin observed in pharmacokinetic studies in man.

1 Exogenous adenosine triphosphate (ATP) stimulates the short circuit current (SCC) in a cultured renal-derived epithelium (MDCK). Half-maximal stimulation is achieved at 1.91 X 10(-5) M ATP. 2 It is suggested that ATP interacts with a P2 purine receptor upon the basis of (a) agonist potency (ATP greater than adenosine diphosphate much greater than adenosine monophosphate, adenosine; ATP greater than uridine triphosphate greater than inosine triphosphate much greater than cytosine triphosphate, guanosine triphosphate); (b) the inhibition of the ATP response by quinidine (1 X 10(-3) M) but not by theophylline (1 X 10(-3) M). 3 Indomethacin (1 X 10(-5) M) inhibits the response of the cultured epithelium to ATP. 4 Prostaglandin E1 (PGE1) stimulates SCC but potentiates the effect of ATP on SCC. The divalent cationic ionophore A23187 (1 X 10(-6) M) transiently stimulates SCC itself and abolishes ATP-induced stimulation of the SCC.