Biomedical subjects

A Guillouzo

Publications and source records attributed to A Guillouzo.

At least 37 records · Page 2Linked to original sources

PubMed2000

Phenobarbital induction of aldehyde dehydrogenase type 2 mRNA in mouse liver: a candidate region on chromosome 7 for a putative regulatory gene.

Phenobarbital (PB) strongly induces in the liver the expression of many genes encoding detoxication enzymes, such as the aldehyde dehydrogenase type 2 in the mouse (Aldh2). With the aim of identifying genes involved in this response, we have undertaken an approach based on a genetic analysis in mice. In a previous report, the genetic analysis of both the C57BL/6J (B6) x DBA/2J (D2) F1 and the (F1 x F1) F2 led us to the hypothesis that Aldh2 responsiveness to PB was under the control of one major locus independent of the structural gene. In the present study, the genetic analysis of the inducibility by PB of Aldh2 in the backcross population B6D2F1 x D2 has allowed us to confirm the involvement of a major regulatory gene in this mechanism. By searching for genetic linkage between this locus and a series of microsatellites DNA markers, we obtained indicative evidence for a region on chromosome 7, which may carry this gene.

Aldehyde Dehydrogenase↗

PubMed2000

The induction of the human hepatic CYP2E1 gene by interleukin 4 is transcriptional and regulated by protein kinase C.

Cytochrome P4502E1 (CYP2E1) plays a key role in the metabolism of numerous drug substrates, mostly in mammalian liver. Both the apoprotein and mRNA levels are increased in response to interleukin 4 (IL-4) in primary human hepatocyte cultures. We developed a human hepatoma cell model that faithfully reproduces the responsiveness of the CYP2E1 gene to IL-4 at least in part through transcriptional activation, upon treatment with 150 U/ml of IL-4. As expected, IL-4 induced tyrosine phosphorylation of the STAT6 transcription factor, an effect prevented by the tyrosine kinase inhibitor tyrphostin A25. However, this inhibitor as well as genistein (another inhibitor of tyrosine kinases) had no effect on the IL-4 induction of CYP2E1. Similarly, protein kinase A activators (forskolin and dibutyryl-cAMP) and inhibitor (H89) did not influence the response to IL-4. However, PKC inhibitors (H7 and calphostin C) strongly blocked any induction of the gene, as well as the IL-4-dependent translocation of PKCS. Taken together, our results show that IL-4 coordinately induces CYP2E1 transcription, mRNA and apoprotein levels in human hepatoma cells in a PKC-dependent manner, potentially through the activity of the PKCzeta isoform.

1-Methyl-3-isobutylxanthine↗

PubMed2000

Viability and drug metabolism capacity of alginate-entrapped hepatocytes after cryopreservation.

In the present study we evaluated viability and detoxifying enzyme capacity of cryopreserved hepatocytes from various species, including man, immobilized in calcium alginate gels. Ethoxyresorufin O-deethylase, phenacetin deethylase, pentoxyresorufin O-dealkylase, tolbutamide hydroxylase, S-mephenytoin hydroxylase, dextromethorphan demethylase, and nifedipine oxidation corresponding to the major cytochromes P450 (CYP) involved in xenobiotic metabolism as well as whole glutathione S-transferase (GST) activity were measured using specific substrates and after exposure or not to prototypical inducers. After deep-freeze storage, viability of immobilized hepatocytes was only slightly reduced and most CYP-related monooxygenase activities were well preserved, being expressed at levels close to those measured in unfrozen hepatocyte monolayers. By contrast, total GST activity was decreased by around 50%. However, as did CYP1A- and 3A-related enzymes, rat GST remained capable of responding to prototypical inducers. The fold increases were comparable in unfrozen and frozen immobilized hepatocytes and in unfrozen hepatocyte monolayers. The duration of storage, even when exceeding one year, did not affect viability and functions. In conclusion, after cryopreservation, alginate-entrapped hepatocytes remain highly viable and capable of expressing most detoxifying enzymes at levels close to those expressed in corresponding unfrozen hepatocyte monolayers and of responding to prototypical inducers.

Alginates↗

PubMed2000

Regulation of phenobarbital-induction of CYP2B and CYP3A genes in rat cultured hepatocytes: involvement of several serine/threonine protein kinases and phosphatases.

We investigated the involvement of diverse protein kinases and phosphatases in the transduction pathways elicited by phenobarbital (PB), a well-known inducer of some hepatic cytochromes P450 (CYP). Different inhibitors or activators of protein kinases or phosphatases were assessed for their ability to modulate PB-induction of CYP2B and CYP3A mRNA expression. Rat hepatocytes in primary culture were treated with the test compounds one hour prior to, and then continuously, in the absence or presence of 1 mmol/L PB for 24 h. By northern blot analysis of CYP2B1/2 and 3A1/2 gene expression, we first confirmed the negative role of the adenosine 3':5' cyclic monophosphate (cAMP)/protein kinase A pathway and the positive role of some serine/threonine protein phosphatases in the mechanism of PB-induction. The present data further suggested that Ca2+/calmodulin-dependent protein kinases II (independently of Ca2+) and extracellular signal-regulated kinases 1/2 (ERK1/2) might function respectively as positive and negative regulator in the PB-induction of CYP2B and CYP3A. In contrast, protein kinases C and phosphatidylinositol-3-kinase did not appear to be involved, while the role of tyrosine kinases remained unclear. We conclude that a complex network of phosphorylation/dephosphorylation events might be crucial for PB-induction of rat CYP2B and CYP3A.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2000

Regulation of phenobarbital induction of the cytochrome P450 2b9/10 genes in primary mouse hepatocyte culture. Involvement of calcium- and cAMP-dependent pathways.

Phenobarbital (PB) has long been known as an inducer of drug-metabolizing enzymes in liver, but the molecular mechanism underlying this induction is still poorly understood. Using primary mouse hepatocyte culture, we have investigated the possible involvement of different regulatory pathways in PB action, by exposing PB-treated cells to various protein kinase/phosphatase modulators. Our results showed a negative role of the cAMP-dependent pathway, as treatment with cAMP-dependent protein kinase (PKA) activators (10 microM dibutyryl-cAMP and 50 microM forskolin) dramatically inhibited PB-induced Cyp2b9/10 mRNA accumulation, whereas PKA inhibitor potentiated the PB responsiveness of this gene. The cGMP-dependent protein kinase (PKG) seems to play a positive role as PKG inhibitor reduced the PB-induced level of Cyp2b9/10 mRNA. We also obtained two lines of evidence for the involvement of Ca2+ in modulating PB action. Firstly, measurements of intracellular Fura-2 fluorescence ratio in murine hepatocytes showed that long-term PB incubation (24 and 48 h) led to a significant increase of [Ca2+]i. Secondly, treatment with an intracellular Ca2+ chelator (BAPTA-AM) nearly completely abolished PB-induced Cyp2b9/10 expression. Ca2+ thus appeared to mediate PB action likely via Ca2+/calmodulin-dependent protein kinase II, as KN62, a specific inhibitor of this enzyme, also dramatically inhibited PB induction of the Cyp2b9/10 genes.

Animals↗

PubMed2000

Hepatotoxicity of tacrine: occurrence of membrane fluidity alterations without involvement of lipid peroxidation.

Tacrine (THA), used in the treatment of Alzheimer's disease, is known to induce hepatotoxicity, the mechanisms of which remain to be fully established. We have previously shown that THA reduced intracellular glutathione concentration in rat hepatocytes in primary culture, thus pointing to a possible role for oxidative stress in THA toxicity. To test this, the effects of antioxidant molecules, namely, the flavonoids silibinin, silibinin dihydrogensuccinate, and silymarin, were evaluated on the toxicity of THA in cultured rat hepatocytes. This toxicity was investigated after a 24-h treatment over a concentration range from 0 to 1 mM, in the presence or absence of antioxidant (1 and 10 microM). We found that simultaneous treatment of hepatocytes with any of the antioxidants and THA remained ineffective on the lactate dehydrogenase release induced by THA. Then, the production of lipid-derived radicals (to estimate lipid peroxidation) was measured in THA (0.05-0.50 mM)-treated cells using a spin-trapping technique coupled to electron paramagnetic resonance (EPR) spectroscopy. No increase of the EPR signal was observed over the period of 30 min to 24 h. In contrast, treatment of cells with the spin label 12-doxyl stearic acid followed by EPR spectroscopy showed that THA (0.05 and 0.25 mM) rapidly increased hepatocyte membrane fluidity. Extracellular application of GM1 ganglioside (60 microM) both reversed this increase in fluidity and partially reduced lactate dehydrogenase release on THA exposure. In conclusion, this work indicates that early alterations of membrane fluidity, not resulting from lipid peroxidation, are likely to play an important role in the development of THA toxicity.

Animals↗

PubMed1999

Detoxifying activity in pig livers and hepatocytes intended for xenotherapy.

BACKGROUND: Both livers and hepatocytes from pigs have been proposed for the treatment of end-stage liver diseases, as an alternative to allogeneic liver transplants. However, little is known of the capability of porcine hepatocytes to fulfill the biotransformation pathways of toxic compounds, including those released from livers in acute failure. We have studied the activity and expression of detoxifying enzymes in porcine livers and in cultured hepatocytes and their induction by phenobarbital. METHODS: Cytochromes P450 (CYP) 1A, 2B, and 3A and GST-like activities were tested with the following specific substrates: 7-ethoxyresorufin, 7-pentoxyresorufin, nifedipine, testosterone, 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, and ethacrinic acid. CYP 1A1/2-, 2B1/2-, 2E1- and 3A4-related and GSTalpha proteins were analyzed by Western blotting and CYP 1A1/2, 2B1/2, 2C6, 2E1, and 3A4, aldehyde dehydrogenase, epoxide hydrolase, and GSTalpha-like RNA by Northern blotting. RESULTS: Enzymatic activities reflecting the expression of CYP 1A-, CYP 2B-, CYP 2E1-, and CYP 3A-like genes, that is, ethoxyresorufin-O-deethylase, pentoxyresorufin-O-deethylase, nifedipine oxidase and testosterone 6beta-hydroxylase, and chlorzoxazone 6-hydroxylase, were identified in pig livers. CYP 1A and CYP 2E1, GSTalpha-like proteins, CYP 1A, 2C, and 2E, epoxide hydrolase, aldehyde dehydrogenase, and GST like RNA were expressed in vivo and in vitro. CYP 2B and CYP 3A RNA and proteins, and their associated activities were induced by phenobarbital. CONCLUSIONS: Porcine hepatocytes express the most important biotransformation enzymes and their corresponding activities and RNA. Thus, livers and hepatocytes from pigs can detoxify a large spectrum of exogenous and endogenous compounds, which makes them a convenient substitute for allogeneic transplants for patients with liver failure.

Aldehyde Dehydrogenase↗

PubMed1999

Up-regulation of multidrug resistance-associated protein 2 (MRP2) expression in rat hepatocytes by dexamethasone.

Regulation of multidrug resistance-associated protein (MRP2) expression in response to dexamethasone (DEX) was analyzed using mainly primary rat hepatocytes. Enhanced levels of MRP2 mRNAs associated with increased amounts of a 190 kDa MRP2 were found in cultured DEX-treated hepatocytes; similarly, administration of DEX to rats (100 mg/kg, i.p.) led to a marked increase of hepatic amounts of MRP2 mRNAs. Maximal induction of MRP2 expression in DEX-treated primary hepatocytes was reached with 10(-5) M DEX, a concentration higher than that (10(-7) M) required for maximal up-regulation of tyrosine aminotransferase (TAT), a typical glucocorticoid receptor-regulated enzyme. In addition, the anti-glucocorticoid compound RU486 failed to inhibit MRP2 induction caused by DEX whereas it fully blocked that of TAT. These findings therefore demonstrate that DEX is a potent inducer of MRP2 expression in rat hepatocytes through a mechanism that seems not to involve the classical glucocorticoid receptor pathway.

Animals↗

PubMed1999

Survival and function of isolated hepatocytes after cryopreservation.

Cryopreservation in liquid nitrogen is presently the only way for long-term storage of isolated hepatocytes. Freeze-thaw conditions are not well defined yet; the most critical parameters appear to be the choice of the cryoprotectant, composition of the freezing medium, and cooling and thawing rates. Comparable results have been obtained with hepatocytes from various species, including man. Cryopreservation usually results in low cell recovery and early alterations of functional activities. However, both phase I and phase II xenobiotic metabolism is still active after thawing, at least during a short period. Moreover, survival and function of cryopreserved hepatocytes can be improved when these cells have a high energy status, are cryopreserved after immobilization in a gel, separated from dead cells on a Percoll gradient or placed in more favorable culture conditions (e.g. in coculture with liver non parenchymal cells). Additional studies are needed to improve freeze-thaw protocols and to better characterize liver parenchymal cells after storage, including evaluation of their responsiveness to specific inducers.

Animals↗

PubMed1999

A multi-laboratory evaluation of cryopreserved monkey hepatocyte functions for use in pharmaco-toxicology.

Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.

Adenosine Triphosphate↗

PubMed1999

Reversal of MRP-mediated multidrug resistance in human lung cancer cells by the antiprogestatin drug RU486.

Multidrug resistance-associated protein (MRP) and P-glycoprotein (P-gp) are drug efflux pumps conferring multidrug resistance to tumor cells. RU486, an antiprogestatin drug known to inhibit P-gp function, was examined for its effect on MRP activity in MRP-overexpressing lung tumor GLC4/Sb30 cells. In such cells, the antihormone compound was found to increase intracellular accumulation of calcein, a fluorescent compound transported by MRP, in a dose-dependent manner, through inhibition of cellular export of the dye; in contrast, it did not alter calcein levels in parental GLC4 cells. RU486, when used at 10 microM, a concentration close to plasma concentrations achievable in humans, strongly enhanced the sensitivity of GLC4/Sb30 cells towards two known cytotoxic substrates of MRP, the anticancer drug vincristine and the heavy metal salt potassium antimonyl tartrate. Vincristine accumulation levels were moreover up-regulated in RU486-treated GLC4/Sb30 cells. In addition, such cells were demonstrated to display reduced cellular levels of glutathione which is required for MRP-mediated transport of some anticancer drugs. These findings therefore demonstrate that RU486 can down-modulate MRP-mediated drug resistance, in addition to that linked to P-gp, through inhibition of MRP function.

ATP-Binding Cassette Transporters↗

PubMed1999

The multidrug resistance-associated protein (MRP) is over-expressed and functional in rat hepatoma cells.

Expression of multidrug-resistance-associated protein (MRP), a drug efflux pump transporting a wide range of xenobiotics, including anti-cancer drugs and chemical carcinogens, and present at low levels in normal hepatocytes, was investigated in rat hepatoma cells. Northern-blot analysis allowed detection of high levels of MRP mRNA in rat diethylnitrosamine-induced hepatocarcinomas when compared with normal liver. Similarly, elevated expression of MRP transcripts were evidenced in 6 rat hepatoma cell lines of different origins, especially in HTC cells, that, in contrast, failed to express mRNA of the canalicular multispecific organic anion transporter (cMOAT), an efflux pump sharing numerous substrates with MRP. HTC cells were also found by Western blotting to display much higher amounts of MRP than those observed in normal hepatocytes. In contrast, the MRP gene copy number was similar both in hepatoma HTC cells and in hepatocytes, as assessed by Southern blotting. Analysis of MRP-related transport using 3 types of MRP substrates, namely, the fluorescent glutathione-bimane, the anionic dye calcein and the cationic anti-cancer drug vincristine, demonstrated that HTC cells displayed cellular efflux of these 3 compounds, an efflux strongly inhibited by MRP modulators such as indomethacin. These results indicate that MRP is over-expressed and functional in rat hepatoma cells and may therefore be included in the de-toxifying pathways that are altered during hepatocarcinogenesis and are thus thought to contribute to the known multidrug resistance of liver tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Inhibition of multidrug resistance-associated protein (MRP) activity by rifampicin in human multidrug-resistant lung tumor cells.

The multidrug resistance-associated protein (MRP) is a drug efflux membrane pump conferring multidrug resistance on tumor cells. In order to look for compounds that can lead to reversal of such a resistance, the antituberculosis compound rifampicin, belonging to the chemical class of rifamycins, was examined for its effect on MRP activity in human multidrug resistant lung cancer GLC4/ADR cells. Rifampicin was shown to increase accumulation of the MRP substrate calcein in GLC4/ADR cells in a dose-dependent manner by inhibiting its MRP-mediated efflux from the cells; it also enhanced intracellular retention of another substrate of MRP such as the anticancer drug vincristine in the resistant cells. By contrast, the antituberculosis drug did not alter cellular levels of accumulation of either calcein or vincristine in parental drug-sensitive GLC4 cells. Other rifamycins such as rifamycin B and rifamycin SV were also demonstrated to increase intracellular accumulation of calcein in GLC4/ADR cells. These results therefore indicate that rifamycins, including rifampicin, probably constitute a new chemical class of modulators down-regulating MRP-mediated drug transport.

ATP-Binding Cassette Transporters↗

PubMed1999

Isolation of a cyp2b10-like cDNA and of a clone derived from a cyp2b10-like pseudogene.

By screening Balb/c male mouse liver cDNA library with a rat CYP2B1 cDNA probe, we have isolated a 1795 bp cyp2b10-like clone, referred to as P16. Its sequence exhibited 34 base differences (98% similarity) with the cyp2b10 published sequence, together with a 97% identity at the amino acid sequence level. By RT-PCR and PCR analyses with Balb/c female and male liver RNA and genomic DNA, using a region showing 8 base differences between the P16 and the cyp2b10 sequences, we have confirmed the identity of our cloned cDNA, and failed in finding a PCR product exhibiting a sequence 100% identical with that of cyp2b10. Our results therefore suggest that the P16 sequence is the authentic cyp2b10 sequence. We have also isolated a partial clone, P21, which 1609 bp sequence overlapped with that of P16, except for a T-->G transversion, giving rise to a premature TGA stop codon, indicating that it was derived from a pseudogene.

Amino Acid Sequence↗

PubMed1999

Overexpression of the multidrug resistance-associated protein (MRP1) in human heavy metal-selected tumor cells.

Cellular and molecular mechanisms involved in the resistance to cytotoxic heavy metals remain largely to be characterized in mammalian cells. To this end, we have analyzed a metal-resistant variant of the human lung cancer GLC4 cell line that we have selected by a step-wise procedure in potassium antimony tartrate. Antimony-selected cells, termed GLC4/Sb30 cells, poorly accumulated antimony through an enhanced cellular efflux of metal, thus suggesting up-regulation of a membrane export system in these cells. Indeed, GLC4/Sb30 cells were found to display a functional overexpression of the multidrug resistance-associated protein MRP1, a drug export pump, as demonstrated by Western blotting, reverse transcriptase-polymerase chain reaction and calcein accumulation assays. Moreover, MK571, a potent inhibitor of MRP1 activity, was found to markedly down-modulate resistance of GLC4/Sb30 cells to antimony and to decrease cellular export of the metal. Taken together, our data support the conclusion that overexpression of functional MRP1 likely represents one major mechanism by which human cells can escape the cytotoxic effects of heavy metals.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Primary cultures of heart cells from the scallop Pecten maximus (mollusca-bivalvia).

Primary cultures of Pecten maximus heart cells, isolated by an enzymatic procedure, were routinely obtained with a high level of reproducibility in a simple medium based on sterile seawater. Cells attached to the plastic substratum without the need to add a special factor. The number of adhering cells gradually increased with the time of culture. Two types of adhering cells were observed: epitheliallike cells and fibroblastlike cells, which were more numerous. The latter cells were identified as myocytes by electron microscopy and immunofluorescent staining. Results obtained by autoradiography, after incorporation of [14C]leucine, [3H]thymidine, and [14C]acetate, confirmed functional activity of the cells. These cultures were maintained viable in vitro during at least 1 mo.

Animals↗

PubMed1999

Evidence for a multidrug resistance-associated protein 1 (MRP1)-related transport system in cultured rat liver biliary epithelial cells.

Cellular accumulation and efflux of the anionic fluorescent dye carboxy-2',7'-dichlorofluorescein (CF) were studied in rat liver SDVI cells thought to derive from primitive bile ductules, in order to characterize carrier-related membrane transport of organic anions in epithelial cells. Probenecid, a common blocker of anion transport, was found to strongly enhance CF levels in SDVI cells in a dose-dependent manner through inhibition of dye efflux. Such an outwardly-directed transport was demonstrated to be temperature-dependent and down-regulated by various metabolic inhibitors, therefore outlining its requirement for energy; it was shown to be Na+- and membrane potential-independent and inhibited by anionic drugs such as indomethacin, indoprofen and rifamycin B. These functional features are closed to those described for multidrug resistance-associated protein 1 (MRP1) that was furthermore demonstrated, in contrast to P-glycoprotein, to be expressed in SDVI cells and to lower CF accumulation in MRP1-overexpressing drug-resistant tumor cells. These data therefore suggest that active membrane transport of organic anions such as CF occurs in epithelial cells like cultured liver biliary SDVI cells through a MRP1-related efflux system.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Intracellular pH alterations induced by tacrine in a rat liver biliary epithelial cell line.

1. The effects of tacrine (THA) on intracellular pH (pH(i)) were examined in a rat liver biliary epithelial cell line (RLEC) in HEPES-buffered medium. pH(i) was recorded using the pH-sensitive fluoroprobe, carboxy-SNARF-1 (carboxy-seminaphtorhodafluor). 2. In the steady state, short-term exposures to THA resulted in alkalinization and re-acidification at 0.1 and 0.25 mM. Following a 24 h-treatment, no significant difference in pH(i) could be detected at 0.1 and 0.25 mM THA, whereas at 0.05 mM, pH(i) was slightly more acid (7.17+/-0. 02, n=16 versus 7.21+/-0.02, n=24 [control]). 3. In control and short-term treated cells, intracellular intrinsic buffering power (beta(i)) increased roughly linearly as pH(i) decreased. This dependence was not seen following long-term treatment. In all cases, beta(i) was increased by THA (by 1.6 to 3.5 fold). 4. Following an acid load (induced by 20 mM NH(4)Cl removal), pH(i) recovery in RLEC relied upon Na(+)/H(+) exchange. A short-term treatment (0.25 mM THA) did not affect total acid extrusion. In contrast, a 24 h-treatment with 0.05 mM THA reduced it (by approximately 36% at a pH(i) of 6.73) while at 0.25 mM, a large increase was detected (by approximately 109% at a pH(i) of 6.75). In Na(+)-free medium, THA (0. 25 mM) still induced an alkalinization in the steady state. Following an acid load, THA stimulated a Na(+)-independent acid efflux in a dose-dependent manner, inhibitable by alpha-cyano-4-hydroxy cinnamate (CHC, 4 mM) but not by quercetin (0. 125 mM). 6. In conclusion, this work demonstrates that THA affects pH(i) in RLEC, through a decrease in Na(+)/H(+) exchange and an increase in beta(i). Stimulation of a CHC-inhibitable, Na(+)-independent acid efflux is also detected.

Animals↗