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Biomedical subjects

K Haga

Publications and source records attributed to K Haga.

At least 109 records · Page 6Linked to original sources
PubMed1986

Functional interaction of purified muscarinic receptors with purified inhibitory guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles.

The GTP binding regulatory protein (Ni involved in adenylate cyclase inhibition was purified from rat brain and reconstituted, together with muscarinic cholinergic receptors purified from porcine brain, into phospholipid vesicles. Guanosine 5'-O-(3-[35S]thio)-triphosphate ([35S]GTP gamma S) binding and GTP hydrolyzing activities of reconstituted Ni were stimulated by the addition of a muscarinic agonist, carbachol. The effect of carbachol was to increase the Vmax values of these activities, but the Km values were also increased slightly in most cases. Carbachol bound to vesicles with the same order of magnitude of Km as that for stimulation of GTPase. The affinity of this binding was reduced by GTP gamma S, indicating that the high-affinity receptor-Ni complex was formed in a GTP-dependent manner in reconstituted vesicles. Incubation of Ni with NAD and islet-activating protein (IAP), pertussis toxin, caused ADP-ribosylation of the alpha-subunit of Ni. The criteria for the receptor-Ni interaction, i.e. carbachol stimulation of the activities of Ni and the GTP gamma S effect on carbachol binding, were no longer observed, when this IAP-treated Ni, instead of the nontreated Ni, was reconstituted into vesicles, though there was no difference between IAP-treated and nontreated Ni in their basal activities observable without carbachol. No, the protein with a character very similar to Ni in rat brain, was also coupled to muscarinic receptors when they were reconstituted into vesicles under the same conditions. Thus, GTP-binding proteins serving as the substrate of IAP-catalyzed ADP-ribosylation are capable of interaction functionally with muscarinic receptors in phospholipid vesicles.

Adenylate Cyclase Toxin↗
PubMed1986

Primary structure of the alpha-subunit of bovine adenylate cyclase-inhibiting G-protein deduced from the cDNA sequence.

The primary structure of the alpha-subunit of the adenylate cyclase-inhibiting G-protein (Gi) has been deduced from the nucleotide sequence of cloned DNA complementary to the bovine cerebral mRNA encoding the polypeptide. A much higher degree of amino acid sequence homology is observed between the alpha-subunits of Gi and transducin (68%) than between those of Gi and the adenylate cyclase-stimulating G-protein (Gs) (43%) or between those of transducin and Gs (42%).

Adenylyl Cyclase Inhibitors↗
PubMed1985

Purification of the muscarinic acetylcholine receptor from porcine brain.

The muscarinic acetylcholine receptor of porcine cerebrum has been purified to apparent homogeneity by affinity chromatography, with conjugated 3-(2'-aminobenzhydryloxy)tropane (ABT) as described previously (Haga, K., and Haga, T. (1983) J. Biol. Chem. 258, 13575-13579). In a single step purification using 900 ml of digitonin/cholate-solubilized preparations and 300 ml of the ABT-agarose gel, we obtained, in a yield of 10-15%, more than 250 pmol of muscarinic receptors which bind [3H]N-methylscopolamine with a specific activity of 1,000-5,000 pmol/mg of protein (1,000-5,000-fold purification). The muscarinic receptors eluted from the ABT-agarose gel with 0.1 mM atropine were adsorbed to hydroxylapatite and then recovered as a concentrated solution. Muscarinic receptors were further purified by rechromatography with the same gel or by gel permeation high pressure liquid chromatography. The amino acid composition of the purified receptor was determined, and the specific activity of the purified preparation was estimated to be 13,100 pmol/mg of protein on the basis of amino acid composition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified receptors with or without radioiodination revealed a single, major band with an apparent Mr of 70,000 either by silver staining or radioautogram. The major band corresponded to the band which specifically bound [3H]propylbenzylcholine mustard (irreversible muscarinic ligand). The purified receptor showed essentially the same specificity for muscarinic ligands as unpurified receptors.

Animals↗
PubMed1984

Hydrodynamic properties of muscarinic acetylcholine receptors solubilized from rat forebrain.

Muscarinic receptors from rat forebrain have been solubilized by Lubrol PX, lysophosphatidylcholine (LPC), digitonin and cholate/1 M sodium chloride. The overall level of solubilization was characterized using receptors prelabelled with an irreversible antagonist. The recovery of nondenatured soluble binding activity was estimated using reversible tritiated antagonists. All these detergents solubilized 60-85% of the total binding sites. In Lubrol PX most of the receptors were recovered in a denatured form. In the other detergents 30-90% of the solubilized receptors were stable and capable of binding reversible [3H]-antagonists with high affinity. The hydrodynamic properties of the soluble receptors have been examined by gel filtration and sucrose gradient centrifugation in H2O and D2O. The soluble receptors in Lubrol PX, lysophosphatidylcholine and cholate were, in general, heterogeneous as regards their molecular size. Estimates of the molecular weight after correction for bound detergent, varied from 82,000 to 134,000. Conditions were identified under which the receptor was largely monodisperse, and the estimates of molecular weight agreed with values (ca. 83,000) from sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis. The amount of bound detergent could not be calculated for the digitonin-muscarinic receptor complex which had an estimated overall median molecular weight of about 290,000. It is concluded that a subpopulation of muscarinic receptors from the rat forebrain is capable of existing in a monomeric soluble form and binding ligands. There is also evidence that complexes with other proteins can exist, but their specificity and functional relevance are not known.

Animals↗
PubMed1984

Antiulcer activity of clotiazepam in rats.

Effect of the anti-anxiety drug clotiazepam on the experimental gastric ulceration induced by restraint and water-immersion stress or aspirin was studied in rats. Clotiazepam prevented the development of each gastric ulcer. From the effect of clotiazepam on aspirin-induced ulceration, we presumed that clotiazepam should have some other antiulcer mechanism in addition to its action on the central nervous system. There was an appreciable correlation between the decrease in the hexosamine level of gastric tissue and associated ulceration. After treatment with aspirin, the hexosamine level was abruptly reduced and was maintained at a low level for several hours. In the clotiazepam -pretreated group, the hexosamine level reduced by aspirin was progressively restored to the intact level. By histological examination with periodic acid-Schiff (PAS)/alcian-blue (AB) staining, clotiazepam increased the amount of gastric mucopolysaccharides decreased by aspirin. Clotiazepam did not affect gastric secretion in pylorus-ligated rats. Atropine and cimetidine inhibited ulceration induced by stress or aspirin and gastric secretion, but did not affect the hexosamine level reduced by aspirin. These results indicate that the antiulcer efficacy of clotiazepam may be attributed to its action not only on the central nervous system, but also on the mucus in gastric mucosa.

Animals↗
PubMed1983

Affinity chromatography of the muscarinic acetylcholine receptor.

A novel compound, 3-(2'-aminobenzhydryloxy)-tropane (ABT), and an ABT-agarose gel were synthesized and used for the purification of solubilized muscarinic receptors. ABT had a high affinity with an apparent dissociation constant (Kd) of 7 nM for the muscarinic receptors solubilized from the porcine brain by digitonin. An ABT-agarose gel was prepared by coupling ABT with epoxy-activated Sepharose 6B, and the degree of substitution to the gel was determined to be 4-5 mumol/ml of the gel by UV absorption spectrum. During affinity chromatography using 10 ml of the ABT-agarose gel and 100 ml of the digitonin-solubilized preparation, 70% of muscarinic receptors were adsorbed to the gel, in marked contrast with the adsorption of only 2% of proteins. Approximately 25% of muscarinic receptors applied to the gel were eluted biospecifically with 1 mM muscarinic ligands. The purified fraction showed a high affinity for [3H]quinuclidinyl benzylate with a Kd of 0.4 nM and similar specificity for muscarinic ligands to that of unpurified soluble receptors. The protein concentration of the purified fraction was too low to be determined accurately, but very approximately a purification of 10(3)-fold was indicated.

Animals↗
PubMed1983

[General pharmacology of traxanox sodium. I. Effects on the central, somatic, and autonomic nervous systems, digestive system, urologic organs, and reproductive system].

Traxanox at the upper dose of 300 mg/kg, p.o., showed no effect on the somatic and autonomic nervous systems in the various tests. This agent caused some relaxation of the guinea pig tracheal strip in the resting tone at a concentration of 10(-6)M or more; however, its activity was less potent than that of isoproterenol and paraverine. Traxanox had no competitive antagonistic action against chemical mediators. Treatment of rats with this agent (3 mg/kg, i.v., or 10 mg/kg, s.c.) resulted in an inhibition of gastric (acid) secretion. Intravenous injections of traxanox (1-10 mg/kg) in dogs caused an immediate but transient inhibition of gastric and jejunal movement, and after a short time, slight potentiation of the latter; however, pretreatment with atropine prevented this latter potentiation. In the in vitro test, 10(-4)M traxanox caused contraction of the guinea pig ileum, a response which was inhibited by atropine. Traxanox (300 mg/kg, p.o.), however, did not show any effects on gastrointestinal propulsion in mice, nor did it have any effect on the gastrointestinal mucosa, gastric ulcers or bile secretion in rats. Traxanox (300 mg/kg, p.o.) showed a diuretic action in both normal and adrenoectomized rats. This action, however, was not observed in the rats treated with indomethacin (1 mg/kg, i.p.). This agent (10 mg/kg, i.v.) suppressed the spontaneous uterine contractions of pregnant rats in some cases. These findings suggest that traxanox at doses (1-5 mg/kg, p. o.) showing antiallergic activity has little effect on the central, somatic and autonomic nervous systems, digestive system, urinary organs and reproductive organs.

Animals↗
PubMed1981

Characterization by [3H]dihydroergocryptine binding of alpha-adrenergic receptors in neuroblastoma X glioma hybrid cells.

[3H]Dihydroergocryptine ([3H]DHE) was shown to bind to sites in membranes from neuroblastoma X glioma hybrid cells (NG 108-15) that had the characteristics expected of alpha-adrenergic receptors. The binding was saturable with 0.3 pmol [3H]DHE bound per mg of protein and of high affinity, with an apparent dissociation constant (KD) of 1.8 nM. The specificity of the binding site for various ligands was more similar to that of alpha 2 receptors than to that of alpha 1. No specific binding of [3H]WB-4101 was found in the membranes derived from NG 108 cells. This finding also indicated that the [3H]DHE binding site in the cell is the alpha 2 receptor. GTP lowered the affinity of agonists for the [3H]DHE binding site, although the nucleotide hardly affected the affinity of antagonists including [3H]DHE.

Adrenergic alpha-Antagonists↗
PubMed1981

Dual contrast echocardiography following peripheral venous injection.

Contrast echocardiography was carried out using a dual method in which two ways of ultrasonic beam were recorded on a cross-sectional plane of the heart. The advantage of this method was to make it possible to demonstrate the right and left cardiac cavities simultaneously and to catch the flow of contrast echoes from many-sided viewpoints, whereas single beam echocardiography could not make it. Dual contrast studies following peripheral venous injection were performed on patients with congenital heart diseases. These studies revealed accurate hemodynamics in 26 of 27 patients.

Child↗
PubMed1980

Comparison of three blood-clotting substances in Staphylococcus aureus strains.

Besides the two well-known blood-clotting substances, coagulase and clumping factor, a third one has been identified from the staphylococci which is a cell surface polysaccharide, is alkali stable, and induces compact-colony formation in serum soft agar. Using some 97 clinical strains of Staphylococcus aureus, we found that in production and activity the substances were distinctly different.

Agar↗
PubMed1979

Studies on the interference between the allergic reactions caused by different antigen-antibody systems.

Interference between two different in vivo antigen-antibody reactions which occurred simultaneously was studied in guinea pigs. Animals were presensitized with an aqueous fraction (CP) extracted from cells of Corynebacterium equi, strain KO-85. Skin reactions of the Arthus type elicited by the CP fraction inhibited passive cutaneous anaphylaxis (PCA) induced by bovine serum albumin (BSA) and rabbit anti-BSA serum. PCA reactions of the CP-sensitized animals were also inhibited upon simultaneous challenges of BSA and the CP as evidence by reduction of PCA titer of the anti-BSA serum.

Animals↗