Biomedical subjects

J L Boyer

Publications and source records attributed to J L Boyer.

At least 37 records · Page 2Linked to original sources

PubMed2000

Integrins regulate the linkage between upstream and downstream events in G protein-coupled receptor signaling to mitogen-activated protein kinase.

Receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) can both activate mitogen-activated protein kinase (MAPK), a critical intermediate in the transduction of proliferative signals. Numerous observations have demonstrated that integrin-mediated cell anchorage can regulate the efficiency of signaling from RTKs to MAPK. Recently, a relationship between integrins and GPCR signaling has also emerged; however, little is understood concerning the mechanisms involved. Here, we investigate integrin regulation of GPCR signaling to MAPK, focusing on the P2Y class of GPCRs that function through activation of phospholipase Cbeta. P2Y receptor signaling to the downstream components mitogen-activated protein kinase kinase and MAPK is highly dependent on integrin-mediated cell anchorage. However, activation of upstream events, including inositol phosphate production and generation of calcium transients, is completely independent of cell anchorage. This indicates that integrins regulate the linkage between upstream and downstream events in this GPCR pathway, just as they do in some aspects of RTK signaling. However, the P2Y pathway does not involve cross-activation of a RTK, nor a role for Shc or c-Raf; thus, it is quite distinct from the classical RTK-Ras-Raf-MAPK cascade. Rather, integrin-modulated P2Y receptor stimulation of MAPK depends on calcium and on the activation of protein kinase C.

Adenosine Triphosphate↗

PubMed2000

Synthesis, biological activity, and molecular modeling of ribose-modified deoxyadenosine bisphosphate analogues as P2Y(1) receptor ligands.

The structure-activity relationships of adenosine-3', 5'-bisphosphates as P2Y(1) receptor antagonists have been explored, revealing the potency-enhancing effects of the N(6)-methyl group and the ability to substitute the ribose moiety (Nandanan et al. J. Med. Chem. 1999, 42, 1625-1638). We have introduced constrained carbocyclic rings (to explore the role of sugar puckering), non-glycosyl bonds to the adenine moiety, and a phosphate group shift. The biological activity of each analogue at P2Y(1) receptors was characterized by measuring its capacity to stimulate phospholipase C in turkey erythrocyte membranes (agonist effect) and to inhibit its stimulation elicited by 30 nM 2-methylthioadenosine-5'-diphosphate (antagonist effect). Addition of the N(6)-methyl group in several cases converted pure agonists to antagonists. A carbocyclic N(6)-methyl-2'-deoxyadenosine bisphosphate analogue was a pure P2Y(1) receptor antagonist and equipotent to the ribose analogue (MRS 2179). In the series of ring-constrained methanocarba derivatives where a fused cyclopropane moiety constrained the pseudosugar ring of the nucleoside to either a Northern (N) or Southern (S) conformation, as defined in the pseudorotational cycle, the 6-NH(2) (N)-analogue was a pure agonist of EC(50) 155 nM and 86-fold more potent than the corresponding (S)-isomer. The 2-chloro-N(6)-methyl-(N)-methanocarba analogue was an antagonist of IC(50) 51.6 nM. Thus, the ribose ring (N)-conformation appeared to be favored in recognition at P2Y(1) receptors. A cyclobutyl analogue was an antagonist with IC(50) of 805 nM, while morpholine ring-containing analogues were nearly inactive. Anhydrohexitol ring-modified bisphosphate derivatives displayed micromolar potency as agonists (6-NH(2)) or antagonists (N(6)-methyl). A molecular model of the energy-minimized structures of the potent antagonists suggested that the two phosphate groups may occupy common regions. The (N)- and (S)-methanocarba agonist analogues were docked into the putative binding site of the previously reported P2Y(1) receptor model.

Animals↗

PubMed2000

Acyclic analogues of deoxyadenosine 3',5'-bisphosphates as P2Y(1) receptor antagonists.

P2Y(1) receptors are activated by ADP and occur on endothelial cells, smooth muscle, epithelial cells, lungs, pancreas, platelets, and in the central nervous system. With the aid of molecular modeling, we have designed nucleotide analogues that act as selective antagonists at this subtype. The present study has tested the hypothesis that acyclic modifications of the ribose ring, proven highly successful for nucleoside antiviral agents such as gancyclovir, are generalizable to P2Y receptor ligands. Specifically, the binding site of the P2Y(1) receptor was found to be sufficiently accommodating to allow the substitution of the ribose group with acyclic aliphatic and aromatic chains attached to the 9-position of adenine. Three groups of adenine derivatives having diverse side-chain structures, each containing two symmetrical phosphate or phosphonate groups, were prepared. Biological activity was demonstrated by the ability of the acyclic derivatives to act as agonists or antagonists in the stimulation of phospholipase C in turkey erythrocyte membranes. An acyclic N(6)-methyladenine derivative, 2-[2-(6-methylamino-purin-9-yl)-ethyl]-propane-1, 3-bisoxy(diammoniumphosphate) (10), containing an isopentyl bisphosphate moiety, was a full antagonist at the P2Y(1) receptor with an IC(50) value of 1.60 micro¿. The corresponding 2-Cl derivative (11) was even more potent with an IC(50) value of 0.84 microM. Homologation of the ethylene group at the 9-position to 3-5 methylene units or inclusion of cis- or trans-olefinic groups greatly reduced antagonist potency at the P2Y(1) receptor. Analogues containing a diethanolamine amide group and an aryl di(methylphosphonate) were both less potent than 10 as antagonists, with IC(50) values of 14 and 16 microM, respectively, and no agonist activity was observed for these analogues. Thus, the ribose moiety is clearly not essential for recognition by the turkey P2Y(1) receptor, although a cyclic structure appears to be important for receptor activation, and the acyclic approach to the design of P2 receptor antagonists is valid.

Adenine↗

PubMed2000

Expression of the bile salt export pump is maintained after chronic cholestasis in the rat.

BACKGROUND & AIMS: This study assessed the expression of the recently identified adenosine triphosphate-dependent bile salt export pump and the functional ability to excrete bile salts in cholestatic models in the rat. METHODS: The effects of common bile duct ligation, endotoxin, and ethinylestradiol on bile salt export pump messenger RNA levels, protein expression, and tissue localization were determined. Changes in the expression of 3 other hepatocyte membrane transporters (Na(+) taurocholate cotransporter, multispecific organic anion transporter, and P-glycoprotein) were also determined for comparison. Functional assessment of bile salt excretion was determined after bile duct ligation. RESULTS: Expression of the bile salt export pump was diminished but relatively preserved compared with other membrane transporters. Tissue localization of the bile salt export pump persisted at the canalicular domain in all 3 models. In contrast, expressions of the Na(+) taurocholate cotransporter and multispecific organic anion transporter were more profoundly diminished. P-glycoprotein levels increased severalfold with common bile duct ligation but were unchanged with either endotoxin or ethinylestradiol. The capacity to excrete bile salts was relatively maintained 3 and even 14 days after bile duct ligation. CONCLUSIONS: Alterations in expression of the bile salt export pump may account for the functional alterations of bile salt secretion observed in cholestasis. However, relative preservation of expression is associated with persistent bile salt excretion and may lessen the extent of liver injury produced by bile salt retention.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Molecular identification and functional characterization of Mdr1a in rat cholangiocytes.

BACKGROUND & AIMS: The multidrug resistance P-glycoprotein 170 gene products (mdr1a and 1b) are glycosylated plasma membrane proteins that function as adenosine triphosphate-dependent transmembrane export pumps for lipophilic xenobiotics of widely different structure. We assessed whether these P-glycoproteins are functionally expressed in cholangiocytes. METHODS: A reverse-transcription polymerase chain reaction was performed on RNA from a normal rat cholangiocyte cell line using mdr1-specific primers. Northern and Western blot analyses were performed on cholangiocytes immunoisolated from 2-week bile duct-ligated rats and cholangiocytes and isolated cholangiocyte membrane subfractions, respectively. Functional assays were performed in isolated bile duct units from bile duct-ligated rats and incubated with rhodamine 123, a P-glycoprotein substrate, with or without the P-glycoprotein inhibitors verapamil or GF120918. RESULTS: A 400-base pair fragment with 99% homology to the cytosolic domain of rat intestinal mdr1a (5' 1953-2350 3') was identified that hybridized to a 5.2-kilobase RNA transcript in a normal rat cholangiocyte cell line, isolated rat cholangiocytes, and ileum. Western analysis localized mdr1 to the apical membrane of cholangiocytes. Confocal microscopy showed active secretion of rhodamine 123 into the lumen of isolated bile duct units that was abolished by vanadate and P-glycoprotein competitive antagonists, verapamil and GF120918, in a dose-dependent manner. CONCLUSIONS: These findings provide the first molecular and functional evidence for the expression of mdr1a on the luminal membrane of cholangiocytes, where it may have a protective role.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2000

Induction of murine hepatocyte death by membrane-bound CD95 (Fas/APO-1)-ligand: characterization of an in vitro system.

Hepatocytes constitutively express CD95 (also called Fas/APO-1) and are therefore potential targets for CD95-ligand (CD95L)-mediated injury. To study this mechanism of cell death in hepatocytes we developed an in vitro model of liver cell apoptosis using membrane-bound CD95L as the inducing agent. Primary mouse hepatocytes were cocultured with NIH 3T3 fibroblasts, stably transfected with mouse CD95L (F(CD95L+)). Fibroblasts stably transfected with vector only (F(CD95L-)) served as controls. Hepatocytes from mice expressing low levels of CD95 (Fas(lpr) mice) served as controls for effects unrelated to CD95. Morphologic and biochemical studies indicate that CD95 is expressed in cultured mouse hepatocytes. Membrane-bound CD95 from transfected fibroblasts destroyed all cocultured hepatocytes within 24 hours in the absence of protein synthesis inhibitors. Characteristic features of apoptosis were observed in dying hepatocytes and occurred in the following sequence: formation of cytoplasmic blebs and nuclear condensation after 3 hours; nuclear fragmentation and DNA strand breaks after 4 hours. These changes were observed only when normal hepatocytes were cocultured with F(CD95L+) and were not observed with F(CD95L-) or in hepatocytes from Fas(lpr) mice. Anti-CD95 antibody (Jo2) evoked similar changes in hepatocytes, although to a much lesser extent. We conclude that coculture of mouse hepatocytes with F(CD95L+) is a useful in vitro model for CD95-mediated apoptosis induced by CD95L. The high incidence of apoptosis caused by membrane-bound CD95L differs from the much smaller effects induced by the Jo2 antibody. In view of the high sensitivity of hepatocytes towards CD95L we speculate that CD95L-induced liver damage in vivo may be minimized by restricting exposure of hepatocytes to CD95L.

3T3 Cells↗

PubMed2000

Molecular alterations in hepatocyte transport mechanisms in acquired cholestatic liver disorders.

Recent advances in the molecular cloning of membrane transport systems that determine bile formation have facilitated studies of the molecular mechanisms of cholestatic liver disease. The present review summarizes what has been learned about the molecular alterations of these membrane transporters in hepatocytes and cholangiocytes in acquired cholestatic liver disorders. Much of this information has been obtained from the study of animal models of cholestasis and from more limited studies in clinical cholestatic liver diseases. Many of these responses may be interpreted as adaptations that serve to diminish cholestatic liver injury.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Cholestatic syndromes.

Continued advances in the field of liver cell biology and molecular biology have provided further insights into the normal physiology of bile secretion and the pathogenesis and therapy for cholestatic syndromes. Important new data have also been published about pathogenesis, clinical features, and treatment of primary biliary cirrhosis, primary sclerosing cholangitis, drug-induced cholestasis, and cholestatic syndromes caused by viral infections.

Journal Article↗

PubMed2000

A molecularly identified P2Y receptor simultaneously activates phospholipase C and inhibits adenylyl cyclase and is nonselectively activated by all nucleoside triphosphates.

We recently cloned and expressed a novel P2Y receptor (tp2y receptor) from a turkey cDNA library. Expression of this receptor in 1321N1 human astrocytoma cells confers nucleotide-dependent stimulation of phospholipase C activity; however, as we demonstrate here, it also confers nucleotide-dependent inhibition of adenylyl cyclase. Both the phospholipase C and adenylyl cyclase responses were promoted by receptor agonists over a similar range of concentrations. Moreover, not only did UTP and ATP activate the avian receptor but ITP, GTP, xanthosine 5'-triphosphate, and CTP were also agonists, with EC(50) values ranging between 0.1 and 1 microM. Similar potencies, rank-order, and selectivity of nucleotide agonists were also demonstrated for intracellular Ca(2+) mobilization measured during a 30-s stimulation under constant superfusion conditions. This observation indicates that receptor activation by nucleoside 5'-triphosphates is not produced by interconversion of these nucleotides into ATP or UTP. Pretreatment of cells with pertussis toxin completely abolished the inhibitory effect of nucleotide agonists on adenylyl cyclase, whereas the activation of phospholipase C was only partially inhibited. These results demonstrate that the avian P2Y receptor is a nucleoside triphosphate receptor of broad agonist selectivity that interacts with both pertussis toxin-insensitive and -sensitive G proteins to activate phospholipase C and to inhibit adenylyl cyclase. This is the first cloned P2Y receptor that is clearly Gi/adenylyl cyclase-linked.

Adenosine Triphosphate↗

PubMed2000

Bile salt excretion in skate liver is mediated by a functional analog of Bsep/Spgp, the bile salt export pump.

Biliary secretion of bile salts in mammals is mediated in part by the liver-specific ATP-dependent canalicular membrane protein Bsep/Spgp, a member of the ATP-binding cassette superfamily. We examined whether a similar transport activity exists in the liver of the evolutionarily primitive marine fish Raja erinacea, the little skate, which synthesizes mainly sulfated bile alcohols rather than bile salts. Western blot analysis of skate liver plasma membranes using antiserum raised against rat liver Bsep/Spgp demonstrated a dominant protein band with an apparent molecular mass of 210 kDa, a size larger than that in rat liver canalicular membranes, approximately 160 kDa. Immunofluorescent localization with anti-Bsep/Spgp in isolated, polarized skate hepatocyte clusters revealed positive staining of the bile canaliculi, consistent with its selective apical localization in mammalian liver. Functional characterization of putative ATP-dependent canalicular bile salt transport activity was assessed in skate liver plasma membrane vesicles, with [(3)H]taurocholate as the substrate. [(3)H]taurocholate uptake into the vesicles was mediated by ATP-dependent and -independent mechanisms. The ATP-dependent component was saturable, with a Michaelis-Menten constant (K(m)) for taurocholate of 40+/-7 microM and a K(m) for ATP of 0.6+/-0.1 mM, and was competitively inhibited by scymnol sulfate (inhibition constant of 23 microM), the major bile salt in skate bile. ATP-dependent uptake of taurocholate into vesicles was inhibited by known substrates and inhibitors of Bsep/Spgp, including other bile salts and bile salt derivatives, but not by inhibitors of the multidrug resistance protein-1 or the canalicular multidrug resistance-associated protein, indicating a distinct transport mechanism. These findings provide functional and structural evidence for a Bsep/Spgp-like protein in the canalicular membrane of the skate liver. This transporter is expressed early in vertebrate evolution and transports both bile salts and bile alcohols.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

ATP-dependent GSH and glutathione S-conjugate transport in skate liver: role of an Mrp functional homologue.

Multidrug resistance-associated proteins 1 and 2 (Mrp1 and Mrp2) are thought to mediate low-affinity ATP-dependent transport of reduced glutathione (GSH), but there is as yet no direct evidence for this hypothesis. The present study examined whether livers from the little skate (Raja erinacea) express an Mrp2 homologue and whether skate liver membrane vesicles exhibit ATP-dependent GSH transport activity. Antibodies directed against mammalian Mrp2-specific epitopes labeled a 180-kDa protein band in skate liver plasma membranes and stained canaliculi by immunofluorescence, indicating that skate livers express a homologous protein. Functional assays of Mrp transport activity were carried out using (3)H-labeled S-dinitrophenyl-glutathione (DNP-SG). DNP-SG was accumulated in skate liver membrane vesicles by both ATP-dependent and ATP-independent mechanisms. ATP-dependent DNP-SG uptake was of relatively high affinity [Michaelis-Menten constant (K(m)) = 32 +/- 9 microM] and was cis-inhibited by known substrates of Mrp2 and by GSH. Interestingly, ATP-dependent transport of (3)H-labeled S-ethylglutathione and (3)H-labeled GSH was also detected in the vesicles. ATP-dependent GSH transport was mediated by a low-affinity pathway (K(m) = 12 +/- 2 mM) that was cis-inhibited by substrates of the Mrp2 transporter but was not affected by membrane potential or pH gradient uncouplers. These results provide the first direct evidence for ATP-dependent transport of GSH in liver membrane vesicles and support the hypothesis that GSH efflux from mammalian cells is mediated by members of the Mrp family of proteins.

ATP-Binding Cassette Transporters↗

PubMed2000

2-Thioether-5'-O-(1-thiotriphosphate)-adenosine derivatives: new insulin secretagogues acting through P2Y-receptors.

P2-receptors (P2-Rs) represent significant targets for novel drug development. P2-Rs were identified also on pancreatic B cells and are involved in insulin secretion. The aim of our study was to synthesize and evaluate pharmacologically the novel P2Y-R ligands, 2-thioether-5'-O-phosphorothioate adenosine derivatives, as potential insulin secretagogues. An efficient synthesis of these nucleosides and a facile method for separation of the chiral products is described. The enzymatic stability of the compounds towards pig-pancreas NTPDase was evaluated. The rate of hydrolysis of 2-hexylthio-5'-O-(1-thiotriphosphate)-adenosine (2-hexylthio-ATP-alpha-S) isomers by NTPDase was 28% that of ATP. The apparent affinity of the compounds to P2Y1-R was determined by measurement of P2Y-receptor-promoted phospholipase C activity in turkey erythrocyte membranes. 2-RS-ATP-alpha-S derivatives were agonists, stimulating the production of inositol phosphates with K0.5 values in the nM range. 2-RS-AMP-S derivatives were full agonists although 2 orders of magnitude less potent. All the compounds were more potent than ATP. The effect on insulin secretion and pancreatic flow rate was evaluated on isolated and perfused rat pancreas. A high increase, up to 500%, in glucose-induced insulin secretion was due to addition of 2-hexylthio-ATP-alpha-S in the nM concentration range, which represents 100-fold enhancement of activity relative to ATP. 2-Hexylthio-AMP-S was 2.5 orders of magnitude less effective. A high chemical hydrolytic stability was observed for 2-hexylthio-ATP-alpha-S. Hydrolysis of the phosphoester bond, which was the only detectable degrading reaction under the investigation conditions (pH 7.4, 37 degrees C), was slow, with a half-life of 264 hours. Moreover, even at gastric juice conditions (pH 1.4, 37 degrees C), hydrolysis of the terminal phosphate was the only detectable reaction, with a half-life of 17.5 hours. 2-Hexylthio-ATP-alpha-S isomers are enzymatically and chemically stable. These isomers are highly potent and effective insulin secretagogues, increasing, however, pancreatic vascular resistance.

Acid Anhydride Hydrolases↗

PubMed1999

Molecular recognition of modified adenine nucleotides by the P2Y(1)-receptor. 1. A synthetic, biochemical, and NMR approach.

The remarkably high potencies of 2-thioether-adenine nucleotides regarding the activation of the P2Y(1)-receptor (P2Y(1)-R) in turkey erythrocyte membranes represent some of the largest substitution-promoted increases in potencies over that of a natural receptor ligand. This paper describes the investigation regarding the origin of the high potency of these P2Y(1)-R ligands over that of ATP. For this study, an integrated approach was employed combining the synthesis of new ATP analogues, their biochemical evaluation, and their SAR analysis involving NMR experiments and theoretical calculations. These experiments and calculations were performed to elucidate the conformation and to evaluate the electronic nature of the investigated P2Y(1)-R ligands. ATP analogues synthesized included derivatives where C2 or C8 positions were substituted with electron-donating groups such as ethers, thioethers, or amines. The compounds were tested for their potency to induce P2Y(1)-R-mediated activation of phospholipase C in turkey erythrocytes and Ca(2+) response in rat astrocytes. 8-Substituted ATP and AMP derivatives had little or no effect on phospholipase C or on calcium levels, whereas the corresponding 2-substituted ATP analogues potently increased the levels of inositol phosphates and ¿Ca(2+)(i). AMP analogues were ineffective except for 2-butylthio-AMP which induced a small Ca(2+) response. P2Y(1)-R activity of these compounds was demonstrated by testing these ligands also on NG108-15 neuroblastoma x glioma hybrid cells. NMR data together with theoretical calculations imply that steric, rather than electronic, effects play a major role in ligand binding to the P2Y(1)-R. Hydrophobic interactions and H-bonds of the C2 substituent appear to be important determinants of a P2Y(1)-R ligand affinity.

Adenine Nucleotides↗

PubMed1999

Hepatic toxicity and persistence of ser/thr protein phosphatase inhibition by microcystin in the little skate Raja erinacea.

Microcystin-induced ser/thr protein phosphatase (PP) inhibition and toxicity were examined in the little skate (Raja erinacea), an evolutionarily primitive marine vertebrate. As in mammals, PP inhibition and toxicity were exclusively hepatocellular, but were much more persistent in the skate. A dose of 63 microg/kg given iv to adult male skates resulted in the near complete inhibition of hepatic PP activity at 24 h. PP activity was still 95% inhibited 7 days after dosing in skates given 125 microg/kg microcystin. Mortality occurred at doses of 500 microg/kg or more. Hepatic lesions were only seen in animals with fully inhibited PP activity in liver. The histological changes seen at 125 microg/kg were mild periportal inflammatory changes increasing in severity together with hepatocyte necrosis at higher doses of microcystin. Microcystin persisted and could be detected in plasma up to 7 days after dosing. This finding shows that, in the skate, as in mammals, the liver is the only organ capable of uptake of microcystin, since there was no significant inhibition of PP activity in the rectal gland and small decreases in PP activity of the kidney that were not time or dose dependent. In vitro microcystin caused dose-dependent inhibition of PP activity in isolated skate hepatocytes, while it was without effect in cultured rectal glands. Uptake of microcystin and the accompanying inhibition of PP activity in skate hepatocytes was prevented by the addition of a series of organic dyes and bile acids. The spectrum of inhibitors of microcystin uptake in skate is similar to that seen in the rat, indicating common features of the carrier(s) in these diverse species.

Animals↗

PubMed1999

Canalicular export pumps traffic with polymeric immunoglobulin A receptor on the same microtubule-associated vesicle in rat liver.

Basolateral to apical vesicular transcytosis in the hepatocyte is an essential pathway for the delivery of compounds from the sinusoidal blood to the bile and to traffic newly synthesized resident apical membrane proteins to their site of function at the canalicular membrane front. To characterize this pathway better, microtubules in a hepatocyte homogenate were polymerized by addition of taxol, and associated membrane-bound vesicles were isolated. This fraction was enriched in polymeric immunoglobulin A receptor and contained apical membrane proteins. Immunoelectron microscopy demonstrated that polymeric immunoglobulin A receptor was localized predominantly on vesicles ranging from 100 to 160 nm and that the multidrug resistance protein 2 and the bile salt export pump co-localized on these vesicles. The minus-ended microtubule motor, dynein, was highly enriched in the fraction, and its intermediate chain could be released effectively by incubation with 1 mM ATP or GTP. However, the association of the transcytotic vesicles with the microtubules was not sensitive to hydrolyzable or non-hydrolyzable nucleotides. This study characterizes a fraction of microtubule-associated vesicles from rat hepatocytes and demonstrates that several resident apical membrane transport proteins and the polymeric immunoglobulin A receptor traffic on the same vesicle.

Animals↗

PubMed1999

2-thioether 5'-O-(1-thiotriphosphate)adenosine derivatives as new insulin secretagogues acting through P2Y-Receptors.

P2-Receptors (P2-Rs) represent significant targets for novel drug development. P2-Rs were identified also on pancreatic B cells and are involved in insulin secretion. Therefore, novel P2Y-R ligands, 2-thioether 5'-O-phosphorothioate adenosine derivatives (2-RS-ATP-alpha-S), were synthesized as potential insulin secretagogues. An efficient synthesis of these nucleotides and a facile method for separation of the chiral products are described. The enzymatic stability of the compounds toward pig pancreas type I ATPDase was evaluated. The rate of hydrolysis of 2-hexylthio-5'-O-(1-thiotriphosphate)adenosine (2-hexylthio-ATP-alpha-S) isomers by ATPDase was 28% of that of ATP. Some 2-thioether 5'-(monophosphorothioate)adenosine derivatives (2-RS-AMP-S) exerted an inhibitory effect on ATPDase. The apparent affinity of the compounds to P2Y(1)-R was determined by measurement of P2Y-R-promoted phospholipase C activity in turkey erythrocyte membranes. 2-RS-ATP-alpha-S derivatives were agonists, stimulating the production of inositol phosphates with K(0.5) values in the nanomolar range. 2-RS-AMP-S derivatives were full agonists, although 2 orders of magnitude less potent. All the compounds were more potent than ATP. The effect on insulin secretion and pancreatic flow rate was evaluated on isolated and perfused rat pancreas. A high increase, up to 500%, in glucose-induced insulin secretion was due to addition of 2-hexylthio-ATP-alpha-S in the nanomolar concentration range, which represents 100-fold enhancement of activity relative to ATP. 2-Hexylthio-AMP-S was 2.5 orders of magnitude less effective.

Adenosine↗

PubMed1999

Structure-activity relationships of bisphosphate nucleotide derivatives as P2Y1 receptor antagonists and partial agonists.

The P2Y1 receptor is present in the heart, in skeletal and various smooth muscles, and in platelets, where its activation is linked to aggregation. Adenosine 3',5'- and 2',5'-bisphosphates have been identified as selective antagonists at the P2Y1 receptor (Boyer et al. Mol. Pharmacol. 1996, 50, 1323-1329) and have been modified structurally to increase receptor affinity (Camaioni et al. J. Med. Chem. 1998, 41, 183-190). We have extended the structure-activity relationships to a new series of deoxyadenosine bisphosphates with substitutions in the adenine base, ribose moiety, and phosphate groups. 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-(methylthio)adenosine 5'-diphosphate (antagonist effect). 2'-Deoxyadenosine bisphosphate analogues containing halo, amino, and thioether groups at the 2-position of the adenine ring were more potent P2Y1 receptor antagonists than analogues containing various heteroatom substitutions at the 8-position. An N6-methyl-2-chloro analogue, 6, was a full antagonist and displayed an IC50 of 206 nM. Similarly, N6-methyl-2-alkylthio derivatives 10, 14, and 15 were nearly full antagonists of IC50 < 0.5 microM. On the ribose moiety, 2'-hydroxy, 4'-thio, carbocyclic, and six-membered anhydrohexitol ring modifications have been prepared and resulted in enhanced agonist properties. The 1,5-anhydrohexitol analogue 36 was a pure agonist with an EC50 of 3 microM, i.e., similar in potency to ATP. 5'-Phosphate groups have been modified in the form of triphosphate, methyl phosphate, and cyclic 3',5'-diphosphate derivatives. The carbocyclic analogue had enhanced agonist efficacy, and the 5'-O-phosphonylmethyl modification was tolerated, suggesting that deviations from the nucleotide structure may result in improved utility as pharmacological probes. The N6-methoxy modification eliminated receptor affinity. Pyrimidine nucleoside 3', 5'-bisphosphate derivatives were inactive as agonists or antagonists at P2Y receptor subtypes.

Animals↗

PubMed1999

Hepatic sequestration and modulation of the canalicular transport of the organic cation, daunorubicin, in the Rat.

In contrast to organic anions, substrates for the canalicular mdr1a and b are usually organic cations and are often sequestered in high concentrations in intracellular acidic compartments. Because many of these compounds are therapeutic agents, we investigated if their sequestration could be regulated. We used isolated perfused rat liver (IPRL), isolated rat hepatocyte couplets (IRHC), and WIF-B cells to study the cellular localization and biliary excretion of the fluorescent cation, daunorubicin (DNR). Despite rapid (within 15 minutes) and efficient (>90%) cellular uptake in the IPRL, only approximately 10% of the dose administered (0.2-20 micromol) was excreted in bile after 85 minutes. Confocal microscopy revealed fluorescence predominantly in vesicles in the pericanalicular region in IPRL, IRHC, and WIF-B cells. Treatment of these cells with chloroquine and bafilomycin A, agents that disrupt the pH gradient across the vesicular membrane, resulted in a loss of vesicular fluorescence, reversible in the case of bafilomycin A. Taurocholate (TC) and dibutyryl cAMP (DBcAMP), stimulators of transcytotic vesicular transport, increased the biliary recovery of DNR significantly above controls, by 70% and 35%, respectively. The microtubule destabilizer, nocodazole, decreased biliary excretion of DNR. No effect on secretion was noted in TR- mutant rats deficient in mrp2. Coadministration of verapamil, an inhibitor of mdr1, also decreased DNR excretion. While TC and DBcAMP did not affect the fluorescent intensity or pattern of distribution in IRHC, nocodazole resulted in redistribution of DNR to peripheral punctuate structures. These findings suggest that the organic cation, DNR, is largely sequestered in cells such as hepatocytes, yet its excretion can still be modulated.

ATP Binding Cassette Transporter, Subfamily B, Mem↗