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J L Boyer

Publications and source records attributed to J L Boyer.

At least 55 records · Page 3Linked to original sources
PubMed1999

Modulation of protein kinase C by taurolithocholic acid in isolated rat hepatocytes.

The protein kinase C (PKC) family of isoenzymes plays a key role in the regulation of hepatocellular secretion. The hydrophobic and cholestatic bile acid, taurolithocholic acid (TLCA), acts as a potent Ca++ agonist in isolated hepatocytes. However, its effect on PKC isoforms has not been elucidated. Here we investigate the effects of TLCA at low micromolar concentrations on the distribution of PKC isoforms and on membrane-associated PKC activity. The distribution of PKC isoforms was determined in isolated rat hepatocytes in short-term culture using Western blotting and immunofluorescence techniques. PKC activity was measured radiochemically. TLCA (10 micromol/L) induced selective translocation of epsilon-PKC by 47.9% +/- 20.5% (P <.02 vs. controls; n = 7), but not of alpha-, delta-, and zeta-PKC to the hepatocellular membranes, whereas the phorbol ester, phorbol 12-myristate 13-acetate (PMA) (1 micromol/L) caused translocation of all mobile isoforms, alpha-, delta-, and epsilon-PKC, as shown by immunoblotting. Immunofluorescence studies demonstrated selective translocation of epsilon-PKC to the canalicular membranes of isolated rat hepatocyte couplets by TLCA (10 micromol/L), but predominant translocation to intracellular and basolateral membranes by PMA (1 micromol/L). Both TLCA (10 micromol/L) and PMA (1 micromol/L) stimulated membrane-bound PKC activity by 60.5% +/- 45. 8% (P <.05 vs. controls; n = 5) and 72.4% +/- 37.2% (P <.05; n = 5), respectively. TLCA at lower concentrations (5 micromol/L) was less effective. Because activation of epsilon-PKC has been associated with impairment of vesicle-mediated targeting and insertion of membrane proteins in secretory cells, it is attractive to speculate that TLCA reduces bile secretory capacity of the liver cell by activation of epsilon-PKC at the canalicular membrane.

Animals↗
PubMed1999

Biliary bile acids in primary biliary cirrhosis: effect of ursodeoxycholic acid.

Bile acid composition in fasting duodenal bile was assessed at entry and at 2 years in patients with primary biliary cirrhosis (PBC) enrolled in a randomized, double-blind, placebo-controlled trial of ursodeoxycholic acid (UDCA) (10-12 mg/kg/d) taken as a single bedtime dose. Specimens were analyzed by a high-pressure liquid chromatography method that had been validated against gas chromatography. Percent composition in bile (mean +/- SD) for 98 patients at entry for cholic (CA), chenodeoxycholic (CDCA), deoxycholic (DCA), lithocholic (LCA), and ursodeoxycholic (UDCA) acids, respectively, were 57.4 +/- 18.6, 31.5 +/- 15.5, 8.0 +/- 9.3, 0.3 +/- 1.0, and 0.6 +/- 0.9. Values for CA were increased, whereas those for CDCA, DCA, LCA, and UDCA were decreased when compared with values in normal persons. Bile acid composition of the major bile acids did not change after 2 years on placebo medication. By contrast, in patients receiving UDCA for 2 years, bile became enriched with UDCA on average to 40.1%, and significant decreases were noted for CA (to 32.2%) and CDCA (to 19.5%). No change in percent composition was observed for DCA and LCA. Percent composition at entry and changes in composition after 2 years on UDCA were similar in patients with varying severity of PBC. In patients whose bile was not enriched in UDCA (entry and placebo-treated specimens), CA, CDCA, DCA, and the small amount of UDCA found in some of these specimens were conjugated to a greater extent with glycine (52%-64%) than with taurine (36%-48%). Treatment with UDCA caused the proportion of all endogenous bile acids conjugated with glycine to increase to 69% to 78%, while the proportion conjugated with taurine (22%-31%) fell (P <.05). Administered UDCA was also conjugated predominantly with glycine (87%).

Bile↗
PubMed1999

The effect of ursodeoxycholic acid on the florid duct lesion of primary biliary cirrhosis.

The frequency with which florid duct lesions are seen in needle-biopsy specimens of the liver was assessed in patients with primary biliary cirrhosis (PBC) enrolled in a 2-year randomized, double-blind, placebo-controlled trial of ursodeoxycholic acid (UDCA) versus placebo. Paired biopsy specimens obtained at entry and after 2 years on medication were reviewed blindly and mostly simultaneously by a panel of 5 hepatopathologists who, earlier, had characterized the florid duct lesion, which has been well described in the pathology literature. Florid duct lesions at entry were identified in approximately 36%. Patients with earlier disease showed florid duct lesions much more frequently than those with more advanced disease. The prevalence of florid duct lesions in 60 patients receiving placebo medication fell from 38.3% to 21.7%, P =. 025, over the period of 2 years. The prevalence of florid duct lesions also decreased in the 55 patients receiving UDCA, from 32.7% to 18.2%, P =.046. The prevalences of these lesions in the placebo and UDCA patients at entry and at 2 years were not significantly different from each other. The findings suggest that UDCA does not prevent ongoing bile duct destruction in patients with PBC. Instead, they support the impression that UDCA exerts its beneficial effects by protecting against the consequences of bile duct destruction.

Bile Ducts↗
PubMed1999

Characterization of ion transport mechanisms involved in bombesin-stimulated biliary secretion in rat cholangiocytes.

BACKGROUND/AIMS: Bombesin is a neuropeptide which stimulates fluid and bicarbonate secretion from cholangiocytes by stimulating Cl-/HCO3- exchange. However, the underlying regulation and interactions of ion transporters and channels mediating this bombesin-stimulated biliary secretion are not well characterized. The aim of the study was to characterize the ion transport processes involved in bombesin-stimulated secretion in polarized cholangiocytes in comparison with those of secretin. METHODS: Isolated bile duct units (IBDU) were prepared from normal rat liver. Biliary secretion induced by bombesin was measured by quantitative video-microscopy in the presence and absence of inhibitors. RESULTS: Bombesin-stimulated secretion was inhibited by H2-DIDS, NPPB, BaCl2, TEA, and acetazolamide. However, in contrast to secretin, bombesin-stimulated secretion was not inhibited by disruption of microtubules. CONCLUSIONS: Bombesin-stimulated biliary secretion is dependent on anion exchangers, Cl- and K+ channels, and carbonic anhydrase but not on microtubules. Bombesin regulates secretion in cholangiocytes by different mechanisms from those established for secretin.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

Functional expression of a cDNA encoding a human ecto-ATPase.

1 The metabolism of extracellular nucleotides plays an important role in nucleotide signalling mediated by P2 receptors. The nucleotide sequence encoding a putative human ecto-ATPase named CD39L1 was reported recently. However, the biological activity of this protein has not been established. 2 Based on the sequence of CD39L1 we isolated from mRNA from human ECV-304 cells a sequence encoding a 495 amino acid protein that is identical to CD39L1, with the exception that this sequence contains a 23 amino acid stretch in the putative extracellular loop that is missing in CD39L1. Partial sequence of a genomic DNA clone indicates that the CD39L1 gene corresponds to an alternative spliced form of the human ecto-ATPase. 3 Stable expression of isolated sequence in NIH-3T3 mouse fibroblasts conferred a marked nucleotide hydrolytic activity consistent with the activity of an ecto-ATPase. 4 The human ecto-ATPase hydrolyzed all naturally occurring nucleoside triphosphates in a Ca(2+)- or Mg(2+)-dependent manner. Nucleoside diphosphates were hydrolyzed at a rate approximately 5% of that of the corresponding triphosphates. The apparent Km and Vmax values were: 394+/-62 microM and 107+/-7 nmol Pi min-1 10(6) cells-1 for the hydrolysis of ATP, and 102+/-33 microM and 4+/-0.4 nmol Pi min-1 10(6) cells-1 for the hydrolysis of ADP, respectively. 5 In conclusion, we report here the cloning and functional expression of a human ecto-ATPase. The study of the biochemical properties and the regulatory mechanisms of ecto-ATPases of defined sequence will be valuable in the definition of their role in nucleotide signalling.

Adenosine Triphosphatases↗
PubMed1999

Vasoactive intestinal polypeptide is a potent regulator of bile secretion from rat cholangiocytes.

BACKGROUND & AIMS: Vasoactive intestinal polypeptide (VIP) is a neuropeptide with diverse biological functions including stimulatory effects on bile secretion. The effects of VIP on bile secretion and its site of action were examined. METHODS: Choleretic effects of VIP were examined using isolated perfused livers, hepatocyte couplets, isolated bile duct units, and cholangiocytes from rat liver. RESULTS: VIP (100 nmol/L) produced a small increase in bile flow and bile salt output in taurocholate-supplemented isolated perfused livers but had no significant effect on bile flow in the absence of bile salt supplements or on fluid secretion in isolated hepatocyte couplets. In addition, VIP significantly increased bile pH, bicarbonate concentration, and output in the isolated perfused livers from both normal and 2 week bile duct-ligated rats, although bile flow increased only in the bile duct-ligated model. VIP also produced a dose-dependent increase in fluid secretion in isolated bile duct units, which was inhibited significantly by VIP antagonist, a specific VIP receptor inhibitor. This VIP-stimulated secretory response in isolated bile duct units was more potent than those produced by bombesin or secretin. Neither somatostatin nor substance P inhibited the VIP response in isolated bile duct units. In contrast to secretin, VIP had no significant effect on adenosine 3', 5'-cyclic monophosphate (cAMP) levels in isolated cholangiocytes. CONCLUSIONS: VIP is a potent stimulant of fluid and bicarbonate secretion from cholangiocytes via cAMP-independent pathways, suggesting that this neuropeptide plays a major regulatory role in biliary transport and secretion.

Amino Acid Sequence↗
PubMed1999

Ageing of the cardiovascular system during 33 years of aerobic exercise.

BACKGROUND: increasing age affects aerobic capacity, with an average loss of 10% or more per decade. AIM: to determine the effect of ageing on the circulatory system in middle-aged men during 33 years of physical training. METHODS: 15 men initially aged 45 years took part in an exercise training programme for 25-33 years. Nine serial measurements were made at rest and during maximal effort. Aerobic training consisted of swimming, jogging, walking and cycling 3-4 times per week. Sessions were for 61-70 min at 77-84% of heart rate reserve. RESULTS: there was no change in resting heart rate, blood pressure, percentage fat or body composition. Minimal cardiovascular losses at maximal work included 5.8-6.8% in maximal oxygen uptake per decade, 25 beats in maximum heart rate and 26 beats in heart rate reserve. CONCLUSION: exercise training has a favourable effect on ageing of the cardiovascular system in older men, resulting in minimal loss of oxygen uptake, no rise in resting blood pressure and no change in body composition.

Adult↗
PubMed1999

Cholestatic syndromes.

Recent advances in cholestatic liver disease have occurred in several areas. Molecular cloning of hepatobiliary transport systems has resulted in the identification of the molecular basis of hereditary and acquired cholestatic syndromes. Apoptosis has been identified as an important mechanism of cholestatic liver injury and bile duct loss. New insights into the pathogenesis of pruritus and fatigue have resulted in new treatment strategies for these debilitating symptoms. Important new studies have been published about pathogenesis, clinical features, and treatment of primary biliary cirrhosis, primary sclerosing cholangitis, cholestasis of pregnancy, and drug-induced cholestasis.

Journal Article↗
PubMed1999

Carrier-mediated uptake of lucifer yellow in skate and rat hepatocytes: a fluid-phase marker revisited.

Uptake of lucifer yellow (LY), a fluorescent disulfonic acid anionic dye, was studied in isolated skate (Raja erinacea) perfused livers and primary hepatocytes to evaluate its utility as a fluid-phase marker in these cells. However, our findings demonstrated that LY is transported across the plasma membrane of skate hepatocytes largely via carrier-mediated mechanisms. Isolated perfused skate livers cleared 50% of the LY from the recirculating perfusate within 1 h of addition of either 22 or 220 microM LY, with only 4.5 and 9% of the LY remaining in the perfusate after 7 h, respectively. Most of the LY was excreted into bile, resulting in high biliary LY concentrations (1 and 10 mM at the two doses, respectively), indicating concentrative transport into bile canalicular lumen. LY uptake by freshly isolated skate hepatocytes was temperature sensitive, exhibited saturation kinetics, and was inhibited by other organic anions. Uptake was mediated by both sodium-dependent [Michaelis-Menten constant (K(m)), 125 +/- 57 microM; maximal velocity (V(max)), 1.5 +/- 0.2 pmol. min(-1). mg cells(-1)] and sodium-independent (K(m), 207 +/- 55 microM; V(max), 1.7 +/- 0.2 pmol. min(-1). mg cells(-1)) mechanisms. Both of these uptake mechanisms were inhibited by various organic anions and transport inhibitors, including furosemide, bumetanide, sulfobromophthalein, rose bengal, probenecid, N-ethylmaleimide, taurocholate, and p-aminohippuric acid. Fluorescent imaging techniques showed intracellular vesicular compartmentation of LY in skate hepatocyte clusters. Studies in perfused rat livers also indicated that LY is taken up against a concentration gradient and concentrated in bile. LY uptake in isolated rat hepatocytes was saturable, but only at high concentrations, and demonstrated a K(m) of 3.7 +/- 1.0 mM and a V(max) of 1.75 +/- 0.16 nmol. min(-1). mg wet wt(-1). These results indicate that LY is transported into skate and rat hepatocytes and bile largely by carrier-mediated mechanisms, rather than by fluid-phase endocytosis.

Animals↗
PubMed1998

Caspase-3 controls both cytoplasmic and nuclear events associated with Fas-mediated apoptosis in vivo.

Both caspase-1- and caspase-3-like activities are required for Fas-mediated apoptosis. However, the role of caspase-1 and caspase-3 in mediating Fas-induced cell death is not clear. We assessed the contributions of these caspases to Fas signaling in hepatocyte cell death in vitro. Although wild-type, caspase-1(-/-), and caspase-3(-/-) hepatocytes were killed at a similar rate when cocultured with FasL expressing NIH 3T3 cells, caspase-3(-/-) hepatocytes displayed drastically different morphological changes as well as significantly delayed DNA fragmentation. For both wild-type and caspase-1(-/-) apoptotic hepatocytes, typical apoptotic features such as cytoplasmic blebbing and nuclear fragmentation were seen within 6 hr, but neither event was observed for caspase-3(-/-) hepatocytes. We extended these studies to thymocytes and found that apoptotic caspase-3(-/-) thymocytes exhibited similar "abnormal" morphological changes and delayed DNA fragmentation observed in hepatocytes. Furthermore, the cleavage of various caspase substrates implicated in mediating apoptotic events, including gelsolin, fodrin, laminB, and DFF45/ICAD, was delayed or absent. The altered cleavage of these key substrates is likely responsible for the aberrant apoptosis observed in both hepatocytes and thymocytes deficient in caspase-3.

3T3 Cells↗
PubMed1998

A pyridoxine cyclic phosphate and its 6-azoaryl derivative selectively potentiate and antagonize activation of P2X1 receptors.

Analogues of the P2 receptor antagonists pyridoxal-5'-phosphate and the 6-azophenyl-2',4'-disulfonate derivative (PPADS), in which the phosphate group was cyclized by esterification to a CH2OH group at the 4-position, were synthesized. The cyclic pyridoxine-alpha4, 5-monophosphate, compound 2 (MRS 2219), was found to be a selective potentiator of ATP-evoked responses at rat P2X1 receptors with an EC50 value of 5.9 +/- 1.8 microM, while the corresponding 6-azophenyl-2',5'-disulfonate derivative, compound 3 (MRS 2220), was a selective antagonist. The potency of compound 3 at the recombinant P2X1 receptor (IC50 10.2 +/- 2.6 microM) was lower than PPADS (IC50 98.5 +/- 5.5 nM) or iso-PPADS (IC50 42.5 +/- 17.5 nM), although unlike PPADS its effect was reversible with washout and surmountable. Compound 3 showed weak antagonistic activity at the rat P2X3 receptor (IC50 58.3 +/- 0.1 microM), while at recombinant rat P2X2 and P2X4 receptors no enhancing or antagonistic properties were evident. Compounds 2 and 3 were found to be inactive as either agonists or antagonists at the phospholipase C-coupled P2Y1 receptor of turkey erythrocytes, at recombinant human P2Y2 and P2Y4 receptors, and at recombinant rat P2Y6 receptors. Similarly, compounds 2 and 3 did not have measurable affinity at adenosine A1, A2A, or A3 receptors. The lack of an aldehyde group in these derivatives indicates that Schiff's base formation with the P2X1 receptor is not necessarily required for recognition of pyridoxal phosphate derivatives. Thus, compounds 2 and 3 are relatively selective pharmacological probes of P2X1 receptors, filling a long-standing need in the P2 receptor field, and are also important lead compounds for future studies.

Adenosine Triphosphate↗
PubMed1998

Endotoxin downregulates rat hepatic ntcp gene expression via decreased activity of critical transcription factors.

Sodium-dependent uptake of bile acids across the hepatic basolateral membrane is rapidly and profoundly diminished during sepsis, thus contributing to the pathogenesis of sepsis-associated cholestasis. This effect is mediated by endotoxin or effector cytokines, which reduce expression of several hepatobiliary transporters, including the sodium-dependent bile acid transporter gene, ntcp. We test here the hypothesis that endotoxin treatment leads to impaired binding activity of ntcp promoter trans-acting factors, resulting in reduction of ntcp mRNA expression. After endotoxin administration, ntcp mRNA levels reached their nadir by 16 h, and nuclear run-on assays demonstrated a marked reduction in ntcp gene transcription. At 16 h after treatment, nuclear binding activities of two key factors that transactivate the ntcp promoter, hepatocyte nuclear factor (HNF) 1 and Footprint B binding protein (FpB BP), decreased to 44 and 47% of pretreatment levels, respectively, while levels of the other known ntcp promoter transactivator, signal transducer and activator of transcription 5, were unaffected. In contrast, the universal inflammatory response factors nuclear factor kappaB and activating protein 1 were both upregulated significantly. Examination of nuclear extracts obtained at sequential time points revealed that the maximal decrease in nuclear activities of both HNF1 and FpB BP preceded the nadir of ntcp mRNA expression by 6-10 h. Furthermore, these two nuclear factors returned towards normal levels before the recovery of ntcp mRNA levels observed by 48 h. Since HNF1alpha mRNA levels were unchanged at all time points, HNF1 is likely to be regulated posttranscriptionally by endotoxin. We conclude that the downregulation of ntcp gene expression by endotoxin is mediated at the level of transcription through tandem reductions in the nuclear binding activity of two critical transcription factors. These findings provide new insight into the coordinated downregulation of hepatobiliary transporters during sepsis.

Animals↗
PubMed1998

Human P2Y1 receptor: molecular modeling and site-directed mutagenesis as tools to identify agonist and antagonist recognition sites.

The molecular basis for recognition by human P2Y1 receptors of the novel, competitive antagonist 2'-deoxy-N6-methyladenosine 3', 5'-bisphosphate (MRS 2179) was probed using site-directed mutagenesis and molecular modeling. The potency of this antagonist was measured in mutant receptors in which key residues in the transmembrane helical domains (TMs) 3, 5, 6, and 7 were replaced by Ala or other amino acids. The capacity of MRS 2179 to block stimulation of phospholipase C promoted by 2-methylthioadenosine 5'-diphosphate (2-MeSADP) was lost in P2Y1 receptors having F226A, K280A, or Q307A mutations, indicating that these residues are critical for the binding of the antagonist molecule. Mutation of the residues His132, Thr222, and Tyr136 had an intermediate effect on the capacity of MRS 2179 to block the P2Y1 receptor. These positions therefore appear to have a modulatory role in recognition of this antagonist. F131A, H277A, T221A, R310K, or S317A mutant receptors exhibited an apparent affinity for MRS 2179 that was similar to that observed with the wild-type receptor. Thus, Phe131, Thr221, His277, and Ser317 are not essential for antagonist recognition. A computer-generated model of the human P2Y1 receptor was built and analyzed to help interpret these results. The model was derived through primary sequence comparison, secondary structure prediction, and three-dimensional homology building, using rhodopsin as a template, and was consistent with data obtained from mutagenesis studies. We have introduced a "cross-docking" procedure to obtain energetically refined 3D structures of the ligand-receptor complexes. Cross-docking simulates the reorganization of the native receptor structure induced by a ligand. A putative nucleotide binding site was localized and used to predict which residues are likely to be in proximity to agonists and antagonists. According to our model TM6 and TM7 are close to the adenine ring, TM3 and TM6 are close to the ribose moiety, and TM3, TM6, and TM7 are near the triphosphate chain.

Adenosine Diphosphate↗
PubMed1998

Deoxyadenosine bisphosphate derivatives as potent antagonists at P2Y1 receptors.

Adenosine 3',5'- and 2',5'-bisphosphates previously were demonstrated to act as competitive antagonists at the P2Y1 receptor (Boyer et al. Mol. Pharmacol. 1996, 50, 1323-1329). 2'- and 3'-Deoxyadenosine bisphosphate analogues containing various structural modifications at the 2- and 6-positions of the adenine ring, on the ribose moiety, and on the phosphate groups have been synthesized with the goal of developing more potent and selective P2Y1 antagonists. Single-step phosphorylation reactions of adenosine nucleoside precursors were carried out. The activity of each analogue at P2Y1 receptors was determined by measuring its capacity to stimulate phospholipase C in turkey erythrocyte membranes (agonist effect) and to inhibit phospholipase C stimulation elicited by 10 nM 2-MeSATP (antagonist effect). Both 2'- and 3'-deoxy modifications were well tolerated. The N6-methyl modification both enhanced antagonistic potency (IC50 330 nM) of 2'-deoxyadenosine 3',5'-bisphosphate by 17-fold and eliminated residual agonist properties observed with the lead compounds. The N6-ethyl modification provided intermediate potency as an antagonist, while the N6-propyl group completely abolished both agonist and antagonist properties. 2-Methylthio and 2-chloro analogues were partial agonists of intermediate potency. A 2'-methoxy group provided intermediate potency as an antagonist while enhancing agonist activity. An N1-methyl analogue was a weak antagonist with no agonist activity. An 8-bromo substitution and replacement of the N6-amino group with methylthio, chloro, or hydroxy groups greatly reduced the ability to interact with P2Y1 receptors. Benzoylation or dimethylation of the N6-amino group also abolished or greatly diminished the antagonist activity. In summary, our results further define the structure-activity of adenosine bisphosphates as P2Y1 receptor antagonists and have led to the identification of the most potent antagonist reported to date for this receptor.

Animals↗