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Biomedical subjects

T Haga

Publications and source records attributed to T Haga.

At least 145 records · Page 8Linked to original sources
PubMed1988

Agonist and antagonist binding of muscarinic acetylcholine receptors purified from porcine brain: interconversion of high- and low-affinity sites by sulfhydryl reagents.

The affinity for muscarinic ligands of a preparation of muscarinic acetylcholine receptors purified from porcine brain was examined by means of competitive binding of [3H]quinuclidinylbenzylate and unlabeled ligands, followed by computer-assisted nonlinear regression analysis. The displacements by antagonists fitted a single-site model. In contrast, the displacements by agonists did not fit the single-site model and could be explained by assuming two populations of binding sites. The proportion of the sites with high affinity for muscarinic agonists (H-sites) ranged from 25 to 35% of the total number of sites. GTP had no effect on the displacements by agonists, a finding indicating that H-sites did not result from interaction between receptors and GTP-binding proteins. In the presence of dithiothreitol, the affinity for muscarinic ligands decreased. The largest effects were observed on the affinity for pirenzepine and that of H-sites for carbachol. Preincubation of the preparation with 5,5'-dithiobis(2-nitrobenzoic acid) resulted in an increase in the proportion of H-sites to 75% of the total number of binding sites. The results of sucrose density gradient centrifugation of the preparation indicated apparent heterogeneity as to molecular size of the receptors, but this heterogeneity did not correlate with that of the affinity for agonists. In addition, the receptors were detected as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparation, regardless of the presence or absence of disulfide-reducing reagents. These results suggest that the redox state of thiol groups in the receptor molecules is relevant to their affinities for ligands.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

The interaction between D-2 dopamine receptors and GTP-binding proteins.

D-2 dopamine receptors were solubilized from porcine striatal membranes with 0.3% sodium cholate/1 M NaCl and separated from the bulk of the guanine nucleotide-binding regulatory proteins (G-proteins) by Ultrogel AcA34 gel filtration chromatography. The partially purified D-2 receptors were reconstituted in phospholipid vesicles with Gi or Go purified from porcine brain. The dissociation constant (Kd) of the D-2 receptors in the reconstituted vesicles for [3H]spiperone binding was 82-89 pm, which was not affected by the presence or absence of G-proteins. The displacement curve for [3H]spiperone/dopamine was analyzed, assuming that there are two populations of binding sites. The Kd values for the binding sites with high affinity for agonists (HAS) and that for the binding sites with low affinity for agonists (LAS) were approximately 1 microM and 100 microM, respectively. The proportion of HAS was 8% when the receptor preparation was reconstituted into phospholipid vesicles without G-proteins, but it increased to 58-64% with increasing G-protein concentrations. The potency of Go was a little higher than that of Gi. The proportion of HAS in the presence of G-proteins decreased to about 11% on addition of GTP. When G-proteins were treated with islet-activating protein, GTP-sensitive HAS were not observed. These results indicate that at least 50% of the partially purified D-2 receptors interact with both Gi and Go.

Animals↗
PubMed1987

[Laboratory and clinical studies of flomoxef].

Laboratory and clinical studies were performed on a new oxacephem antibiotic, flomoxef (FMOX, 6315-S). In vitro antibacterial activity of FMOX was evaluated in comparison to latamoxef (LMOX), cefmetazole (CMZ), cefazolin (CEZ) using clinically isolated strains of Gram-negative and Gram-positive bacteria. Antibacterial activities of FMOX were stronger than LMOX, CMZ, CEZ against Escherichia coli, Klebsiella but only slightly effective against Staphylococcus aureus. This antibiotic drug was administered to 5 patients consisting of 2 cases with pneumonia, one each with pyelonephritis, chronic bronchitis and urinary tract infection. The drug was given in 1 g drip infusion twice a day for 8 to 13 days. Clinical efficacies of FMOX were excellent in 1 case, good in 2, fair in 1, and unevaluable in 1. As for bacteriological effect of FMOX, organisms were eradicated in 3 cases. No side effect was noted and there was no abnormal change in laboratory findings.

Adult↗
PubMed1986

Reconstitution of the muscarinic acetylcholine receptor. Guanine nucleotide-sensitive high affinity binding of agonists to purified muscarinic receptors reconstituted with GTP-binding proteins (Gi and Go).

Muscarinic acetylcholine receptors purified from porcine brain were reconstituted with two kinds of GTP-binding proteins (Gi and Go). The binding of agonists was affected by guanine nucleotides when the receptor was reconstituted with either Gi or Go, but not in the absence of one of the GTP-binding proteins. The displacement curves with agonists for the [3H]quinuclidinyl benzylate [( 3H]QNB) binding were explained by assuming there are two sites with different affinities for a given agonist. The proportion of the high affinity site increased with increasing concentrations of the GTP-binding proteins, and the maximum value represented 50-70% of the total [3H]QNB-binding sites. Reconstitution of the receptor with both Gi and Go did not increase the proportion any further. These results indicate that Gi and Go interact with the same site, which rules out the possibility that there are two kinds of muscarinic receptors, one interacting with Gi and the other with Go. GDP as well as GTP decreased the affinity for the agonists of the muscarinic receptors reconstituted with Gi or Go. The conversion of GDP to GTP during the incubation was less than 1%, indicating that the effect of GDP is not due to its conversion to GTP, and that the binding of either GTP or GDP with the GTP-binding proteins suppresses their interaction with the receptor.

Animals↗
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↗
PubMed1986

Chronic treatment with nifedipine does not change the number of [3H]nitrendipine and [3H]dihydroalprenolol binding sites.

Possible receptor changes occurring after withdrawal of chronic nifedipine treatment or chronic propranolol treatment were examined by administering nifedipine (100 mg/kg per day) or propranolol (45 mg/kg per day) to rats for 2 weeks and then withdrawing treatment [3H]Nitrendipine and [3H]DHA binding were measured in membrane fragments of the ventricle. In propranolol-treated rats, 8 h after the last administration, the maximum binding for [3H]DHA was significantly increased from 79.9 +/- 8.0 to 139.8 +/- 12.8 fmol/mg protein (mean +/- S.E.); the dissociation constant was significantly increased from 4.9 +/- 0.7 to 10.7 +/- 1.2 nM. On the other hand, in nifedipine-treated rats, 12 and 48 h after the last administration, [3H]nitrendipine binding and [3H]DHA binding had not changed significantly. These results indicate that the mechanism of the calcium antagonist withdrawal syndrome may be different from that of beta-blocker withdrawal syndrome.

Animals↗
PubMed1986

Dose-response curves of central and peripheral airways to nicotine injections in dogs.

The dose-response curves of the central and peripheral airways to intravenously injected nicotine were studied in 55 anesthetized dogs. With intact vagi, nicotine caused a dose-dependent increase in central airway resistance (Rc) similar to the increase in peripheral airway resistance (Rp) at concentrations ranging from 4 to 64 micrograms/kg. However, the responses of both Rc and Rp fell progressively when sequential doses of nicotine greater than 256 micrograms/kg were administered. With intact vagi and the administration of propranolol, there was a greater increase in Rp than in Rc at a nicotine dose of 64 micrograms/kg (P less than 0.05). With vagotomy, the responsiveness of both central and peripheral airways to nicotine decreased with doses of nicotine less than 64 micrograms/kg, but with doses of nicotine greater than 256 micrograms/kg the suppressive effect of nicotine on both Rc and Rp was less than that seen with intact vagi. Under conditions in which the vagi were cut and atropine administered, the responsiveness of nicotine was even further depressed. Combinations either of atropine and chlorpheniramine or atropine and phenoxybenzamine also completely blocked reactions to nicotine. Additionally reactions to nicotine were completely blocked by hexamethonium. These results suggest that nicotine increases both Rc and Rp mainly through a vagal reflex and stimulation of the parasympathetic ganglia.

Airway Resistance↗
PubMed1986

[The protocol of informing cancer patients of their condition].

The seven points we must consider in informing cancer patients of their condition are as follows: It is necessary that informing the patient has one or more of the following purposes: Patients have a right to know the truth. Any patient who desires to know the nature of his own disease has a right to be told. To avoid legal action. In cases where information is uncertain, which may cause difficulty for the patient. To avoid the burden of fabricating an untrue explanation. To help the patient bear radical treatment such as surgery, radiation and/or chemotherapy. To allow the patient to put an end to his/her life's work or finalize problems of property. To help the patient see the need to change his/her way of life. Are the conditions sufficient or not? Almost all these conditions must be clear. Is there any constructive purpose in informing the patient? Does the patient have a rational and receptive character? Is the patient in a mentally stable condition? Is these personal experience of death in the patient's family. Does the patient have a sure faith, thoughts and/or views of life and death? Is there enough cooperative work among the medical team? Is there enough cooperative work among the patient's family? Who tells the truth to the patient? Is there a need to inform the patient in stages? At what stage of the disease is the patient told? Where is the patient told? Comprehensive care must be given after informing the patient of his/her condition.

Aftercare↗
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↗
PubMed1985

Bioavailability and diuretic effect of furosemide during long-term treatment of chronic respiratory failure.

The bioavailability and diuretic effect of furosemide 40 mg administered orally for at least 6 months have been compared in patients with chronic respiratory failure and in healthy controls. The mean urinary recovery of unchanged drug was 11.5 mg and 9.41 mg in 24 h after pre- and postprandial administration to 10 patients, whereas the recovery was 14.4 mg in 10 healthy subjects. The diuretic effect, in terms of urine flow and sodium ion excretion in the 6 h after administration, was also less in patients than in healthy subjects. This was ascribed to the lower bioavailability of furosemide in patients, based on the urinary recovery of unchanged drug, and not to a lower level of response to furosemide than in healthy subjects. The mean absolute bioavailability of furosemide in 6 patients was 41.3% and 63.4%, respectively, calculated from unchanged drug and total drug (unchanged plus glucuronide conjugate). Approximately 53.9% of the dose of furosemide was excreted as the glucuronide conjugate after oral administration, and 34.2% after i.v. injection in the 6 patients. In 3 of the 6 patients studied, a distinct first-pass effect for glucuronidation of furosemide was observed after oral administration. In another study, the mean glucuronide fraction recovered in 24-h urine was 20.7% and 7.3% (p less than 0.01) in 38 patients and 12 healthy subjects, respectively. The fraction in urine was not affected by changing the dose of furosemide from 20 to 120 mg. The lower bioavailability in patients as compared to healthy subjects is ascribed to enhanced glucuronidation and incomplete drug absorption.

Administration, Oral↗