Search PubMed⌕ Search

Biomedical subjects

H Fromm

Publications and source records attributed to H Fromm.

At least 37 records · Page 2Linked to original sources
PubMed1998

Two isoforms of glutamate decarboxylase in Arabidopsis are regulated by calcium/calmodulin and differ in organ distribution.

The nucleotide sequences of cDNAs encoding two isoforms of Arabidopsis glutamate decarboxylase, designated GAD1 (57.1 kDa) and GAD2 (56.1 kDa) and sharing 82% identical amino acid sequences, were determined. The recombinant proteins bound [35S] calmodulin (CaM) in the presence of calcium, and a region of 30-32 amino acids from the C-terminal of each isoform was sufficient for CaM binding when fused to glutathione S-transferase. Full-length GAD1 and GAD2 were expressed in Sf9 insect cells infected with recombinant baculovirus vectors. Recombinant proteins were partially purified by CaM affinity chromatography and were found to exhibit glutamate decarboxylase activity, which was dependent on the presence of Ca2+/CaM at pH 7.3. Southern hybridizations with GAD gene-specific probes suggest that Arabidopsis possesses one gene related to GAD1 and one to GAD2. Northern hybridization and western blot analysis revealed that GAD1 was expressed only in roots and GAD2 in roots, leaves, inflorescence stems and flowers. Our study provides the first evidence for the occurrence of multiple functional Ca2+/CaM-regulated GAD gene products in a single plant, suggesting that regulation of Arabidopsis GAD activity involves modulation of isoform-specific gene expression and stimulation of the catalytic activity of GAD by calcium signalling via CaM.

Amino Acid Sequence↗
PubMed1998

Relationship between biochemical and virological responses to interferon therapy in chronic hepatitis C infection. Consensus Interferon Study Group.

We have investigated the relationship between serum alanine aminotransferase (ALT) and hepatitis C virus (HCV) RNA in the assessment of responses to interferon (IFN) therapy in chronic HCV infection. Data from 704 patients with HCV infection who were randomized to receive consensus IFN-alpha (CIFN) 3 micrograms (n = 232 patients) or 9 micrograms (n = 232 patients), or IFN-alpha 2b 3 million units (MU) (n = 240 patients), were used for these analyses. All patients were treated three times weekly. Hepatitis C viral RNA (HCV RNA) was determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) with a lower limit of detection of 100 copies ml-1. Of patients with normal serum ALT concentrations, 53% (120/225) had undetectable HCV RNA at the end-of-treatment period and 47% (51/109) had undetectable HCV RNA at the end of the post-treatment observation period. In contrast, of the patients with undetectable HCV RNA, 75% (120/161) and 84% (51/61) had normal serum ALT activities at the end-of-treatment and post-treatment observations periods, respectively. The majority of patients with undetectable HCV RNA had normal ALT values. In contrast, only half of the patients with normal ALT values were negative for HCV. End-of-treatment HCV RNA response also better predicted sustained virological response than did end-of-treatment ALT response.

Adult↗
PubMed1998

Changes in G protein expression account for impaired modulation of hepatic cAMP formation after BDL.

The regulation of cAMP synthesis by hormones and bile acids is altered in isolated hamster hepatocytes 2 days after bile duct ligation (BDL) [Y. Matsuzaki, B. Bouscarel, M. Le, S. Ceryak, T. W. Gettys, J. Shoda, and H. Fromm. Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G164-G174, 1997]. Therefore, studies were undertaken to elucidate the mechanism(s) responsible for this impaired modulation of cAMP formation. Hepatocytes were isolated 48 h after either a sham operation or BDL. Both preparations were equally devoid of cholangiocyte contamination. Although the basal cAMP level was not affected after BDL, the ability of glucagon to maximally stimulate cAMP synthesis was decreased by approximately 40%. This decreased glucagon effect after BDL was not due to alteration of the total glucagon receptor expression. However, this effect was associated with a parallel 50% decreased expression of the small stimulatory G protein alpha-subunit (GsalphaS). The expression of either the large subunit (GsalphaL) or the common beta-subunit remained unchanged. The expression of Gialpha2 and Gialpha3 was also decreased by 25 and 46%, respectively, and was associated with the failure of ANG II to inhibit stimulated cAMP formation. Therefore, alterations of the expression of GsalphaS and Galphai are, at least in part, responsible for the attenuated hormonal regulation of cAMP synthesis. Because cAMP has been reported to stimulate both bile acid uptake and secretion, impairment of cAMP synthesis and bile acid uptake may represent an initial hepatocellular defense mechanism during cholestasis.

Angiotensin II↗
PubMed1997

Treatment of chronic hepatitis C with consensus interferon: a multicenter, randomized, controlled trial. Consensus Interferon Study Group.

This multicenter, randomized, controlled, double-blind, phase III study in 704 patients with chronic hepatitis C infection compared treatment with consensus interferon (CIFN), a non-natural recombinant type-1 interferon, with a standard regimen of recombinant interferon alfa-2b (IFN-alpha2b). Patients were randomized to receive CIFN at doses of 3 microg or 9 microg, or 15 microg IFN-alpha2b (3 million units), subcutaneously three times weekly for 24 weeks, followed by 24 weeks of observation. Efficacy was assessed by normalization of serum alanine transaminase (ALT) concentration and decrease in serum hepatitis C virus (HCV) RNA concentration below the limit of detection by reverse-transcription polymerase chain reaction (RT-PCR) (100 copies/mL). The beneficial effect of CIFN was greater with the 9-microg dose than the 3-microg dose. The sustained ALT and HCV RNA response rates were 20.3% and 12.1%, respectively, in the 9-microg CIFN cohort and 19.6% and 11.3%, respectively, in the 15-microg IFN-alpha2b cohort. However, patients receiving 9 microg of CIFN had a greater reduction in serum HCV RNA concentrations compared with patients receiving 15 microg IFN-alpha2b over the course of treatment (P < .01). Similarly, analysis of patients infected with HCV genotype 1 showed a greater reduction in serum HCV RNA concentration over the course of treatment for the 9-microg CIFN group when compared with the 15-microg IFN-alpha2b group (P < .01). In addition, a greater percentage of patients infected with HCV genotype 1 treated with 9 microg CIFN had undetectable HCV RNA concentrations when compared with patients in the 15-microg IFN-alpha2b cohort at the end of treatment (24% vs. 15%; P = .04). Improvements in liver histology were noted in all three treatment groups; 52% to 55% of the patients in the three cohorts had at least a 2-unit improvement in the Knodell score at the end of the posttreatment period. The adverse-events profiles were characteristic of treatment with type-1 interferon, and the incidences of anti-interferon antibody formation did not significantly differ among the three treatment groups. These results show that administration of 9 microg CIFN three times weekly for 6 months is safe and is effective in reducing serum HCV RNA concentration.

Adult↗
PubMed1997

Characterization of the plant homologue of prohibitin, a gene associated with antiproliferative activity in mammalian cells.

This report describes the cloning and characterization of a plant cDNA coding for a protein which shows high amino acid sequence similarity with prohibitin, whose gene is associated with antiproliferative activity in mammalian cells. Arabidopsis thaliana and Nicotiana tabacum prohibitin complete cDNAs were isolated, and the expression pattern of prohibitin was examined using polyclonal antibodies raised against the Arabidopsis recombinant prohibitin expressed in Escherichia coli. A single immunoreactive protein was detected in various plant species and in all Arabidopsis organs examined. Subcellular fractionation using tobacco leaves revealed prohibitin in a mitochondrial-enriched fraction. Phylogenetic conservation of prohibitin's amino acid sequence and subcellular localization suggests a similar function in plants, yeast and mammals.

Amino Acid Sequence↗
PubMed1997

Effect of cholestasis on regulation of cAMP synthesis by glucagon and bile acids in isolated hepatocytes.

Previously, we have reported that bile acids can directly inhibit hormone-induced adenosine 3',5'-cyclic monophosphate (cAMP) formation through a protein kinase C (PKC)-dependent mechanism [Bouscarel, B., T.W. Gettys, H. Fromm, and H. Dubner. Am. J. Physiol. 268 (Gastrointest. Liver Physiol. 31): G300-G310, 1995]. Therefore, the regulation of cAMP synthesis by glucagon and bile acids was investigated in hepatocytes isolated after 2-day ligation of the common bile duct in Golden Syrian hamsters. The bile acid concentration was increased 30-fold in the serum, whereas it was not significantly different in the bile of duct-ligated vs. sham-operated hamsters. The glycine/taurine and cholate/chenodeoxycholate ratios were significantly increased fourfold and sevenfold, respectively, only in the serum of bile duct-ligated hamsters. Ligation of the bile duct decreased the efficacy of glucagon-stimulated cAMP synthesis by 40-50% without changing its potency. This attenuation of cAMP synthesis, which was also observed with forskolin, remained in the absence of any detectable amount of bile acids in the hepatocytes. The decrease in glucagon-stimulated cAMP production was also not attributable to changes in either the affinity or the number of receptors for this hormone. The potency and efficacy of the bile acids to inhibit glucagon-induced cAMP formation was also reduced in bile duct-ligated hamsters. The inhibitory regulation of cAMP synthesis through angiotensin II was similarly diminished after bile duct ligation. Although the total expression of PKC-alpha was not affected, an increased translocation by 60% from the cytosol to the membrane fraction was observed in hepatocytes isolated after bile duct ligation. Therefore, during cholestasis and prolonged exposure of the liver to bile acids, both the stimulatory and inhibitory regulatory, mechanisms of cAMP synthesis are compromised in an irreversible manner because the effects persist even after isolation of the hepatocytes. This decreased regulation of cAMP synthesis is possibly mediated through PKC-alpha activation.

Angiotensin II↗
PubMed1996

Characterization of the calmodulin gene family in wheat: structure, chromosomal location, and evolutionary aspects.

Calmodulin is a ubiquitous transducer of calcium signals in eukaryotes. In diploid plant species, several isoforms of calmodulin have been described. Here, we report on the isolation and characterization of calmodulin cDNAs corresponding to 10 genes from hexaploid (bread) wheat (Triticum aestivum). These genes encode three distinct calmodulin isoforms; one isoform is novel in that it lacks a conserved calcium binding site. Based on their nucleotide sequences, the 10 cDNAs were classified into four subfamilies. Using subfamily-specific DNA probes, calmodulin genes were identified and the chromosomal location of each subfamily was determined by Southern analysis of selected aneuploid lines. The data suggest that hexaploid wheat possesses at least 13 calmodulin-related genes. Subfamilies 1 and 2 were both localized to the short arms of homoeologous-group 3 chromosomes; subfamily 2 is located on all three homoeologous short arms (3AS, 3BS and 3DS), whereas subfamily 1 is located only on 3AS and 3BS but not on 3DS. Further analysis revealed that Aegilops tauschii, the presumed diploid donor of the D-genome of hexaploid wheat, lacks a subfamily-1 calmodulin gene homologue, whereas diploid species related to the progenitors of the A and B genomes do contain such genes. Subfamily 3 was localized to the short arm of homoeologous chromosomes 2A, 2B and 2D, and subfamily 4 was mapped to the proximal regions of 4AS, 4BL and 4DL. These findings suggest that the calmodulin genes within each subfamily in hexaploid wheat represent homoeoallelic loci. Furthermore, they also suggest that calmodulin genes diversified into subfamilies before speciation of Triticum and Aegilops diploid species.

Amino Acid Sequence↗
PubMed1996

Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants.

Glutamate decarboxylase (GAD) catalyzes the decarboxylation of glutamate to CO2 and gamma-aminobutyrate (GABA). GAD is ubiquitous in prokaryotes and eukaryotes, but only plant GAD has been shown to bind calmodulin (CaM). Here, we assess the role of the GAD CaM-binding domain in vivo. Transgenic tobacco plants expressing a mutant petunia GAD lacking the CaM-binding domain (GADdeltaC plants) exhibit severe morphological abnormalities, such as short stems, in which cortex parenchyma cells fail to elongate, associated with extremely high GABA and low glutamate levels. The morphology of transgenic plants expressing the full-length GAD (GAD plants) is indistinguishable from that of wild-type (WT) plants. In WT and GAD plant extracts, GAD activity is inhibited by EGTA and by the CaM antagonist trifluoperazine, and is associated with a CaM-containing protein complex of approximately 500 kDa. In contrast, GADdeltaC plants lack normal GAD complexes, and GAD activity in their extracts is not affected by EGTA and trifluoperazine. We conclude that CaM binding to GAD is essential for the regulation of GABA and glutamate metabolism, and that regulation of GAD activity is necessary for normal plant development. This study is the first to demonstrate an in vivo function for CaM binding to a target protein in plants.

Calmodulin↗
PubMed1996

Comparative effect of ursodeoxycholic acid and calcium antagonists on the binding, uptake and degradation of LDL in isolated hamster hepatocytes.

We have shown that ursodeoxycholic acid (UDCA) stimulates low density lipoprotein (LDL) metabolism (Biochem. J. 280 (1991) 589), as well as calcium mobilization (Am. J. Physiol. 264 (1993) G243) in isolated hepatocytes. Therefore, the effect of UDCA and that of different calcium antagonists on hepatic LDL metabolism was compared. Isolated hamster hepatocytes were incubated at 37 degrees C for 60 min in the presence of 125I-labelled hamster LDL, increasing concentrations (25-100 microM) of verapamil, nifedipine, and diltiazem, respectively, and with or without 700 microM ursodeoxycholic acid (UDCA). At concentrations up to 100 microM, neither verapamil nor nifedipine significantly affected cell associated LDL, but both agents decreased LDL degradation in a dose-dependent manner, with almost total inhibition with 100 microM of either agent. In contrast, 25 microM diltiazem stimulated LDL binding and uptake, with a maximum increase of 15-20% of control, while 50 and 100 microM diltiazem stimulated LDL degradation by 50 and 100%, respectively. UDCA increased native LDL binding and uptake by 20%, and degradation by 50%. None of the agents tested had any effect on the binding, uptake and degradation of methylated LDL. The increased hepatic LDL uptake induced by UDCA was not altered in the presence of calcium antagonists, while the increased degradation of LDL by UDCA was abolished by the addition of 50 microM of either verapamil or nifedipine. However, 100 microM diltiazem and 700 microM UDCA stimulated LDL degradation without any additive effect. These studies show that different calcium antagonists have differential effects on hepatic LDL metabolism. The similarities between the effect of diltiazem and UDCA on LDL metabolism and the absence of any additive effect, suggest that these two agents have a similar mechanism of action, which may involve the integration of both agents into the plasma membrane lipid bilayer.

Animals↗
PubMed1996

Activation of a recombinant petunia glutamate decarboxylase by calcium/calmodulin or by a monoclonal antibody which recognizes the calmodulin binding domain.

To date, only plants have been shown to possess a form of glutamate decarboxylase (GAD) that binds calmodulin. In the present study, a recombinant calmodulin-binding 58-kDa petunia GAD produced in Escherichia coli was purified to homogeneity using calmodulin-affinity chromatography, and its responsiveness to calcium and calmodulin was examined in vitro. At pH 7.0-7.5, the purified recombinant enzyme was essentially inactive in the absence of calcium and calmodulin, but it could be stimulated to high levels of activity (Vmax = 30 micromol of CO2 min-1 mg of protein-1) by the addition of exogenous calmodulin (K0.5 = 15 nM) in the presence of calcium (K0.5 = 0.8 microM). Neither calcium nor calmodulin alone had any effect on GAD activity. Recombinant GAD displayed hyperbolic kinetics at pH 7.3 (Km = 8.2 mM). A monoclonal antibody directed against the carboxyl-terminal region, which contains the calmodulin-binding domain of GAD, was able to fully activate GAD in a dose-dependent manner in the absence of calcium and calmodulin, whereas an antibody recognizing an epitope outside of this region was unable to activate GAD. This study provides the first evidence that the activity of the purified 58-kDa GAD polypeptide is essentially calcium/calmodulin-dependent at physiological pH. Furthermore, activation of GAD by two different proteins that interact with the calmodulin-binding domain, a monoclonal antibody or calcium/calmodulin, suggests that this domain plays a major role in the regulation of plant GAD activity.

Amino Acid Sequence↗
PubMed1996

Regulation of taurocholate and ursodeoxycholate uptake in hamster hepatocytes by Ca(2+)-mobilizing agents.

In isolated hamster hepatocytes, the Ca2+ ionophore A-23187 immediately decreased the uptake rate of taurocholic acid (TCA) by 60-70%, whereas it slowly inhibited that of ursodeoxycholic acid (UDCA) by a maximum of 35-45%, with an inhibition constant (Ki) of 0.36 and 1.93 microM, respectively. In contrast to ionomycin, which mimicked the effect of A-23187, vasopressin inhibited the bile acid uptake rate by 40 and 45%, respectively, only after a 5- to 10-min preincubation. The Na(+)-dependent bile acid transport was exclusively inhibited by these agents, and this inhibition was independent of extracellular Ca2+. However, intracellular Ca2+ depletion with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid or chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid resulted in 40-50% inhibition of the uptake rate of both bile acids. The exogenous protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), but not the nonactive 4 alpha-phorbol, significantly inhibited TCA uptake rate. Although both A-23187 and ionomycin immediately increased and decreased the cellular Na+ and K+ concentration, respectively, neither vasopressin nor PMA had a significant effect on the cellular concentration of these cations, even after a 10-min incubation. Furthermore, the effect of A-23187 and ionomycin on TCA uptake and Na+ flux, respectively, disappeared after a 40-min preincubation, and additional ionophore remained without effect. However, after a 40-min incubation with A-23187, PMA was still able to inhibit TCA uptake. Therefore, A-23187 and ionomycin transiently inhibited Na(+)-dependent uptake of both TCA and UDCA, in part because of transient alteration of the cellular Na+ and K+ concentration. Vasopressin and PMA inhibited Na(+)-dependent bile acid uptake, at least in part, through protein kinase C activation.

Animals↗
PubMed1995

PCR-generated cDNA library of transition-stage maize embryos: cloning and expression of calmodulin genes during early embryogenesis.

One hundred maize zygotic embryos microdissected at the transition stage were used to construct a cDNA library after non-selective PCR (NS-PCR) amplification of whole cDNA populations. The library contains 2.3 x 10(5) recombinants and two different calmodulin cDNAs were cloned using a heterologous probe from petunia. Calmodulin expression was confirmed throughout maize embryogenesis at the mRNA, amplified cDNA and protein levels. Sequence analysis suggests a maize origin for both clones and negligible nucleotide changes linked to PCR. This library is the first described for early plant embryos and represents a breakthrough to isolate genes involved in embryo differentiation.

Amino Acid Sequence↗
PubMed1995

Studies on the mechanism of the ursodeoxycholic acid-induced increase in hepatic low-density lipoprotein binding.

Previously, we have shown, in golden Syrian hamsters, that chronic feeding of ursodeoxycholic acid (UDCA), in contrast to that of its 7 alpha-epimer, chenodeoxycholic acid (CDCA), produced a significant increment in hepatic low-density lipoprotein (LDL) uptake, despite similar suppression of bile acid synthesis by both bile acids. Evidence for a direct effect of this bile acid on hepatic LDL metabolism was shown in vitro, with isolated hamster hepatocytes, suggesting that this effect was unique to UDCA and specific for receptor-mediated LDL catabolism. The aim of the present study was to define the cellular mechanism(s) associated with this phenomenon, using male golden Syrian hamsters. Regardless of chronic exposure of the liver to either UDCA or CDCA, acute incubation with UDCA consistently resulted in an increase of LDL binding to isolated hepatocytes by 15 to 40%. Furthermore, chronic treatment with either UDCA or CDCA did not result in alterations in lipoprotein particle composition. Likewise, incubation of hepatocytes with UDCA was not associated with a change of the membrane lipid composition. In isolated liver membrane fractions, UDCA increased both the maximum number of LDL binding sites and the affinity constant for LDL by around 35%, suggesting an interaction of UDCA with the LDL receptor, at the plasma membrane level, independent of an effect on receptor cycling. The results of the studies support a role for UDCA in the recruitment of cryptic LDL receptors from a cellular membrane pool, possibly due to the unique localization of UDCA in the plasma membrane lipid bilayer.

Animals↗
PubMed1995

Molecular and biochemical analysis of calmodulin interactions with the calmodulin-binding domain of plant glutamate decarboxylase.

We previously provided what to our knowledge is the first evidence that plant glutamate decarboxylase (GAD) is a calmodulin (CaM)-binding protein. Here, we studied the GAD CaM-binding domain in detail. A synthetic peptide of 26 amino acids corresponding to this domain forms a stable complex with Ca2+/CaM with a 1:1 stoichiometry, and amino acid substitutions suggest that tryptophan-485 has an indispensable role in CaM binding. Chemical cross-linking revealed specific CaM/GAD interactions even in the absence of Ca2+. However, increasing KCI concentrations or deletion of two carboxy-terminal lysines abolished these interactions but had a mild effect on CaM/GAD interactions in the presence of Ca2+. We conclude that in the presence of Ca(2+)-hydrophobic interactions involving tryptophan-485 and electrostatic interactions involving the carboxy-terminal lysines mediate CaM/GAD complex formation. By contrast, in the absence of Ca2+, CaM/GAD interactions are essentially electrostatic and involve the carboxy-terminal lysines. In addition, a tryptophan residue and carboxy-terminal lysines are present in the CaM-binding domain of an Arabidopsis GAD. Finally, we demonstrate that petunia GAD activity is stimulated in vitro by Ca2+/CaM. Our study provides a molecular basis for Ca(2+)-dependent CaM/GAD interactions and suggests the possible occurrence of Ca(2+)-independent CaM/GAD interactions.

Amino Acid Sequence↗
PubMed1995

Ursodeoxycholic acid inhibits glucagon-induced cAMP formation in hamster hepatocytes: a role for PKC.

The effect of bile acids on adenosine 3',5'-cyclic monophosphate (cAMP) synthesis was investigated in isolated hamster hepatocytes. Bile acids had no direct effect on cAMP production. However, ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid inhibited, by approximately 45%, cAMP formation induced by concentrations of glucagon greater than 1 nM, with a respective half-maximum inhibitory effect observed at 4 +/- 2 microM. Similar inhibition was observed with phorbol 12-myristate 13-acetate (PMA). Chenodeoxycholic, murocholic, and taurodeoxycholic acids were the next most potent bile acids. Taurolithocholic acid was 100-fold less potent than UDCA, whereas both ursocholic and taurocholic acids had no effect at concentrations up to 0.5 mM. Neither bile acids nor PMA affected either the binding of glucagon to its receptor, the cAMP-dependent phosphodiesterase, adenylate cyclase, or the inhibitory and stimulatory (Gs) GTP-binding proteins. The inhibitory effect of PMA and UDCA on glucagon-induced cAMP synthesis was abolished in the presence of the protein kinase C (PKC) inhibitor, staurosporine. Furthermore, UDCA induced PKC translocation from cytosol to membrane and stimulated phosphorylation of an 80-kDa protein substrate for PKC. In conclusion, mediated by PKC activation, bile acids inhibit glucagon-induced cAMP synthesis by uncoupling the glucagon receptor and Gs.

Angiotensin II↗
PubMed1995

Alteration of cAMP-mediated hormonal responsiveness by bile acids in cells of nonhepatic origin.

The present study was undertaken to determine whether bile acids could inhibit hormone-induced adenosine 3',5'-cyclic monophosphate (cAMP) production in cells of nonhepatic origin, as previously reported in the liver [Bouscarel et al., Am. J. Physiol. 268 (Gastrointest. Liver Physiol. 31): G300-G310, 1995]. The bile acids, ursodeoxycholic acid (UDCA), chenodeoxycholic acid, and deoxycholic acid inhibited prostaglandin E1 (PGE1)- and isoproterenol-induced cAMP production by 40-60% in human skin fibroblasts and human umbilical vein endothelial cells, respectively, to a similar extent as that observed in the liver. However, in both models, the taurine conjugates of these respective dihydroxy bile acids were without effect. After permeabilization of fibroblasts with saponin, UDCA, and its taurine conjugates inhibited hormone-induced cAMP production in a similar manner with a maximum inhibition of approximately 55%. The other taurine-conjugated dihydroxy bile acids were also able to inhibit PGE1-induced cAMP production. Furthermore, in human fibroblasts, UDCA was taken up in a dose- and time-dependent manner, whereas there was no uptake of taurocholic acid, even after 30 min of incubation. Therefore these results suggest that plasma membrane crossing of bile acids is a requirement for their inhibition of hormone-induced cAMP production. The ability of certain bile acids to affect hormone-induced cAMP production in extrahepatic tissues may be of pathophysiological significance in certain cholestatic liver diseases.

Alprostadil↗
PubMed1995

The role of sodium in the uptake of ursodeoxycholic acid in isolated hamster hepatocytes.

The uptake of ursodeoxycholic acid (UDCA) was studied in isolated hamster hepatocytes. The uptake was rapid and linear up to 60 seconds for each concentration studied. When the uptake rate was plotted against UDCA concentration, the curve was nonlinear, indicating both saturable and nonsaturable uptake mechanisms. The nonsaturable process had a diffusion constant of 0.01 nmol.s-1.g of cell.mumol/L-1. The saturable component was characterized by a maximum rate of uptake (Vmax) of 5.68 nmol.s-1.g of cell-1 and a Michaelis constant (Km) of 224 mumol/L. In the presence of monensin, ouabain, and amiloride, the uptake of UDCA was significantly decreased by 35% to 55%, whereas the sodium-independent uptake of UDCA was not affected by either monensin or amiloride, thereby confirming sodium dependence of UDCA uptake. The sodium-dependent uptake of UDCA was characterized by a Vmax and a Km of 1.57 nmol.s-1.g of cell-1 and 46 mumol/L, respectively. The rate of uptake of UDCA was maximal at extracellular sodium concentrations > or = 20 mmol/L. Furthermore, the uptake of UDCA was competitively inhibited by both taurocholic acid and cholic acid with an inhibitory constant (Ki) of 60 mumol/L and 48 mumol/L, respectively. Finally, 1 mmol/L of 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) inhibited solely the sodium-dependent uptake of cholic acid and UDCA. These findings confirm that the hepatocellular uptake of UDCA involves, at least in part, a sodium-dependent, ouabain, amiloride, and DIDS-sensitive transporter.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗