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Query: UNIPROT:P06889 (Mol)
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The mechanism for the vasopressin- and epinephrine-induced decrease in bile formation and increase in sinusoidal efflux of glutathione was investigated in rat livers perfused with recirculating fluorocarbon emulsion. Vasopressin and epinephrine transiently decreased bile flow and excretion of endogenous bile acids and glutathione and increased the bile/perfusate ratio of [14C]sucrose, suggesting an increase in junctional permeability, but had no effect on the bile/perfusate ratio of [3H]polyethylene glycol-900. The decreased biliary glutathione was balanced by an increase in sinusoidal efflux, such that total hepatic release remained unchanged. The adrenergic antagonist dihydroergotamine blocked the effects of epinephrine. To examine whether an increase in junctional permeability per se could account for the changes in glutathione efflux, biliary permeability was increased by either bile duct ligation, lowering of perfusate Ca2+ concentration with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or addition of taurolithocholate, a cholestatic bile acid. All three maneuvers produced a decrease in biliary glutathione excretion and a concomitant increase in sinusoidal glutathione efflux, whereas total glutathione release was largely unaffected. The effects of EGTA were partially reversed if CaCl2 was reintroduced into the perfusate. Because the GSH/GSSG ratio in perfusate could not be measured in this experimental system due to the spontaneous oxidation of GSH to GSSG, additional experiments in the nonrecirculating mode examined the effects of vasopressin and bile duct ligation on sinusoidal release of GSH and GSSG. In control livers there was no detectable GSSG in perfusate (less than 0.5 nmol.min-1.g-1). After vasopressin administration, the additional sinusoidal glutathione was mainly as GSH, although there was also a significant amount of GSSG (1-2 nmol.min-1.g-1). The additional glutathione released into perfusate after bile duct ligation was 47% as GSSG. When vasopressin was administered to livers whose bile duct had been ligated, its ability to enhance sinusoidal glutathione release was diminished, suggesting that the effects of vasopressin and bile duct ligation are not additive. These observations support previous findings that vasopressin and epinephrine can modulate hepatocyte tight junctional permeability and demonstrate that these hormones produce cholestasis and inverse changes in sinusoidal and biliary glutathione efflux. Other maneuvers that increased biliary permeability to [14C]sucrose also produced cholestasis and a redistribution of glutathione efflux from bile to perfusate, suggesting that an increase in junctional permeability may allow biliary glutathione to reflux from bile to plasma.
Mol Pharmacol 1990 Jul
PMID:Cholestasis, altered junctional permeability, and inverse changes in sinusoidal and biliary glutathione release by vasopressin and epinephrine. 211 13

The dihydropyridine receptor associated with the L-type Ca2+ channel in adrenal medulla membranes has been identified and characterized. [3H]PN200-110 binds in a stereoselective, saturable manner to a single class of high affinity sites in adrenal medulla membranes, with a Kd of 0.1 nM and a Bmax of 141 fmol/mg of protein. Dihydropyridines inhibited [3H]PN200-110 binding with the rank order (+)-PN200-110 greater than nifedipine greater than nimodipine greater than usoldipine greater than or equal to nitrendipine greater than BayK8644 greater than (-)-PN200-110. [3H] PN200-110 binding was sensitive to divalent cations, as examined by the effects of Ca2+, Mg2+, and the chelators ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and EDTA. [3H]PN200-110 binding was modulated by various classes of L-type Ca2+ channel effectors. Benzothiazepines modulated binding of [3H]PN200-110 in a negative or positive manner that was temperature dependent, whereas phenylalkylamines weakly inhibited [3H]PN200-110 binding. Bepridil stimulated [3H] PN200-110 binding, whereas phencyclidine was without effect. The photoaffinity probe [3H]azidopine labeled a single polypeptide that migrated with an apparent molecular weight of 185,000-190,000 in sodium dodecyl sulfate gel electrophoresis. The dihydropyridine receptor was found to bind specifically to wheat germ agglutinin columns. These results demonstrate the presence of a Ca2+ channel blocker complex in adrenal medulla. The drug receptor sites reside on a glycoprotein complex in which a polypeptide analogous to the alpha 1-subunit of the L-type Ca2+ channel from skeletal muscle has been identified.
Mol Pharmacol 1990 Feb
PMID:Identification, characterization, and photoaffinity labeling of the dihydropyridine receptor associated with the L-type calcium channel from bovine adrenal medulla. 215 69

In designing new DNA recognizing and cleaving reagents, we introduce herein a bisacridine derivative (referred to as bisacridine) in which two acridine heterocycles are connected by a penta(ethylene glycol) bridging chain. This compound offers two possible functions: 1, stabilization of DNA bisacridine intercalator complex by metal ion. The penta(ethylene glycol) chain stabilizes metal ions binding to the phosphate site of DNA, where the penta(ethylene glycol) chain constitutes a part of a pseudomacrocyclic ligand for metal binding; and 2, enhancement of metal-assisted hydrolytic cleavage of DNA by means of a metal concentration effect by the pseudomacrocyclic ethereal chain. The binding isotherms of bisacridine with DNA in the presence of metal ions showed that the binding was mainly governed by the cation exchange reaction on the anionic DNA polymer chain, i.e., the exchange between metal ions and the cationic bisacridine. The bisacridine showed an increase DNA binding ability compared to quinacrine, the monoacridine counterpart, and caused an enhancement of DNA cleavage in the presence of Cu2+ ions. Additional experiments which included DNase 1 footprinting in the presence of bisacridine and the DNA cleavage by Cu2+/bisacridine using a 32P end-labelled DNA fragment, suggested that the Cu2(+)-assisted DNA cleavage sites in the presence of bisacridine were in reasonable overlap with the DNA binding sites of bisacridine.
J Mol Recognit 1990 Aug
PMID:Cleavage of double helical DNA by Cu2+ ion in the presence of bisintercalator containing penta(ethylene glycol) connector chain. 227 32

The effect of 5-hydroxytryptamine (5-HT) receptor stimulation on protein kinase C (PKC) activity and translocation was assessed in slices or synaptosomes obtained from rat brain. Serotonin (0.5-100 microM) and the specific 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (0.01-10 microM) but not the 5-HT1A or 5-HT1B agonists elicited time- and dose-related translocations in cortical slices. The maximal translocation elicited by 5-HT (10-100 microM, 15 min) or DOI (1 microM, 10 min) was similar to that achievable by the phorbol ester phorbol myristate acetate (PMA) (162 nM). In synaptosomes, short exposures to depolarizing concentrations of K+ (45-65 mM) resulted in PKC translocation. In addition, PMA but not serotonin induced enzyme translocation in synaptosomes. In slices, serotonin-stimulated PKC translocation was prevented by 5-HT2 antagonists but not by dopamine or alpha-adrenergic antagonists. PKC translocation induced by serotonin but not by PMA was inhibited by incubation of slices in a Ca2+-free medium. However, addition of 0.5 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to the incubation mixture abolished the effects of both serotonin and PMA. These results indicate that, in cortical slices, serotonin operating via a 5-HT2 postsynaptic receptor can induce the translocation of PKC from cytosol to membrane. This action of the neurotransmitter appears to be dependent on extracellular Ca2+.
Mol Pharmacol 1990 Jan
PMID:Central 5-hydroxytryptamine receptor-linked protein kinase C translocation: a functional postsynaptic signal transduction system. 230 46

The effects of modification of bovine pancreatic ribonuclease A by monomethoxypoly(ethylene glycol) (MPEG) were examined for changes in recognition by antiRNase antibodies, enzymatic activity against low and high molecular weight substrates and conformational stability to temperature elevation. Modified forms of RNase were prepared containing an average of 4, 9, and 11 mol of MPEG/mol protein, by amino group modification. These were analysed by binding to RNase antibodies crosslinked to solid phase-immobilized protein A. The affinity column was incorporated into a high performance liquid chromatograph and the RNase species were studied by both zonal and frontal analytical affinity chromatography. An antibody dissociation constant of 7.6 x 10(-8) M was found for unmodified RNase, as compared to values of 1.3 x 10(-7) and 1.2 x 10(-6) M for RNase with 4 and 9 covalently bound MPEG chains, respectively. Modification also led to progressive loss of enzymatic activity against RNA, down to 3% for the most highly modified enzyme. In contrast, enzymatic activity against cytidine-2',3'-cyclic monophosphate was suppressed to a maximum of only 33% at the highest modification level, and the stability to temperature, as followed by circular dichroism, was reduced only partially, from 67 degrees C for native protein to 57 degrees C for RNase with 11 mol equivalents MPEG incorporated. The above differential effects on enzymatic activity, antibody binding and temperature effects are consistent with the view that MPEG modification has relatively small effects on conformational stability and small molecule accessibility, but more dramatic effects on large molecule (substrate as well as antibody) accessibility.(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Recognit 1990 Apr
PMID:Effects of monomethoxypoly(ethylene glycol) modification of ribonuclease on antibody recognition, substrate accessibility and conformational stability. 236 Oct 62

Disruption of normal calcium homeostasis has been implicated in the development of cell injury by certain chemicals. Furthermore, calcium channel blockers, which may prevent such a disruption, were shown to protect against this type of cellular damage in some excitable and nonexcitable tissues. Therefore, the present work was designed to address the role of calcium in the pachytene cell death caused by ethylene glycol monomethyl ether (EGME) by investigating the effect of the calcium channel blockers, verapamil and diltiazem, on the pathogenesis of such lesions. Male F344 rats were treated with a single gavage dose of 200 or 300 mg EGME/kg. Other groups of rats were treated with the same doses of EGME in combination with one, two, three, or four doses of verapamil or diltiazem. Twenty-four hours after administration of EGME, the animals were sacrificed, and the left testis and epididymis were excised, fixed in Bouin's solution, embedded in paraffin, sectioned, and stained with PAS-H. The sections were evaluated "blind" and scored for the number of lesioned stage XIV tubules. At 200 mg/kg, EGME produced a moderately severe lesion as characterized by pachytene spermatocyte cell death in stage XIV semniferous tubules. Verapamil was protective against this lesion with the protection being directly proportional to the number of verapamil doses administered and was maximum in rats treated with three doses. At 300 mg/kg, EGME caused a severe lesion in the testis, and verapamil was not as effective in protecting against this lesion as against the low dose of EGME. In contrast, diltiazem was not as effective as verapamil at either dose of EGME. These studies show, for the first time, that verapamil protects rats against EGME-induced testicular toxicity.
Exp Mol Pathol 1990 Jun
PMID:Calcium channel blockers protect against ethylene glycol monomethyl ether (2-methoxyethanol)-induced testicular toxicity. 236 34

The low-angle X-ray diffraction pattern from Lethocerus flight muscle fibres was recorded in rigor or under two conditions that modify crossbridge structure and behaviour, aqueous adenylylimidodiphosphate (AMPPNP) and AMPPNP + calcium in an ethylene glycol-water mixture. The effects on the 38.7 nm layer-line peaks (hk.6) of the diffraction patterns were studied in detail. In aqueous AMPPNP at room temperature, a condition in which rigor tension drops to half without loss of stiffness, the peaks remained nearly as intense as in rigor except for the 10.6, which dropped to half. In 20% (v/v) ethylene glycol-AMPPNP + 100 microM-Ca2+ at 23 degrees C (gly + pnp + Ca), a condition which removed muscle tension but left stiffness close to the rigor value, the 10.6 and 11.6 peaks greatly decreased but the 31.6 remained relatively high. The 14.5 nm meridional peak (00.16) became stronger on addition of AMPPNP and again on adding glycol + calcium. Considered in terms of constructively interfering filaments and crossbridges, the X-ray data indicated a transfer of diffracting crossbridge mass towards the thick filament as relaxation proceeds. We compared the X-ray diffraction patterns and crossbridge structure seen with electron microscopy (EM) under the same chemical conditions. EM and X-ray observations were mutually quite consistent overall. However, X-ray data indicated that more crossbridge mass was stereospecifically related to actin before fixation in the partially relaxed state (gly + pnp + Ca) than was suggested by the disordered crossbridge profiles seen by EM. We conclude that myosin heads at the start of the power stroke may both be closely related to their thick filament origins and form actin-determined attachments to the thin filament.
J Mol Biol 1990 Jul 05
PMID:X-ray diffraction and electron microscopy from Lethocerus flight muscle partially relaxed by adenylylimidodiphosphate and ethylene glycol. 237 Jun 60

The aim of the present study was to investigate the possible role played by the Na(+)-Ca2+ exchange system in the modulation of [3H]dopamine ([3H]DA) release from tuberoinfundibular hypothalamic (TIDA) neurons. 2',4'-Dimethylbenzamil (DMB) dose-dependently (10-100 microM) inhibited Na(+)-dependent 45Ca2+ efflux from brain synaptosomes. This compound (30-300 microM), as well as alpha-phenylbenzamil amiloride (30-100 microM), another inhibitor of the Na(+)-Ca2+ antiporter, was also able to stimulate basal release of [3H]DA from superfused TIDA neurons. This stimulation was completely prevented by the removal of extracellular Ca2+ ions, in the presence of 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. In addition, DMB-induced [3H]DA release was unaffected by the dopamine transport inhibitor nomifensine (10 microM). On the other hand, 5-[N-methyl-N-guanidinocarbonylmethyl]amiloride (MGCMA) (100-300 microM), which lacks inhibitory properties on the Na(+)-Ca2+ exchanger but behaves as an inhibitor of the Na(+)-H+ antiporter, failed to modify basal [3H]DA release from TIDA neurons. When the Na(+)-Ca2+ antiporter operates as a Ca2+ influx pathway, as occurs upon the removal of extracellular Na+ ions, Na(+)-dependent 45Ca2+ uptake in brain synaptosomes was dose-dependently (10-300 microM) inhibited by DMB, whereas DMB itself was unable to prevent 55 mM K(+)-induced 45Ca2+ uptake, which mainly reflects the activation of voltage-operated Ca2+ channels. In keeping with these results, ouabain (500 microM)-induced [3H]DA release, which depends on the activation of the Na(+)-Ca2+ exchanger due to inhibition of the Na(+)-K(+)-ATPase pump, was prevented by superfusion of TIDA neurons with DMB (50 microM). By contrast, MGCMA (100 microM) failed to modify either Na(+)-dependent 45Ca2+ influx or ouabain-induced [3H]DA release. In conclusion, the results of the present study appear to suggest that the pharmacological inhibition of the Na(+)-Ca2+ antiporter by amiloride analogues may affect DA release from central neurons. Opposite effects are observed, depending on the direction of operation of the exchanger. In fact, when the Na(+)-Ca2+ exchanger operates as a Ca2+ efflux pathway, its pharmacological blockade can produce a stimulation of DA release. In contrast, when this antiporter operates as a Ca2+ influx pathway, as occurs as a consequence of the inhibition of the Na(+)-K(+)-ATPase pump by ouabain, its pharmacological blockade can prevent ouabain-induced DA release from TIDA neurons.
Mol Pharmacol 1990 Sep
PMID:Na(+)-Ca2+ exchange activity in central nerve endings. II. Relationship between pharmacological blockade by amiloride analogues and dopamine release from tuberoinfundibular hypothalamic neurons. 240 28

Chromosomes were isolated in a preparative scale by synchronisation of CHO cells with a double Thymidine block followed by an arrest in the metaphase by addition of Colcemid. Under proper cultivation conditions a mitotic index of 77% total cells could be routinely achieved. Bulk chromosome preparations free of nuclei and other subcellular particles have been obtained by low speed centrifugation followed by a 60 transfer countercurrent distribution using aqueous two phase systems composed of polyethylenglycol and dextran. The partition of CHO chromosomes previously purified in aqueous two phase systems were studied further to develop a protocol for the separation and isolation of individual chromosomes. Partition experiments with chromosomes changing the electrostatic phase potential by addition of charged PEG-derivatives suggest the existence of relatively highly charged chromosome groups. Most promising results with regard to separation were obtained using two PEG-derivatives, which interact specifically with the bases in DNA. For this affinity partitioning a GC- and AT-specific macroligand were employed. Comparing CCD's using each of these ligands information on the GC and AT content of exposed DNA in the chromosomes groups could be derived, demonstrating that specific sequences of DNA are accessible at the surface of metaphase chromosomes.
Mol Cell Biochem 1986 Feb
PMID:Isolation and fractionation of CHO chromosomes in aqueous two phase systems using charged polymers and base specific macroligands. 242 Nov 51

Stimulation of T cells or the Jurkat T-cell line with soluble antibodies to the CD3/T-cell receptor complex causes mobilization of cytoplasmic Ca2+, which is blocked by pertussis toxin but not by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and translocation of protein kinase C activity from the cytoplasm to the membrane. Such stimulation also causes phosphorylation of pp60c-src at an amino-terminal serine residue. These activities are consistent with induction of phosphatidylinositol metabolism after antibody binding. Anti-CD3 stimulation with antibody in solution, however, does not cause Jurkat cells to release interleukin 2 and blocks rather than induces proliferation of T cells. Induction of interleukin 2 production by Jurkat cells and proliferation by normal T cells requires anti-CD3 stimulation with antibody on a solid support, such as Sepharose beads or a plastic dish. Thus, we examined phosphorylation of pp60c-src after stimulation of Jurkat cells with anti-CD3 in solution or on solid phase. Both of these caused serine phosphorylation of pp60c-src that was indistinguishable even after 4 h of stimulation. These results indicate that the mode of anti-CD3 stimulation (in solution or on solid phase) controls a cellular function that modifies the consequences of signal transduction through phosphatidylinositol turnover.
Mol Cell Biol 1987 Feb
PMID:Stimulation of T cells through the CD3/T-cell receptor complex: role of cytoplasmic calcium, protein kinase C translocation, and phosphorylation of pp60c-src in the activation pathway. 243 33


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