cAMP-guanine exchange factor protection from bile acid-induced hepatocyte apoptosis involves glycogen synthase kinase regulation of c-Jun NH2-terminal kinase.

Cholestatic liver disorders are accompanied by the hepatic accumulation of cytotoxic bile acids that induce cell death. Increases in cAMP protect hepatocytes from bile acid-induced apoptosis by a cAMP-guanine exchange factor (cAMP-GEF)/phosphoinositide-3-kinase (PI3K)/Akt pathway. The aim of these studies was to identify the downstream substrate in this pathway and to determine at what level in the apoptotic cascade cytoprotection occurs. Since inhibitory phosphorylation of glycogen synthase kinase-3 (GSK) occurs downstream of PI3K/Akt and this phosphorylation has been implicated in cell survival, we conducted studies to determine whether GSK was downstream in cAMP-GEF/PI3K/Akt-mediated cytoprotection. Our results show that treatment of hepatocytes with the cAMP-GEF-specific analog, 4-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cAMP, results in PI3K-dependent phosphorylation of GSK. Direct chemical inhibition of GSK in rat hepatocytes or human HUH7-NTCP cells with several structurally and functionally distinct inhibitors including bromoindirubin-3'-oxime (BIO), maleimides (SB216763, SB415286), thiadiazolidine derivatives, and LiCl attenuates apoptosis induced by glycochenodeoxycholate (GCDC). In addition, genetic silencing of the GSK ß isoform with small interfering RNA attenuates GCDC apoptosis in HUH7-NTCP cells. Adenoviral inhibition of the Rap1 blocks both cAMP-GEF-mediated cytoprotection against GCDC-induced apoptosis and Akt/GSK3ß phosphorylation. GCDC-induced phosphorylation of the proapoptotic kinase, c-Jun NH(2)-terminal kinase (JNK) is inhibited by GSK inhibition or cAMP-GEF activation. GCDC-induced apoptosis is accompanied by phosphorylation of the endoplasmic reticulum stress markers pIEF2α and IRE-1, and pretreatment with the cAMP-GEF analog or GSK inhibitors prevents this phosphorylation. Collectively, our results support the presence of a cAMP/cAMP-GEF/Rap1/PI3K/Akt/GSKß survival pathway in hepatocytes that inhibits bile acid-induced JNK phosphorylation.

Phosphoinositide 3-kinase, but not mitogen-activated protein kinase, pathway is involved in hepatocyte growth factor-mediated protection against bile acid-induced apoptosis in cultured rat hepatocytes.

We have previously shown that cAMP protects against hydrophobic bile acid-induced apoptosis in cultured rat hepatocytes through pathways dependent on activation of phosphoinositide 3-kinase and inhibition of mitogen activated protein kinase. Hepatocyte growth factor protects epithelial cells against apoptosis and activates both of these kinases in hepatocytes. We studied the effect of hepatocyte growth factor on glycochenodeoxycholate-induced apoptosis to determine whether hepatocyte growth factor protects hepatocytes against bile acid-induced apoptosis and whether the protective effect is mediated via phosphoinositide 3-kinase and/or mitogen-activated protein kinase pathways. Two-hour exposure of cultured rat hepatocytes to glycochenodeoxycholate resulted in apoptosis in 12.5 +/- 0.49% of the cells. Pretreatment with hepatocyte growth factor (50 ng/mL) decreased apoptosis by 50% to 70%. Hepatocyte growth factor cytoprotection was prevented by pretreatment with the phosphoinositide 3-kinase inhibitors, wortmannin (50 nmol/L) or Ly 294002 (40 micromol/L). Hepatocyte growth factor activated phosphoinositide 3-kinase dependent protein kinase B and mitogen-activated protein kinase. Pretreatment of hepatocytes with a mitogen-activated protein kinase inhibitor, U0126 (40 micromol/L) or an inhibitor of pp70(s6) kinase, rapamycin (100 nmol/L), had no effect on the growth factor's anti-apopotic effect. Treatment with hepatocyte growth factor resulted in mitogen-activated protein kinase-dependent phosphorylation of BAD on serine(112). In summary, hepatocyte growth factor protection against bile acid-induced apoptosis occurs via a phosphoinositide 3-kinase pathway and is not dependent on the mitogen-activated protein kinase pathway, phosphorylation of BAD on serine(112), or activation of p70(S6) kinase.

Cell swelling-induced translocation of rat liver Na(+)/taurocholate cotransport polypeptide is mediated via the phosphoinositide 3-kinase signaling pathway.

Cell swelling stimulates phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) in hepatocytes, and the PI3K signaling pathway is involved in cAMP-mediated translocation of sinusoidal Na(+)/taurocholate (TC) cotransporter (Ntcp) to the plasma membrane. We determined whether cell swelling also stimulates TC uptake and Ntcp translocation via the PI3K and/or MAPK signaling pathway. All studies were conducted in isolated rat hepatocytes. Hepatocyte swelling induced by hypotonic media resulted in: 1) time- and medium osmolarity-dependent increases in TC uptake, 2) an increase in the V(max) of Na(+)/TC cotransport, and 3) wortmannin-sensitive increases in TC uptake and plasma membrane Ntcp mass. Hepatocyte swelling also induced wortmannin-sensitive activation of PI3K, protein kinase B, and p70(S6K). Rapamycin, an inhibitor of p70(S6K), inhibited cell swelling-induced activation of p70(S6K) but failed to inhibit cell swelling-induced stimulation of TC uptake. Because PD98095, an inhibitor of MAPK, did not inhibit cell swelling-induced increases in TC uptake, it is unlikely that the effect of cell swelling on TC uptake is mediated via the MAPK signaling pathway. Taken together, these results indicate that 1) cell swelling stimulates TC uptake by translocating Ntcp to the plasma membrane, 2) this effect is mediated via the PI3K, but not MAPK, signaling pathway, and 3) protein kinase B, but not p70(S6K), is a likely downstream effector of PI3K.

Role of the PI3K/PKB signaling pathway in cAMP-mediated translocation of rat liver Ntcp.

cAMP stimulates Na(+)-taurocholate (TC) cotransport by translocating the Na(+)-TC-cotransporting peptide (Ntcp) to the plasma membrane. The present study was undertaken to determine whether the phosphatidylinositol-3-kinase (PI3K)-signaling pathway is involved in cAMP-mediated translocation of Ntcp. The ability of cAMP to stimulate TC uptake declined significantly when hepatocytes were pretreated with PI3K inhibitors wortmannin or LY-294002. Wortmannin inhibited cAMP-mediated translocation of Ntcp to the plasma membrane. cAMP stimulated protein kinase B (PKB) activity by twofold within 5 min, an effect inhibited by wortmannin. Neither basal mitogen-activated protein kinase (MAPK) activity nor cAMP-mediated inhibition of MAPK activity was affected by wortmannin. cAMP also stimulated p70(S6K) activity. However, rapamycin, an inhibitor of p70(S6K), failed to inhibit cAMP-mediated stimulation of TC uptake, indicating that the effect of cAMP is not mediated via p70(S6K). Cytochalasin D, an inhibitor of actin filament formation, inhibited the ability of cAMP to stimulate TC uptake and Ntcp translocation. Together, these results suggest that the stimulation of TC uptake and Ntcp translocation by cAMP may be mediated via the PI3K/PKB signaling pathway and requires intact actin filaments.

Sodium taurocholate cotransporting polypeptide is a serine, threonine phosphoprotein and is dephosphorylated by cyclic adenosine monophosphate.

Na+/taurocholate (Na+/TC) cotransport in hepatocytes is mediated primarily by Na+/TC cotransporting polypeptide (Ntcp), and cyclic adenosine monophosphate (cAMP) stimulates Na+/TC cotransport by inducing translocation of Ntcp to the plasma membrane. The aim of the present study was to determine if Ntcp is a phosphoprotein and if cAMP alters Ntcp phosphorylation. Freshly prepared hepatocytes from rat livers were incubated with carrier-free 32PO4 for 2 hours, followed by incubation with 10 micromol/L 8-chlorophenylthio adenosin 3':5'-cyclic monophosphate (CPT-cAMP) for 15 minutes. Subcellular fractions isolated from 32P-labeled hepatocytes were subjected to immunoprecipitation using Ntcp antibody, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography to determine if Ntcp is phosphorylated. Ntcp immunoprecipitated from plasma membranes isolated from nonlabeled hepatocytes was subjected to immunoblot analysis using anti-phosphoserine, anti-phosphothreonine, or anti-phosphotyrosine antibody to determine whether Ntcp is a serine, threonine, or tyrosine phosphoprotein. Hepatocytes were loaded with bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid (MAPTA), a Ca2+ buffering agent, and the effect of CPT-cAMP on TC uptake, cytosolic [Ca2+], and ntcp phosphorylation and translocation was determined. In addition, the effect of cAMP on protein phosphatases 1 and 2A (PP1/2A) was determined in homogenates and plasma membranes obtained from CPT-cAMP-treated hepatocytes. Phosphorylation study showed that phosphorylated Ntcp is detectable in plasma membranes, and cAMP treatment resulted in dephosphorylation of Ntcp. Immunoblot analysis with phosphoamino antibodies revealed that Ntcp is a serine/threonine, and not a tyrosine, phosphoprotein, and cAMP inhibited both serine and threonine phosphorylation. In MAPTA-loaded hepatocytes, CPT-cAMP failed to stimulate TC uptake, failed to increase cytosolic [Ca2+], and failed to induce translocation and dephosphorylation of Ntcp. cAMP did not alter the activity of PP1/2A in either homogenates or in plasma membranes. Taken together, these results suggest that Ntcp is a serine/threonine phosphoprotein and is dephosphorylated by cAMP treatment. Activation of PP1/2A is not involved in cAMP-mediated dephosphorylation of Ntcp. Both translocation and dephosphorylation of Ntcp may be involved in the regulation of hepatic Na+/TC cotransport.

Role of protein phosphatases in cyclic AMP-mediated stimulation of hepatic Na+/taurocholate cotransport.

Cyclic AMP has been proposed to stimulate Na+/taurocholate (TC) cotransport in hepatocytes by translocating Na+/TC cotransport polypeptide (Ntcp) to the plasma membrane and to induce Ntcp dephosphorylation. Whether protein phosphatases 1 and 2A (PP1/2A) are involved in the regulation of Na+/TC cotransport by cAMP was investigated in the present study. Okadaic acid and tautomycin, inhibitors of PP1/2A, inhibited cAMP-mediated increases in TC uptake and cytosolic [Ca2+], and only tautomycin inhibited basal TC uptake. Removal of cAMP reversed cAMP-mediated increases in TC uptake and plasma membrane Ntcp mass. Okadaic acid alone increased Ntcp phosphorylation without affecting Ntcp mass in plasma membranes and homogenates. In the presence of okadaic acid, cAMP failed to increase plasma membrane Ntcp mass, induce Ntcp dephosphorylation, and decrease endosomal Ntcp mass. Phosphorylated Ntcp was detectable in endosomes isolated from okadaic acid-treated hepatocytes but not in endosomes from control and cAMP-treated hepatocytes. PP1 was found to be enriched in plasma membranes, whereas PP2A was mostly in the cytosol. Cyclic AMP did not activate either PP1 or PP2A, whereas okadaic acid inhibited primarily PP2A. These results suggest that 1) the effect of cAMP on Na+/TC cotransport is not mediated via either PP1 or PP2A; rather, cAMP-mediated signaling pathway is maintained by PP2A and inhibition of PP2A overrides cAMP-mediated effects, and 2) okadaic acid, by inhibiting PP2A, inhibits cAMP-mediated increases in Na+/TC cotransport by decreasing the ability of cAMP to increase cytosolic [Ca2+]. It is proposed that cAMP-mediated dephosphorylation of Ntcp leads to an increased retention of Ntcp in the plasma membrane, and okadaic acid, by inhibiting PP2A, inhibits cAMP-mediated stimulation of Na+/TC cotransport by reversing the ability of cAMP to increase cytosolic [Ca2+] and to induce Ntcp dephosphorylation.

Cyclic adenosine monophosphate-mediated protection against bile acid-induced apoptosis in cultured rat hepatocytes.

UNLABELLED: Cyclic adenosine monophosphate (cAMP) has been shown to modulate apoptosis. To evaluate the role of cAMP in bile acid-induced hepatocyte apoptosis, we studied the effect of agents that increase cAMP on the induction of apoptosis by glycochenodeoxycholate (GCDC) in cultured rat hepatocytes. GCDC induced apoptosis in 26.5%+/-1.1% of hepatocytes within 2 hours. Twenty-minute pretreatment of hepatocytes with 100 micromol/L 8-(4-chlorothiophenyl) cAMP (CP-cAMP) resulted in a reduction in the amount of apoptosis to 35.2%+/-3.8% of that seen in hepatocytes treated with GCDC alone. Other agents that increase intracellular cAMP, including dibutyryl cAMP (100 micromol/L), glucagon (200 nmol/L), and a combination of forskolin (20 micromol/L) and 3-isobutyl-1-methylxanthine (20 micromol/L), also inhibited GCDC-induced apoptosis to a similar extent. Pretreatment with the protein kinase A (PKA) inhibitor, KT5720, prevented the protective effect of CP-cAMP and inhibited CP-cAMP-induced activation of PKA activity. Inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin (50 nmol/L), or Ly 294002 (20 micromol/L) also prevented the cytoprotective effect of cAMP. PI3K assays confirmed that wortmannin (50 nmol/L) inhibited PI3K activity, while CP-cAMP had no effect on the activity of this lipid kinase. GCDC increased mitogen-activated protein kinase (MAPK) activity, but had no effect on stress-activated protein kinase (SAPK) activity in hepatocytes. cAMP decreased basal and GCDC-induced MAPK activity and increased SAPK activity. The MAPK kinase inhibitor, PD 98059, inhibited both GCDC-mediated MAPK activation and GCDC-induced apoptosis. IN CONCLUSION: 1) agents that increase intracellular cAMP protect against hepatocyte apoptosis induced by hydrophobic bile acids; 2) activation of MAPK by GCDC may be involved in bile acid-induced apoptosis; and 3) cAMP-mediated cytoprotection against bile acid-induced apoptosis appears to involve PKA, MAPK, and PI3K.

Delayed triphenyltetrazolium chloride staining remains useful for evaluating cerebral infarct volume in a rat stroke model.

Sixteen of 24 Sprague-Dawley rats with permanent middle cerebral artery occlusion for 24 hours were subjected to immediate or 8-hour delayed 2,3,5-triphenyltetrazolium chloride (TTC) staining (n = 8 at each time point); the other 8 animals were subjected to immediate or 8-hour delayed measurement of succinate dehydrogenase activity (n = 4 at each time point). The TTC staining was of good quality good in all animals, and the infarcted region could be distinguished easily from normal tissue. There was no significant difference in corrected infarct volume between the two groups (263.8 +/- 43.1 versus 264.4 +/- 54.8 mm3 [mean +/- standard deviation]). The activity of succinate dehydrogenase was not significantly different when normal or infarcted tissue was measured immediately after death or with an 8 hour delay, although less activity was detected at both time points in the infarcted tissue. These results demonstrate that an 8-hour delay of TTC staining is reliable for evaluating brain infarct volume in a rat stroke model and this probably is attributable to the slow deterioration of mitochondrial enzyme activity in nonischemic brain over this time period.

cAMP increases liver Na+-taurocholate cotransport by translocating transporter to plasma membranes.

Adenosine 3',5'-cyclic monophosphate (cAMP), acting via protein kinase A, increases transport maximum of Na+-taurocholate cotransport within 15 min in hepatocytes (S. Grüne, L. R. Engelking, and M. S. Anwer. J. Biol. Chem. 268: 17734-17741, 1993); the mechanism of this short-term stimulation was investigated. Cycloheximide inhibited neither basal nor cAMP-induced increases in taurocholate uptake in rat hepatocytes, indicating that cAMP does not stimulate transporter synthesis. Studies in plasma membrane vesicles showed that taurocholate uptake was not stimulated by the catalytic subunit of protein kinase A but was higher when hepatocytes were pretreated with cAMP. Immunoblot studies with anti-fusion protein antibodies to the cloned Na+-taurocholate cotransport polypeptide (Ntcp) showed that pretreatment of hepatocytes with cAMP increased Ntcp content in plasma membranes but not in homogenates. Ntcp was detected in microsomes, endosomes, and Golgi fractions, and cAMP pretreatment resulted in a decrease only in endosomal Ntcp content. It is proposed that cAMP increases transport maximum of Na+-taurocholate cotransport, at least in part, by translocating Ntcp from endosomes to plasma membranes.

Bile acids in the diagnosis, pathology, and therapy of hepatobiliary diseases.

Bile acids are normally confined in the enterohepatic circulation in which they play an important role in bile formation, biliary lipid excretion, and intestinal lipid absorption. In hepatobiliary diseases, bile acids escape the confinement of the enterohepatic circulation, allowing the measurement of the serum total bile acid concentration as a diagnostic indicator. Accumulation of certain bile acids within the hepatocyte, amplified as a consequence of cholestatic hepatobiliary disease, probably enhances cytotoxicity and leads to secondary pathology. Ursodeoxycholate, a bile acid with atypical physiological effects, may be useful in the treatment of various long-term cholestatic hepatobiliary diseases. Presently, most of the information on the toxicity and therapeutic usefulness of bile acids are based on studies in humans and experimental animals. Further studies, both basic and clinical, are needed to determine the pathologic as well as the therapeutic effects of bile acids in domestic animals.

Mechanism of activation of the Na+/H+ exchanger by arginine vasopressin in hepatocytes.

Arginine vasopressin has been shown to activate the Na+/H+ exchanger in hepatocytes by calcium/calmodulin-dependent processes. Whether this activation also involves protein kinase C and is associated with changes in the intracellular pH setpoint was investigated in this study. Changes in pHi and intracellular Ca++ concentration were measured with the fluorescent probes BCECF and quin-2, respectively. Intracellular pH recovery rate was calculated from time-dependent changes in intracellular pH in hepatocytes acid-loaded with sodium propionate. Arginine vasopressin, phorbol myristate acetate and thapsigargin stimulated intracellular pH recovery but did not increase basal intracellular pH. Arginine vasopressin and thapsigargin, but not phorbol myristol acetate, increased intracellular Ca++ concentration. The protein kinase C inhibitors staurosporine and calphostin C inhibited arginine vasopressin- and phorbol myristol acetate-induced, but not thapsigargin-induced, intracellular pH recovery. Neither staurosporine nor calphostin C affected arginine vasopressin- and thapsigargin-induced increases in intracellular Ca++ concentration, and no inhibitor affected basal intracellular pH recovery. Arginine vasopressin, phorbol myristol acetate and thapsigargin increased intracellular pH dependency of intracellular pH recovery without affecting intracellular pH setpoint. These results indicate that the activation of the Na+/H+ exchanger by arginine vasopressin is mediated both by Ca++/calmodulin and protein kinase C and may be due to enhanced interaction of H+ with the internal modifier site of the exchanger.

Mechanism of ionomycin-induced intracellular alkalinization of rat hepatocytes.

Calcium ionophores such as ionomycin and A23187 are often used to determine the role of intracellular Ca++ in cellular processes. Ionomycin but not Ca+(+)-mobilizing agonists increases basal intracellular pH in hepatocytes. To explain this difference in effects of agents that increase intracellular Ca++ concentration, the mechanism of ionomycin-induced increases in basal intracellular pH in isolated rat hepatocytes was studied. Changes in intracellular pH and intracellular Ca++ concentration were measured with the fluorescent probes BCECF (2',7'-bis-2-[carboxyethyl ester]-5[6]carboxyfluorescein) and quin-2, respectively. Ionomycin produced dose-dependent increases in intracellular pH and intracellular Ca++ concentration, with the increase in intracellular Ca++ concentration preceded by the increase in intracellular pH. Ionomycin-induced increases in intracellular pH were not affected by 1 mmol/L amiloride, 100 mumol/L diisothiocyanostilbene disulfonate or removal of extracellular Na+, indicating that the effect is not mediated by Na+/H+ exchange, Cl-/HCO3- exchange or Na+/HCO3- cotransport. Ionomycin failed to increase intracellular pH or intracellular Ca++ concentration in the absence of extracellular Ca++, and both intracellular pH and intracellular Ca++ concentration increased promptly when extracellular Ca++ was reintroduced. Ionomycin-induced increases in intracellular Ca++ concentration but not intracellular pH were smaller in hepatocytes loaded with the Ca++ buffering agent MAPTA. Thapsigargin increased intracellular Ca++ concentration but failed to increase intracellular pH. Thus the effect of ionomycin is independent of the effect of ionomycin on intracellular Ca++ concentration and dependent on extracellular intracellular Ca++ concentration. Experimental conditions that produce cell depolarization did not increase basal intracellular pH but lowered ionomycin-induced increases in intracellular pH by 25% without affecting increases in intracellular Ca++ concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of intracellular calcium and protein kinases in the activation of hepatic Na+/taurocholate cotransport by cyclic AMP.

Glucagon and dibutyryl cyclic AMP (Bt2cAMP) stimulate Na+/taurocholate (TC) cotransport and increase the intracellular Ca2+ concentration ([Ca2+]i) of hepatocytes. Whether the effect of cAMP is mediated via increases in [Ca2+]i, cAMP-dependent protein kinase (PKA), and/or protein kinase C (PKC) was investigated in this study. TC uptake and [Ca2+]i were determined in isolated rat hepatocytes using [14C]TC and the fluorescent dye quin-2, respectively. Bt2cAMP, forskolin, and 8-bromo-cAMP stimulated Na(+)-dependent, but not Na(+)-independent TC uptake. Bt2cAMP increased the maximal rate of Na+/TC cotransport without affecting the apparent Km. Increases in TC uptake and [Ca2+]i by Bt2cAMP were inhibited in hepatocytes preloaded with bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid (MAPTA) or preincubated with 8-diethylaminooctyl 3,4,5-trimethoxybenzoate (TMB8). Calmodulin antagonists inhibited Bt2cAMP-induced increases in TC uptake, but not [Ca2+]i. Other Ca(2+)-mobilizing agents (thapsigargin, vasopressin, phenylephrine, and ionomycin) increased [Ca2+]i but failed to stimulate TC uptake, indicating that an increase in [Ca2+]i alone is not a sufficient stimulus for TC uptake. However, increases in TC uptake by 1 and 10 microM Bt2cAMP were further increased by thapsigargin, indicating a permissive role for Ca2+/calmodulin. Bt2cAMP-induced increases in TC uptake and [Ca2+]i were inhibited by known inhibitors of PKA and by an activator of PKC, but they remained unaffected by a specific inhibitor of PKC. Unlike thapsigargin, vasopressin inhibited Bt2cAMP-induced increases in TC uptake. Taken together these results indicate that stimulation of hepatic Na+/TC cotransport by cAMP 1) is mediated via PKA; 2) is potentiated, but not mediated, by Ca2+/calmodulin-dependent processes; and 3) may be down-regulated by PKC.

Intracellular calcium-mediated activation of hepatic Na+/H+ exchange by arginine vasopressin and phenylephrine.

The effect of Ca++ mobilizing agonists arginine vasopressin and phenylephrine on Na+/H+ exchange was studied in freshly isolated hepatocytes and isolated perfused rat livers. The activity of Na+/H+ exchange was determined from the rate of H+ efflux, 22Na uptake and pHi recovery. Arginine vasopressin and phenylephrine stimulated H+ efflux and 22Na uptake in isolated rat hepatocytes and increased the rate of pHi recovery from acid-loaded hepatocytes. These effects were inhibited by amiloride. Arginine vasopressin- and phenylephrine-induced increases in H+ efflux were also dependent on extracellular Na+. Arginine vasopressin- and phenylephrine-induced increases in intracellular Ca++ concentration, H+ efflux, 22Na uptake and intracellular pH recovery were decreased in hepatocytes preloaded with the Ca(++)-buffering agent [bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid] (MAPTA). Na+/H+ exchange-dependent intracellular pH recovery from cytosolic acidification was stimulated by thapsigargin, which increases intracellular calcium concentration by inhibiting endoplasmic reticulum Ca++ ATPase. Arginine vasopressin- and phenylephrine-induced increases in intracellular pH recovery were not dependent on extracellular Ca++ and were inhibited by calmidazolium, a calmodulin inhibitor. Arginine vasopressin and phenylephrine also increased H+ efflux in the absence but not in the presence of amiloride in perfused rat livers without affecting biliary HCO3- excretion. These results indicate that arginine vasopressin and phenylephrine activate Na+/H+ exchange in rat hepatocytes, an effect mediated in part by intracellular Ca++ and calmodulin kinase. Furthermore, sinusoidal Na+/H+ exchange does not appear to be involved in biliary HCO3- excretion.

Mechanism of inhibition of hepatic bile acid uptake by amiloride and 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS).

The mechanisms by which amiloride and 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) inhibit hepatic uptake of cholate and taurocholate (TC) were investigated in isolated rat hepatocytes. Amiloride inhibited Na(+)-dependent uptake of cholate and TC only when hepatocytes were preincubated with amiloride, indicating an indirect effect of amiloride. Time-dependent studies showed that the inhibition of bile acid uptake was associated with a parallel increase in intracellular Na+ concentration ([Na+]i). Although amiloride decreased intracellular pH, this decrease preceded amiloride-induced inhibition of bile acid uptake and increase in [Na+]i. Amiloride inhibited bile acid uptake, decreased membrane potential, and increased [Na+]i with comparable concentration dependency. DIDS inhibited Na(+)-dependent uptake of cholate and TC non-competitively. Neither DIDS nor amiloride inhibited Na(+)-independent uptake of cholate and TC. These results indicate that amiloride inhibits Na(+)-dependent cholate and TC uptake by decreasing the transmembrane Na(+)-gradient, and further support the hypothesis that two different transporters may be involved in hepatic bile acid uptake by Na(+)-dependent and Na(+)-independent mechanisms.

Taurine-conjugated bile acids act as Ca2+ ionophores.

The ionophoretic properties of several taurine-conjugated bile acids have been investigated in two experimental systems: in a two-phase bulk partitioning system and in proteoliposomes. In the former, a bile acid/Ca2+ complex was extracted into the bulk organic phase and had an experimental stoichiometry of 1.75. Extraction was specific for Ca2+ over Mg2+; Na+ and K+ did not compete with the extraction of Ca2+. In the second system, bile acids at concentrations as low as 5-100 molecules/vesicle lowered the steady-state Ca2+ gradient maintained by a reconstituted sarcoplasmic reticulum Ca(2+)-ATPase. The effect was not due to nonspecific membrane perturbation. In addition to releasing intravesicular Ca2+ in a transmembraneous process, bile acids caused partition of Ca2+/bile acid complexes into the hydrophobic core of the bilayer. In both experimental systems, the Ca2+ ionophoretic activity correlated well with the concentration and the hydrophobicity of the bile acid. Taurolithocholate was most active, with a significant effect measurable at 10 microM in either system. Since bile acid concentrations equal to those used in our experiments can occur in the blood in certain liver diseases, the results support the notion that bile acids can increase the intracellular Ca2+ concentration bypassing the regulatory systems that maintain cellular Ca2+ homeostasis.