metiamide and tiotidine

1. Rat histamine H2 receptors were epitope-tagged with six histidine residues at the C-terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope-tagged H2 receptor were prepared and were used to infect insect Sf9 cells. 2. The His-tagged H2 receptors expressed in insect Sf9 cells showed typical H2 receptor characteristics as determined with [125I]-aminopotentidine (APT) binding studies. 3. In Sf9 cells expressing the His-tagged H2 receptor histamine was able to stimulate cyclic AMP production 9 fold (EC50=2.1+/-0.1 microM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with Ki values of 0.60+/-0.43 microM, 0.25+/-0.15 microM and 28+/-7 nM, respectively (mean+/-s.e.mean, n=3). 4. The expression of the His-tagged H2 receptors in infected Sf9 cells reached functional levels of 6.6+/-0.6 pmol mg(-1) protein (mean+/-s.e.mean, n=3) after 3 days of infection. This represents about 2 x 10(6) copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol-beta-cyclodextrin complex resulted in an increase of [125I]-APT binding up to 169+/-5% (mean+/-s.e.mean, n=3). 5. The addition of 0.03 mM cholesterol-beta-cyclodextrin complex did not affect histamine-induced cyclic AMP production. The EC50 value of histamine was 3.1+/-1.7 microM in the absence of cholesterol-beta-cyclodextrin complex and 11.1+/-5.5 microM in the presence of cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 microM histamine was identical, 85+/-18 pmol/10(6) cells in the absence and 81+/-11 pmol/10(6) cells in the presence of 0.03 mM cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). 6. Immunofluorescence studies with an antibody against the His-tag revealed that the majority of the His-tagged H2 receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well. 7. These experiments demonstrate the successful expression of His-tagged histamine H2 receptors in insect Sf9 cells. The H2 receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His-tag reveal that only a limited amount of H2 receptor protein is functional. These functional receptors are targeted to the plasma membrane.

Topics: Adenylyl Cyclases; Animals; Baculoviridae; beta-Cyclodextrins; Blotting, Western; Cell Line, Transformed; Cholesterol; Cimetidine; Cyclic AMP; Cyclodextrins; Epitopes; Fluorescent Antibody Technique; Guanidines; Histamine H2 Antagonists; Histidine; Insecta; Microscopy, Confocal; Oligonucleotides; Piperidines; Ranitidine; Rats; Receptors, Histamine H2; Transfection

Histamine and the guanosine 3',5'-cyclic monophosphate (cGMP)-inducing agent sodium nitroprusside both increased serotonin (5-HT) uptake and cGMP levels in isolated human platelets in vitro. Histaminergic stimulation was observed at concentrations ranging from 10 nM to 0.25 microM [mean effective concentration (EC50) = 0.1 microM histamine]. The inhibition produced by the H2-receptor antagonists tiotidine, metiamide, and cimetidine was 10-10(5) times more potent on histamine receptors regulating 5-HT uptake and cGMP generation in human platelets than on the histaminergic receptors H1, HIC, H2, and H3 in other tissues. The in vitro histamine-induced 5-HT uptake was prevented by preincubation of isolated human platelets in the presence of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine or the cGMP-lowering agent LY-83583. Histamine was ineffective in stimulating cAMP generation in human platelets and did not interact with effector sites known to downregulate 5-HT uptake, including imipramine, gamma-aminobutyric acid A, peripheral type benzodiazepine-binding sites, and V1a vasopressin receptors inducing human platelet shape change and aggregation. These atypical human platelet histaminergic receptors differ from the previously classified histamine receptors by their apparent high affinity to histamine H2-receptor antagonists and their apparent link with the soluble, nitric oxide-dependent guanylate cyclase. These findings suggest that human platelets express a new subtype H2h of histamine receptors.

Topics: Adult; Aminoquinolines; Biological Transport; Blood Platelets; Cimetidine; Cyclic AMP; Dimaprit; Famotidine; Female; Histamine Antagonists; Histidine; Humans; Imipramine; Impromidine; In Vitro Techniques; Male; Metiamide; Middle Aged; Models, Biological; Platelet Aggregation; Receptors, Histamine; Serotonin

The characteristics of histamine-stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in slices of rabbit cerebral cortex have been investigated. The selective H2-receptor antagonists, cimetidine, tiotidine, metiamide and ranitidine appeared to antagonize the stimulation of cyclic AMP accumulation elicited by histamine in a competitive manner consistent with an interaction with histamine H2-receptors. The H1-receptor antagonist mepyramine (0.8 microM) produced only a weak inhibition of the response to histamine. The inhibition appeared to be non-competitive producing a decrease in the maximal response with little effect on the EC50 value. The specific H2-receptor agonist, impromidine, produced a maximum response of only 31 +/- 2% of that obtained with histamine. Studies with histamine and impromidine in combination indicated that impromidine was not acting as a partial agonist. 2-Thiazolylethylamine, a selective H1-agonist, produced only a weak response (EC50 approximately 1mM) yielding a relative potency with respect to histamine (= 100) of 2.5. In the presence of a supramaximal concentration of impromidine, histamine and 2-thiazolylethylamine further elevated the response to impromidine. In these conditions the relative potency of 2-thiazolylethylamine was increased to 59 (histamine = 100), a value which was comparable with that reported for H1-receptor-mediated contractions of guinea-pig ileum. The H1-receptor antagonists mepyramine, promethazine, triprolidine and chlorpheniramine competitively antagonized the potentiation of impromidine-stimulated cyclic AMP accumulation elicited by histamine and 2-thiazolylethylamine in rabbit cerebral cortex without affecting the response to impromidine alone. (+)-Chlorpheniramine was some 150 fold more potent than the (-)-isomer in this respect. Histamine and adenosine in combination had a much greater than additive effect on the accumulation of cyclic AMP in rabbit cerebral cortical slices. The potentiation of the adenosine response could be partially but not completely antagonized by either cimetidine or mepyramine. In the presence of H2-receptor blockade with 0.02 mM tiotidine, histamine elicited a significant potentiation (EC50 44 microM) of the response to adenosine. This response was antagonized competitively by mepyramine yielding a KB value of 0.05 microM similar to that obtained from inhibition of the potentiation of impromidine-stimulated accumulation of cyclic AMP (0.02 microM). Thes

Topics: Adenosine; Animals; Cerebral Cortex; Chlorpheniramine; Cimetidine; Cyclic AMP; Dose-Response Relationship, Drug; Drug Synergism; Imidazoles; Impromidine; Kinetics; Mathematics; Metiamide; Rabbits; Ranitidine; Receptors, Histamine; Receptors, Histamine H1; Receptors, Histamine H2; Thiazoles

Cumulative concentration-response curves to histamine or dimaprit were constructed on guinea-pig isolated right atria and agonist dose-ratios were determined following addition of ranitidine, cimetidine, metiamide or tiotidine alone or a combination of any two of these H2-receptor blocking drugs. The observed histamine or dimaprit dose-ratios for combinations of any two of the H2-antagonists tested were consistent with results predicted from the equation, DR1+2 = DR1 + DR2 - 1, for two antagonists competing for the same receptor sites. Therefore we conclude that all four H2-antagonists compete for the same histamine H2-receptor.

Topics: Animals; Cimetidine; Dimaprit; Drug Interactions; Furans; Guanidines; Guinea Pigs; Heart; Heart Rate; Histamine; In Vitro Techniques; Metiamide; Ranitidine; Receptors, Histamine; Receptors, Histamine H2; Thiazoles; Thiourea