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J L Boyer

Publications and source records attributed to J L Boyer.

At least 73 records · Page 4Linked to original sources
PubMed1998

Maternal cholestasis does not affect the ontogenic pattern of expression of the Na+/taurocholate cotransporting polypeptide (ntcp) in the fetal and neonatal rat liver.

To assess the effects of cholestasis during pregnancy on fetal and neonatal mRNA expression, protein mass, and function of the Na+/taurocholate cotransporting polypeptide (Ntcp), common bile duct ligation (BDL) was performed in pregnant rats on day 14 of pregnancy (maternal cholestasis [MC] group), and livers were harvested at days 20 and 21 of fetal life, as well as at days 4, 7, 14, 21, and 28 after birth. Sham-operated rats and their litters were used as controls. Ntcp steady-state mRNA levels, protein mass, and function were determined by Northern blotting, immunoblotting, and taurocholate (TC) transport studies in isolated short-term cultured hepatocytes, respectively. In addition, protein mass and function of the organic anion transporting polypeptide (Oatp1), another sinusoidal bile acid transporter, were studied at 4 weeks of age. The majority of pregnant cholestatic rats (94%) were able to carry pregnancy to term. Body and liver weights of the offspring from the MC group were lower than those from sham-operated animals at all postnatal time points. Ntcp steady-state mRNA levels, protein mass, and function were unaffected by MC. The ontogenic pattern of expression was identical in offspring from MC and controls with detection of the Ntcp mRNA at day 21 of fetal life. There was a significant increase in mRNA postnatally, reaching adult levels by 7 days of age. Protein mass and function of Ntcp as well as of Oatp1 were similar in offspring from MC and control groups at 4 weeks of age. In conclusion, maternal obstructive cholestasis during the last third of pregnancy does not affect the fetal/neonatal expression of the basolateral bile acid transporters, Ntcp and Oatp1. This suggests that the impaired bile acid excretion described in this experimental model is not related to altered uptake of bile acids in the affected neonate.

Animals↗
PubMed1998

Nitric oxide and guanosine 3',5'-cyclic monophosphate stimulate bile secretion in isolated rat hepatocyte couplets, but not in isolated bile duct units.

Nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cGMP) have recently been shown to stimulate bile acid-independent bile flow in the isolated perfused rat liver (IPRL). However, the cellular origin and mechanisms of this choleresis have not yet been determined. To address these questions, we examined the effects of NO and cGMP on bile secretion in isolated rat hepatocyte couplets (IRHC) and in isolated bile duct units (IBDU), both of which are isolated cell systems in which cell polarity is maintained and secretion can be measured directly. Changes in the area of the canalicular and ductular lumens were determined in IRHC and IBDU, respectively, as indicators of the rate of fluid secretion using video microscopy. In addition, Cl-/HCO3- exchanger activity in IBDU was evaluated by measuring changes in intracellular pH (pHi) after Cl- removal/readmission by microfluorometric methods. In the presence of HCO3-, both the NO donor, S-nitroso-acetyl-penicillamine (SNAP), and the cell-permeant cGMP analogue, dibutyryl cGMP (DBcGMP), stimulated canalicular bile secretion (P <.05), as did the cell-permeant cAMP analogue, dibutyryl cAMP (DBcAMP) (P <.05). Removal of HCO3- from the buffer completely abolished the choleretic effects of DBcGMP, but had no effect on NO-induced choleresis. In contrast, secretion in IBDU was not stimulated following incubations with SNAP or DBcGMP over 30 minutes, whereas DBcAMP and secretin, a cholangiocyte secretagogue and cAMP agonist, both had a marked effect on ductular secretion over this same time interval (P <.05). SNAP also had no effect on Cl-/HCO3- exchanger activity in IBDU, and inhibition of endogenous NO synthesis by NG-monomethyl-L-arginine (L-NMMA) did not alter secretin-induced stimulation of ductular bile secretion and Cl-/HCO3- exchanger activity. In summary, NO and cGMP stimulate bile secretion exclusively at the the level of hepatocytes, whereas cAMP mediates choleresis at both hepatocyte and bile duct levels. These findings may have important implications for the regulation of ductular bile secretion by hormones and neuropeptides, as well as under pathological conditions with increased hepatic NO synthesis.

Animals↗
PubMed1998

Role of glutathione in hepatic bile formation during reperfusion after cold ischemia of the rat liver.

BACKGROUND/AIMS: Liver reperfusion following cold ischemia is frequently associated with diminished bile flow in patients undergoing liver transplantation. Glutathione is a major determinant of bile-acid independent bile flow, and the effects of cold ischemia on biliary glutathione excretion are unknown. METHODS: We examined the effects of cold ischemia (University of Wisconsin solution (4 degrees C), 24 h) with subsequent reperfusion (100 min) on biliary glutathione excretion in a recirculating system. Since glutathione might represent an important antioxidant within the biliary tract and oxidative stress in the biliary tract during reperfusion could contribute to the pathogenesis of bile duct injury after liver transplantation, we also assessed bile duct morphology in reperfused livers of mutant TR- -rats, in whom biliary excretion of glutathione is already impaired. RESULTS: Hepatic bile formation was diminished in reperfused Wistar rat livers after cold ischemia. Biliary glutathione concentrations and output were significantly decreased and correlated with postischemic changes in bile secretion. An increased biliary oxidized glutathione/glutathione ratio, indicating oxidative stress, was detected only immediately after the onset of reperfusion. Basal bile flow rates in TR- -rat livers which were already markedly reduced in control-perfused livers, decreased further during the early but not the later reperfusion period. Reperfusion of both Wistar and TR- -rat livers was not associated with electron microscopic evidence of bile duct damage. CONCLUSIONS: We conclude that impaired biliary excretion of glutathione contributes to decreased bile flow after cold ischemia. The absence of biliary glutathione does not appear to promote ultrastructural evidence of bile duct injury during reperfusion in the isolated perfused rat liver.

Adenosine↗
PubMed1998

Competitive and selective antagonism of P2Y1 receptors by N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate.

The antagonist activity of N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate (N6MABP) has been examined at the phospholipase C-coupled P2Y1 receptor of turkey erythrocyte membranes. N6MABP antagonized 2MeSATP-stimulated inositol phosphate hydrolysis with a potency approximately 20 fold greater than the previously studied parent molecule, adenosine 3',5'-bisphosphate. The P2Y1 receptor antagonism observed with N6MABP was competitive as revealed by Schild analysis (pK(B) = 6.99 +/- 0.13). Whereas N6MABP was an antagonist at the human P2Y1 receptor, no antagonist effect of N6MABP was observed at the human P2Y2, human P2Y4 or rat P2Y6 receptors.

Adenosine Diphosphate↗
PubMed1998

Intracellular pH regulation in bombesin-stimulated secretion in isolated bile duct units from rat liver.

Bombesin, a neuropeptide, stimulates fluid and HCO-3 secretion from cholangiocytes, but the underlying mechanisms are poorly understood. In this study, we aimed to examine the effects of bombesin on ion transport processes involved in the regulation of intracellular pH (pHi) and HCO-3 secretion in polarized cholangiocytes. Isolated bile duct units from normal rat liver were used to measure pHi by 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein 495 nm-to-440 nm dual ratio methods. Bombesin increased Cl--HCO-3 exchange activity but did not affect basal pHi or the activities of Na+/H+ exchange or Na+-HCO-3 symport. Depolarization of cholangiocytes increased basal pHi and the activity of Cl-/HCO-3 exchange, suggesting that an electrogenic Na+-HCO-3 symport might function as a counterregulatory pHi mechanism. Na+-independent acid-extruding mechanisms were not observed. We conclude that bombesin stimulates biliary secretion from cholangiocytes by activating luminal Cl-/HCO-3 exchange, which may be coupled to basolateral electrogenic Na+-HCO-3 symport.

Animals↗
PubMed1998

Cyclic AMP stimulates sorting of the canalicular organic anion transporter (Mrp2/cMoat) to the apical domain in hepatocyte couplets.

The canalicular membrane of rat hepatocytes contains an ATP-dependent multispecific organic anion transporter, also named multidrug resistance protein 2, that is responsible for the biliary secretion of several amphiphilic organic anions. This transport function is markedly diminished in mutant rats that lack the transport protein. To assess the role of vesicle traffic in the regulation of canalicular organic anion transport, we have examined the redistribution of the transporter to the canalicular membrane and the effect of cAMP on this process in isolated hepatocyte couplets, which retain secretory polarity. The partial disruption of cell-cell contact, due to the isolation procedure, leaves the couplet with both remnant apical membranes, as a source of apical proteins, and an intact apical domain and lumen, to which these proteins are targeted. The changes in distribution of the transporter were correlated to the apical excretion of a fluorescent substrate, glutathione-methylfluorescein. The data obtained in this study show that the transport protein, endocytosed from apical membrane remnants, first is redistributed along the basolateral plasma membrane. Then it is transcytosed to the remaining apical pole in a microtubule-dependent fashion, followed by the fusion of transporter-containing vesicles with the apical membrane. The cAMP analog dibutyrylcAMP stimulates all three steps, resulting in increased apically located transport protein, glutathione-methylfluorescein transport activity and apical membrane circumference. These findings indicate that the organic anion transport capacity of the apical membrane in hepatocyte couplets is regulated by cAMP-stimulated sorting of the multidrug resistance protein 2 to the apical membrane. The relevance of this phenomenon for the intact liver is discussed.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Agonist action of adenosine triphosphates at the human P2Y1 receptor.

The agonist selectivity for adenosine di- and triphosphates was determined for the human P2Y1 receptor stably expressed in human 1321N1 astrocytoma cells and was studied under conditions in which nucleotide metabolism was both minimized and assessed. Cells were grown at low density on glass coverslips, encased in a flow-through chamber, and continuously superfused with medium, and Ca2+ responses to nucleotides were quantified. Superfusion with high performance liquid chromatographically purified ADP, ATP, 2-methylthio-ADP, and 2-methylthio-ATP resulted in rapid Ca2+ responses, with EC50 values of 10 +/- 5, 304 +/- 51, 2 +/- 1, and 116 +/- 50 nM, respectively. Similar peak responses were observed with maximal concentrations of these four agonists and with the hydrolysis-resistant adenine nucleoside triphosphate adenosine-5'-O-(3-thiotriphosphate). No conversion of [3H]ATP to [3H]ADP occurred under these conditions. Similar full agonist activities of ATP, 2-methylthio-ATP, and ADP were observed in human embryonic kidney 293 cells, which natively express the P2Y1 receptor. In contrast to these results, Leon et al. [FEBS Lett 403:26-30 (1997)] and Hechler et al. [Mol Pharmacol 53:727-733 (1998)] recently reported that, whereas ADP and 2-methylthio-ADP were agonists, ATP and 2-methylthio-ATP were weak antagonists in studies of the human P2Y1 receptor expressed in human Jurkat cells. To assess whether differences in the degree of receptor reserve might explain this discrepancy of results, P2Y1 receptor-expressing 1321N1 cells were incubated for 24 hr with adenosine-5'-O-(2-thiodiphosphate), with the goal of down-regulating the level of functional receptors. Pretreatment with adenosine-5'-O-(2-thiodiphosphate) resulted in a 10-fold rightward shift in the concentration-effect curve for ADP; in contrast, the agonist activity of ATP was completely abolished. Taken together, our results indicate that adenosine di- and triphosphates are agonists at the human P2Y1 receptor. However, the intrinsic efficacy of ATP is less than that of ADP, and the capacity of ATP to activate second messenger responses through this receptor apparently depends on the degree of P2Y1 receptor reserve.

Adenosine Diphosphate↗
PubMed1997

Selection of peptides that functionally replace a zinc finger in the Sp1 transcription factor by using a yeast combinatorial library.

We have developed a strategy for the identification of peptides able to functionally replace a zinc finger domain in a transcription factor. This strategy could have important ramifications for basic research on gene regulation and for the development of therapeutic agents. In this study in yeast, we expressed chimeric proteins that included a random peptide combinatorial library in association with two zinc finger domains and a transactivating domain. The library was screened for chimeric proteins capable of activating transcription from a target sequence in the upstream regulatory regions of selectable or reporter genes. In a screen of approximately 1.5 x 10(7) transformants we identified 30 chimeric proteins that exhibited transcriptional activation, some of which were able to discriminate between wild-type and mutant DNA targets. Chimeric library proteins expressed as glutathione S-transferase fusions bound to double-stranded oligonucleotides containing the target sequence, suggesting that the chimeras bind directly to DNA. Surprisingly, none of the peptides identified resembled a zinc finger or other well-known transcription factor DNA binding domain.

Amino Acid Sequence↗
PubMed1997

ATP regulation of a swelling-activated osmolyte channel in skate hepatocytes.

Hypotonic swelling of isolated skate hepatocytes activates a regulatory volume decrease (RVD) which is achieved in part by the release of taurine and other intracellular organic osmolytes. Volume-activated taurine efflux appears to be mediated by an anion channel that exhibits a taurine/chloride permeability ratio of approximately 0.2. Of significance, this channel was shown to be regulated by intracellular nucleotide. When intracellular ATP was decreased to about 50% of control levels, channel opening was completely prevented. Many putative ion channel blockers were found to inhibit the channel indirectly, by depleting intracellular ATP, rather than by directly interacting with the channel. Investigators using these channel blockers in whole cell preparations should be aware of this alternative mechanism. Cell swelling-activated taurine efflux was also inhibited by HgCl2, DIDS, and pyridoxal 5-phosphate, at concentrations of these agents that had no effect on intracellular ATP levels, suggesting additional mechanisms of inhibition and regulation of the volume-sensitive osmolyte channels.

Adenosine Triphosphate↗
PubMed1997

Nitric oxide donors stimulate bile flow and glutathione disulfide excretion independent of guanosine 3',5'-cyclic [corrected] monophosphate in the isolated perfused rat liver.

Nitric oxide (NO) modulates several metabolic functions in hepatocytes, but the role of NO in bile secretion has not been clearly defined. In the present study, we examined the effects of NO on bile flow and biliary HC03- and glutathione excretion in the isolated perfused rat liver and assessed the role of guanosine 3',5'-cyclic monophosphate (cGMP) in mediating these effects. The NO donors sodium nitroprusside (SNP) and S-nitroso-acetyl-penicillamine stimulated bile flow and increased both HCO3- and glutathione excretion. Increases in bile flow were linearly related to increases in biliary glutathione concentration and output (P < .0001), which were almost entirely caused by glutathione disulfide, whereas the excretion of reduced glutathione remained unchanged. NO donors increased cGMP concentrations in bile and perfusate, and the membrane-permeant cGMP analogue dibutyryl cGMP was also found to stimulate bile flow and HCO3- excretion. However, in contrast to the NO donors, dibutyryl cGMP did not increase glutathione excretion. Furthermore, the NO donors failed to stimulate bile flow in mutant TR- rats in which the canalicular transport of glutathione and glutathione conjugates is deficient, although dibutyryl cGMP increased bile flow and HCO3- excretion in the mutant rats as in normals. These findings indicate that exogenous sources of NO increase bile acid-independent bile flow by stimulating glutathione disulfide excretion, effects that are independent of cGMP.

Animals↗
PubMed1997

Expression of the rat liver Na+/taurocholate cotransporter is regulated in vivo by retention of biliary constituents but not their depletion.

Expression and function of the hepatic Na+/taurocholate cotransporter (ntcp) are down-regulated in several models of experimental cholestasis. To test whether retention and/or depletion of biliary constituents are involved in ntcp regulation, ntcp expression was quantified in several animal models with altered levels of these constituents. In choledochocaval fistula rats (CCF) (retention model), ntcp mRNA expression specifically declined after 1 and 3 days by 76 +/- 4% (P < .005) and 31 +/- 9% (P < .05), respectively, returning to control levels by 7 days. However, protein expression as assessed by Western blotting remained unchanged for up to 7 days of CCF. In rats with bile fistulas (depletion model) for 0.5, 1, 2, 4, and 7 days, both ntcp protein and mRNA expression remained unaltered. Infusion of either taurocholate or taurochenodeoxycholate for 12 hours also did not effect ntcp mRNA expression in intact animals, probably because of its inability to increase serum and intrahepatic bile acid levels. In rats with selective bile duct ligation (SBDL), ntcp mRNA levels were down-regulated by 40 +/- 10% (P < .05) only after 12 and 24 hours in ligated lobes, and mRNA levels returned to control values in these lobes after 2 and 4 days. ntcp mRNA expression remained unchanged in the nonobstructed lobes at any time. When data from CCF and SBDL rats were combined, serum bile acids correlated linearly with ntcp mRNA (r = .62, P < .0005) over a 0 to 110-micromol/L range. Our results indicate that ntcp is constitutively expressed and remains uneffected by either depletion or increased flux of biliary constituents. However, retention of biliary constituents results in rapid down-regulation of ntcp mRNA, consistent with the concept that hepatocytes may be protected from bile acid toxicity during cholestasis by this mechanism.

Animals↗
PubMed1997

Endotoxin impairs biliary glutathione and HCO3- excretion and blocks the choleretic effect of nitric oxide in rat liver.

Cholestasis in patients with sepsis has been attributed to the effects of endotoxin (lipopolysaccharides, LPS) and LPS-induced cytokines, which are also potent stimulators of systemic and hepatic nitric oxide (NO) synthesis. NO donors stimulate bile acid-independent bile flow in normal rat liver, but the effects of LPS-induced NO on bile formation remain unclear. To address this question we examined the effects of NO and its mediator guanosine 3',5'-cyclic monophosphate (cGMP) on bile flow and biliary HCO3- and glutathione excretion in isolated perfused rat livers (IPRL) from LPS-treated rats. Portal and systemic NO2- + NO3- plasma levels were increased 47-fold in LPS-treated rats and were also elevated in perfusate (6-fold) and bile (9-fold) after isolating and perfusing livers from these animals. Bile flow, HCO3-, and glutathione output were decreased by 33%, 25%, and 81% in these IPRL, respectively. Stimulation of NO synthesis with L-arginine or inhibition of inducible NO synthesis with aminoguanidine did not change bile flow, although pretreatment with aminoguanidine inhibited NO production by 85%. Moreover, the choleretic effects of infusions of the NO donors sodium nitroprusside (SNP) and S-nitroso-acetyl-penicillamine were markedly reduced in endotoxemic IPRL compared with normal controls, and SNP-induced HCO3- and glutathione excretion were reduced by 61% and 86%, respectively. SNP-induced cyclic GMP production was 2.3-fold lower than in normals, but the choleretic effect of dibutyryl cGMP was only slightly reduced in endotoxemic livers. These findings indicate that LPS reduces bile acid-independent bile flow primarily by inhibiting biliary excretion of glutathione and to a lesser extent HCO3-, whereas LPS-induced NO does not modulate bile formation in endotoxemia. Thus, impairment of the major determinants of bile acid-independent bile flow by LPS may contribute significantly to the pathogenesis of the cholestasis of sepsis.

Animals↗
PubMed1997

Extrahepatic biliary obstruction impairs microvascular perfusion and increases leukocyte adhesion in rat liver.

To determine if disturbances of the liver microcirculation may be of pathophysiological relevance for liver damage during acute biliary obstruction, we studied the effects of bile duct ligation (BDL) on hepatic microhemodynamics and leukocyte adhesion in rat liver in vivo. Male Wistar rats were subjected to BDL for 3 days and 7 days, respectively. Sham-operated controls underwent laparotomy without BDL. After 3 days, intravital fluorescence microscopy (IVM) and hydrogen gas (H2) clearance were performed to study hepatic microvascular perfusion. Furthermore, leukocyte-endothelial cell interactions were assessed by IVM. Intercellular adhesion molecule 1 (ICAM-1) protein expression was studied by Western blot analysis and tissue immunofluorescence after 3 and 7 days, respectively. Analysis of microvascular perfusion by IVM revealed a marked impairment of sinusoidal perfusion after 3 days. Assessment of H2 clearance confirmed that overall hepatic microvascular perfusion was decreased. In addition, increased leukocyte adhesion in sinusoids and venules could be observed. A concomitant increase of ICAM-1 expression in liver tissue was also noted within the first week after BDL. Our results show that BDL is followed by a marked depression of the hepatic microcirculation and increased leukocyte adhesion in vivo within 3 to 7 days. Together, these findings suggest that deficits in microvascular perfusion and increased neutrophil infiltration may represent a potential source of liver injury during acute biliary obstruction.

Animals↗