European Journal of Pharmacology (v.421, #1)

The present study examined how the multidrug resistance protein (MRP1), which is an ATP-dependent anionic conjugate transporter, also mediates the transport of reduced glutathione (GSH) and the co-transport of the cationic drug, daunorubicin, with GSH in living GLC4/Adr cells. To obtain information on the affinity of GSH for the multidrug resistance protein in GLC4/Adr cells, we investigated the GSH concentration dependence of the ATP-dependent GSH efflux. The intracellular GSH concentration was modulated by preincubation of the cells with 25 μM buthionine sulfoximine, an inhibitor of GSH synthetase, for 0–24 h. The transport of GSH was related to the intracellular GSH concentration up to ∼5 mM and then plateaued. Fitting of the obtained data according to the Michaelis–Menten equation revealed a K m of 3.4±1.4 mM and a V max of 1.5±0.2×10−18 mol/cell/s. The ATP-dependent transport of GSH was inhibited by 3-([{3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl}-{(3-dimethylamino-3-oxopropyl)-thio}-methyl]thio)propanoic acid (MK571), with 50% inhibition being obtained with 1.4 μM MK571. We investigated the GSH concentration dependence of the MRP1-mediated ATP-dependent transport of daunorubicin under conditions where the transport of daunorubicin became saturated. The daunorubicin transport was related to the intracellular GSH concentration up to ∼5 mM and then plateaued. We were therefore in the situation where GSH acted as an activator: its presence was necessary for the binding and transport of daunorubicin by MRP1. However, GSH was also transported by the multidrug resistance protein. The concentration of GSH that gave half the maximal rate of daunorubicin efflux was 2.1±0.8 mM, very similar to the K m value obtained for GSH. In conclusion, the rate of daunorubicin efflux, under conditions where the transport of daunorubicin became saturated, and the rate of GSH efflux determined at any intracellular concentration of GSH were very similar, yielding a 1:1 stoichiometry with respect to GSH and daunorubicin transport. These results support a model in which daunorubicin is co-transported with GSH.
Keywords: Multidrug resistance; MRP1 (multidrug resistance protein); Efflux; Anthracycline; Glutathione;

Nature of the oligomers formed by muscarinic m2 acetylcholine receptors in Sf9 cells by Paul Park; Chi Shing Sum; David R Hampson; Hubert H.M Van Tol; James W Wells (11-22).
Wild-type, FLAG-tagged, and c-myc-tagged muscarinic m2 receptors extracted in digitonin–cholate from singly and co-infected Sf9 (Spodoptera frugiperda) cells were indistinguishable in their binding of [3H]quinuclidinylbenzilate, either before or after purification. The FLAG epitope was found to coimmunoprecipitate with the c-myc epitope when co-infected cells were solubilised in digitonin–cholate, n-dodecyl-β-d-maltoside or Lubrol-PX. The degree of coprecipitation in digitonin–cholate was unaffected by preincubation of the extract for up to 60 min at 30°C, with or without muscarinic receptor ligands; no coimmunoprecipitation occurred in mixed extracts from singly infected cells. As measured by [3H]quinuclidinylbenzilate, the efficiency of immunoprecipitation from co-infected cells was 87% of that from singly infected cells. The amount of receptor immunoprecipitated from the latter, as determined by densitometry, was 2.3-fold that expected from the loss of binding from the extract. The data suggest that at least some of the receptors were trimeric or larger and that oligomers neither formed nor dissociated under the conditions of the experiments. Also, some receptors appear to be non-functional or latent in digitonin-solubilised extracts.
Keywords: Muscarinic receptor; Oligomer; Oligomeric integrity; G protein-linked receptor; Sf9 cell;

We tested the hypothesis that in isolated rabbit cardiac myocytes, the negative functional effects of cyclic GMP are partly mediated by cyclic GMP-dependent protein kinase activity, and that these effects are altered in thyroxine (T4, 0.5 mg/kg/day for 16 days)-induced hypertrophic myocytes. Using isolated ventricular myocytes from control (N=8) and T4 (N=8) hypertrophic hearts, data for percent cell shortening (%) and maximum rate of contraction (μm/s) were collected using a video edge detector at baseline, after the addition of 10−6 M 8-bromo-cyclic GMP (8-Br-cGMP), 10−5 M 8-Br-cGMP, and 10−6 M KT5823 (10-methoxy-10-methoxycarbonyl-9, 10, 11, 12-tetrahydro-9, 12-epoxy-(1H)-diinidolo [1, 2, 3, f–g: 3′, 2′, 1′-k-j]-pyrrolidino-[3,4-i] [1,6]-benzodiazocin-2-methyl-1-one, cyclic GMP protein kinase inhibitor). Protein phosphorylation was determined autoradiographically after gel electrophoresis. In both control and T4 myocytes, 8-Br-cGMP caused a significant decrease in percent shortening (5.56±0.49% to 3.02±0.47% in control and 4.34±0.33% to 3.13±0.17% in T4 myocytes) and maximal rate of contraction 57.35±6.05 to 36.82±3.17 μm/s in control and 58.49±3.28 to 42.88±2.29 μm/s in T4 myocytes). KT5823 significantly increased percent shortening to 3.77±0.28% and rate to 48.68±4.71 μm/s after 8-Br-cGMP only in control myocytes. In T4 myocytes, the changes in percent shortening and rate after KT5823 were not significant. Protein phosphorylation was increased by 8-Br-cGMP in control and to a lesser extent in T4 myocytes, but the increment was reduced by KT-5823 in control only. These data demonstrated that cyclic GMP had negative functional effects partially mediated by cyclic GMP protein kinase in control myocytes. Cyclic GMP also exerted negative functional effects in thyroxine-induced hypertrophic myocytes, but cyclic GMP protein kinase activity was not an important regulator of these effects in T4 ventricular myocytes.
Keywords: Nitric oxide (NO); Second messenger; Myocyte; Hypertrophy; (Rabbit);

Increased behavioral neurosteroid sensitivity in a rat line selectively bred for high alcohol sensitivity by Esa R. Korpi; Riikka Mäkelä; Elena Romeo; Alessandro Guidotti; Mikko Uusi-Oukari; Carmelo Furnari; Flavia di Michele; Maija Sarviharju; Mei Xu; Per H. Rosenberg (31-38).
Acute administration of a neurosteroid 5β-pregnan-3α-ol-20-one induced a greater impairment in motor performance of the selectively bred alcohol-sensitive (ANT) than alcohol-insensitive (AT) rats. This difference was not associated with the sensitivity of γ-aminobutyrate type A (GABAA) receptors, as 5α-pregnan-3α-ol-20-one (allopregnanolone) decreased the autoradiographic signals of t-butylbicyclophosphoro[35S]thionate binding to GABAA receptor-associated ionophores more in the brain sections of AT than ANT rats. Nor was the difference associated with baseline levels of neuroactive progesterone metabolites, as 5α-pregnan-3,20-dione (5α-DHP) and 5α-pregnan-3α-ol-20-one were lower in the ANT rats. After ethanol (2 g/kg, i.p.) administration and the subsequent motor performance test, the increased brain concentrations of these metabolites were still lower in the ANT than AT rats, although especially in the cerebellum the relative increases were greater in the ANT than AT rats. The present data suggest that the mechanisms mediating neurosteroid-induced motor impairment are susceptible to genetic variation in rat lines selected for differences in ethanol intoxication.
Keywords: Selected rat line; GABAA receptor; Progesterone; Pregnanolone; Motor impairment; Allopregnanolone;

Antisera against endogenous opioids increase the nocifensive response to formalin: demonstration of inhibitory β-endorphinergic control by Hsiang-en Wu; Kuei-chun Hung; Masahiro Ohsawa; Hirokazu Mizoguchi; Leon F Tseng (39-43).
The roles of endogenous opioid peptides in the brain in the modulation of nocifensive responses to formalin in ICR mice were studied. Mice were pretreated intracerebroventricularly (i.c.v.) with rabbit antiserum against β-endorphin, [Leu5]enkephalin, [Met5]enkephalin or dynorphin A-(1–17) 1 h prior to intraplantar injection of formalin (0.5%, 25 μl) and the nocifensive licking responses were then observed. Pretreatment of mice with antiserum against β-endorphin enhanced the second phase, but not the first phase of the nocifensive responses to formalin. Pretreatment with antiserum against [Leu5]enkephalin also caused a small but statistically significant enhancement of the second phase, but not the first phase of nocifensive responses to formalin. On the other hand, pretreatment with antiserum against [Met5]enkephalin or dynorphin A-(1–17) did not affect the nocifensive response to formalin. Our results indicate that β-endorphinergic, and to a lesser extent, [Leu5]enkephalinergic systems are activated at the supraspinal sites to attenuate the nocifensive responses to formalin stimulation.
Keywords: Tonic pain; Nocifensive response; Formalin-induced; Antiserum against β-endorphin; Pain control descending; (Mouse);

Hypoxia impairs endothelium-dependent relaxation in organ cultured pulmonary artery by Takahisa Murata; Hideyuki Yamawaki; Masatoshi Hori; Koichi Sato; Hiroshi Ozaki; Hideaki Karaki (45-53).
In intrapulmonary arteries cultured under hypoxic conditions (5% oxygen) for 7 days, endothelium-dependent relaxation and cGMP accumulation induced by substance P were decreased as compared to those of a normoxic control (20% oxygen). In rabbit mesenteric arteries exposed to chronic hypoxia, however, endothelial dysfunction was not observed. Furthermore, in endothelium-denuded pulmonary arteries exposed to hypoxia, neither relaxation nor cGMP accumulation due to sodium nitroprusside differed from those of the normoxic control. Hypoxia did not change the mRNA expression of endothelial NO synthase (eNOS), the protein expression of eNOS or the eNOS regulatory protein caveolin-1 as assessed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) or whole-mount immunostaining. Morphological study revealed atrophy of endothelial cells and condensation of the eNOS protein in many cells. These results suggest that chronic hypoxia impaired NO-mediated arterial relaxation without changing either the eNOS protein expression or the NO-sensitivity of smooth muscle cells in pulmonary arteries. Changes in cell structure and organization may be involved in endothelial dysfunction.
Keywords: Hypoxia; Organ culture; Endothelium-dependent relaxation;

Effect of sodium tauroursodeoxycholate on phalloidin-induced cholestasis in rats by Kaoru Ishizaki; Shigemasa Kinbara; Norio Hirabayashi; Kazuyuki Uchiyama; Minoru Maeda (55-60).
We investigated the therapeutic effect of tauroursodeoxycholate on phalloidin-induced cholestasis in rats. Intrahepatic cholestasis was induced by administration of phalloidin (500 μg/kg, i.p.) for 7 days. From the day of the last phalloidin injection, tauroursodeoxycholate (60–360 μmol/kg) was given intravenously twice a day for 4 days. On the next day after the last tauroursodeoxycholate administration, bile flow, serum biochemical parameters and biliary lipid excretion rates were determined. Tauroursodeoxycholate significantly suppressed the decrease in bile flow and increases in serum alkaline phosphatase, leucine aminopeptidase and glutamic pyruvic transaminase activities, cholesterol, phospholipid and bile acid concentrations observed in phalloidin-induced cholestasis in rats. Furthermore, tauroursodeoxycholate significantly improved the biliary cholesterol and phospholipid excretion rates in phalloidin-induced cholestasis in rats. These results demonstrate the usefulness of tauroursodeoxycholate as a therapeutic agent in intrahepatic cholestasis.
Keywords: Bile flow; Tauroursodeoxycholate; Phalloidin; Cholestasis; (Rat);

Receptor constants for endomorphin-1 and endomorphin-1-ol indicate differences in efficacy and receptor occupancy by Mahmoud Al-Khrasani; György Orosz; László Kocsis; Viktor Farkas; Anna Magyar; Imre Lengyel; Sándor Benyhe; Anna Borsodi; András Z Rónai (61-67).
The opioid properties of endomorphin derivatives containing a C-terminal alcoholic(-ol) function were compared to the parent amidated compounds in isolated organs (longitudinal muscle strip of guinea-pig ileum and mouse vas deferens). Similar data were also generated for the μ-opioid receptor selective agonist synthetic peptide (d-Ala2, MePhe4, Gly5-ol)-enkephalin (DAMGO) and its Gly5-NH2 congener (DAMGA). Endomorphin-1-ol (Tyr-Pro-Trp-Phe-ol) had an IC50 of 80.6 nM in mouse vas deferens and 61.2 nM in guinea-pig ileum; the corresponding values for endomorphin-2-ol (Tyr-Pro-Phe-Phe-ol) were 49.6 and 48.2 nM, for DAMGO 59.8 and 29.2 nM, respectively. As it was indicated by the antagonism by naltrexone, the agonist actions were exerted exclusively at μ-opioid receptors in both organs. The -ol derivatives were slightly (2.3–4.3 times) less potent than the parent amides in the bioassays: all peptides had, apparently, full agonist properties in intact preparations. With the aim of revealing potential partial agonist properties among the investigated peptides, we partially inactivated the μ-opioid receptor pool in mouse vas deferens by 5×10−7 M β-funaltrexamine. The calculated receptor constants indicated a “high-affinity, low intrinsic efficacy” profile (i.e. a potential partial agonist property) for endomorphin-1, an intermediate character for endomorpin-1-ol and full agonism for DAMGA and DAMGO. Apparently, a higher receptor fraction remained accessible for endomorphin-1 (42.8%) than for the -ol congener (14.0%), DAMGO (20.2%) and DAMGA (14.1%) after partial inactivation.
Keywords: Endomorphin-1-ol; Endomorphin-2-ol; DAMGO ((d-Ala2, MePhe4, Gly5-ol)-enkephalin); DAMGA ((d-Ala2, MePhe4, Gly5-NH2)-enkephalin); Ileum; Vas deferens; β-Funaltrexamine; Receptor constant;