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Biomedical subjects

T Asakawa

Publications and source records attributed to T Asakawa.

At least 109 records · Page 6Linked to original sources
PubMed1983

Catecholamine sensitivity and cardiac myosin ATPase activity in dogs treated with propranolol.

The effects of propranolol treatment on adrenoceptor reactivity and on cardiac myosin ATPase activity have been studied. Eight mongrel dogs weighing 8 to 14 Kg were given propranolol orally 3 times daily (10 mg/Kg/day) for 2 weeks. Although the levels of propranolol were effective and there was a significant decrease in resting heart rate, there was no evidence of a rebound adrenoceptor hypersensitivity after abrupt withdrawal of propranolol treatment. The responsiveness of cardiovascular systems to 1-isoproterenol (1-ISO) was significantly attenuated 15 hrs after the last medication, but it did not differ from the premedication levels 6 days after discontinuation of medication. The effects of 1-ISO on adenylate cyclase activity in the myocardial membrane preparations were not significantly different between the 3 groups (no medication, 1 day and 6 days after discontinuation). With propranolol treatment, though a rise in total plasma catecholamine (norepinephrine, epinephrine, dopamine) levels was the predominant change, there were no definite changes in respective catecholamines. Serum triiodothyronine (T3) increased during medication in 7 of the 8 animals and decreased after medication. Calcium-activated and K+-activated myosin ATPase activity in the inner and outer layers of the left ventricular wall were uniform in unmedicated dogs, and propranolol produced no significant effects. No reliable evidence for propranolol withdrawal syndromes was provided in the present study.

Adenosine Triphosphatases↗
PubMed1983

Separation and properties of a regulatory GTPase activity associated with the adenylate cyclase system in rat brain synaptic plasma membranes.

A GTPase activity having a Km value of 0.5 microM was present in synaptic plasma membranes from rat brain. The activity was inhibited 63% and 24% by NaF and cholera toxin respectively and solubilized from the membranes together with the adenylate cyclase activity. Upon gel chromatography, the solubilized GTPase activity was co-eluted with the GTP-binding regulatory protein of adenylate cyclase. A similar GTPase activity was also associated with the regulatory protein from myelin. The regulatory protein fraction enhanced the activity of the catalytic unit of adenylate cyclase in the presence of 5'-guanylylimidodiphosphate but inhibited the adenylate cyclase activity by 70% in the presence of GTP. These results indicate the possible involvement of the GTPase activity in the regulation of the adenylate cyclase activity.

Adenylyl Cyclases↗
PubMed1983

Labeling of a GTP-binding regulatory protein of rat brain adenylate cyclase system by cholera toxin-catalyzed ADP-ribosylation.

Cholera toxin ADP-ribosylated several proteins in synaptic plasma membranes of rat brains such as peptides of 66,000, 48,000, 38,000, 25,000, and 18,000 daltons. A 42,000-dalton protein reported to be the GTP-binding regulatory protein in several tissues was faintly labeled and its content varied from preparation to preparation. Adenylate cyclase activity was concomitantly enhanced by more than twice in proportion to the ADP-ribosylation. Accumulated evidence indicated that a protein of 48,000 daltons (48K protein) was a component which constituted the GTP-binding regulatory protein of adenylate cyclase system in rat brain. Among ADP-ribosylated proteins, the 48K protein was the major substrate for the ADP-ribosylation, and the content of this protein was estimated to be about 15 pmol/mg membrane protein. When synaptic membranes were solubilized and applied on a Ultrogel AcA34 gel filtration column to separate the regulatory protein from other components of adenylate cyclase, the 48K protein--but not other ADP-ribosylated proteins--co-eluted with the regulatory protein activity assayed by the reconstitution with catalytic unit of adenylate cyclase. The 48K protein was also detected in myelin fraction which lack adenylate cyclase activity. When solubilized proteins from myelin were subjected to the gel filtration in the AcA34 column, the regulatory protein activity eluted in the 48K protein fractions where the regulatory protein from synaptic membranes appeared. Myelin contained basic protein-like peptides of 20,000 and 18,000 daltons ADP-ribosylated by cholera toxin. These peptides were solubilized from the membranes with acid. Soluble proteins in brain cytosol were ADP-ribosylated by cholera toxin, which was dependent on the presence of nucleotides.

Adenosine Diphosphate Ribose↗
PubMed1982

Chromosomal analyses after in vitro fertilization of squirrel monkey (Saimiri sciureus) oocytes.

The effect of 1 microM dibutyril cAMP (cAMP media) on the chromosomal normality of in vitro fertilized squirrel monkey oocytes was examined. A total of 877 oocytes were collected laparoscopically 15-16 h after hCG injection from hormonally-induced follicles of 65 squirrel monkeys. Of these, 330 (37.6%) were prepared between 6 and 30 h after insemination and analyzed. Sperm penetration and the presence of a swollen sperm head in the ooplasm were observed by 6 h, and the nucleate stage by 10 h. Addition of 1 microM dbcAMP did not affect the oocyte maturation rate, however the in vitro fertilization rate was increased 26.5% over controls (46.3 vs. 36.6%). The first cleavage metaphase was found at 16 h after insemination. Between 24 and 30 h post-insemination, 76.6% of fertilized oocytes had reached the first cleavage metaphase. An incidence of triploidy (including dispermy) of 11.7% was observed. After chromosomal analysis of metaphase I, univalents were observed in 16.7% of the cases. Aneuploidy (8.9%) was also found in metaphase II. Of the first cleavage metaphases, 88.0% had the diploid number of chromosomes. Aneuploidy occurred 12.0% of the time and 5 cases of triploidies were observed.

Animals↗
PubMed1981

Light chains of abalone myosin. UV absorption difference spectrum and resensitization of desensitized scallop myosin.

1. Abalone myosin consisted of one kind of heavy chain and two kinds of light chain according to SDS gel electrophoresis. The molecular weights of the light chains were estimated as 16,600 daltons (Light Chain-1: LC-1) and 14,500 daltons (Light Chain-2: LC-2). 2. The amino acid composition of LC-2 was not appreciably different from that of LC-1 except that the valine residue was 1 mol per mol of LC-2. 3. both LC-1 and LC-2 showed a calcium-induced UV absorption difference spectrum through the apparent binding constants for Ca2+ were low (2.5 x 10(-3) M for LC-1 and 3.2 x 10(-4) M for LC-2). 4. The modification of carboxyl groups of LC-2 by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide caused the disappearance of the calcium-induced UV absorption difference spectrum. 5. Manganese ion could also induce a UV absorption difference spectrum with these light chains although the concentration of Mn2+ required to produce the difference spectrum was very high. 6. Abalone LC-2 bound to desensitized scallop myosin and restored the calcium sensitivity of the myosin but LC-1 did not.

Actomyosin↗
PubMed1979

Difference UV-absorption spectrum of scallop adductor myosin induced by ATP.

A difference UV-absorption spectrum induced by ATP was observed with scallop myosins purified from both striated and smooth adductor muscles. The difference spectra showed a positive peak at 289 nm with a shoulder around 295 nm, and two small negative troughs around 280 nm. Tryptophanyl movement similar to that in rabbit skeletal myosin is indicated. Some tyrosyl movements in scallop myosins, however, may be in the opposite direction.

Adenosine Triphosphate↗
PubMed1978

Epinephrine-induced elevation of guanosine 3':5'-cyclic monophosphate in isolated fat cells of rat.

The effects of epinephrine (as low as 0.1 muM) on guanosine 3':5'-cyclic monophosphate (cGMP) and adenosine 3':5'-cyclic monophosphate (cAMP) in isolated fat cells were examined. Epinephrine increased both cGMP and cAMP levels, with the elevation of cAMP preceding the rise of cGMP. Maximal elevation was obtained with 1 muM epinephrine for each nucleotide. The increase in content of cGMP and cAMP due to epinephrine was completely blocked by a beta-adrenergic antagonist (5 muM propranolol). Phentolamine (10-100 muM), an alpha-adrenergic antagonist, enhanced the response to epinephrine resulting in elevation of cAMP levels, whereas a high concentration (100 muM) of phentolamine suppressed the elevation of cGMP. The ability of epinephrine to increase cGMP and cAMP levels was markedly diminished by "feedback regulator" partially purified from the incubation mixtures of isolated fat cells exposed to epinephrine [Ho, R.J. & Sutherland, E. W. (1971) J. Biol. Chem. 246, 6822-6827], whereas an increase in cGMP, but not cAMP, levels was observed in isolated fat cells incubated with "feedback regulator" alone (without epinephrine). These observations suggest the possibility that the epinephrine-induced elevation of cGMP levels in isolated fat cells might be mediated by an increase in formation of intracellular "feedback regulator" due to an elevation of cAMP by epinephrine.

Adipose Tissue↗
PubMed1976

F-induced changes and its reversal by ITP in membrane turbidity and adenylate cyclase activity of chick brain microsomes.

The activation of adenylate cyclase by NaF was dependent on the previous incubation time and the concentration of F-. The activation by F- was irreversible and Mg2+ was required for the maximum effect. Turbidity of microsome suspension was also greatly increased by F- plus Mg2+. These effects on adenylate cyclase and membrane turbidity were specific for F- and F- saturation curves for both were similar, though Mg2+-saturation curves for both were dissimilar. The increase in turbidity induced by F- plus Mg2+ was rapidly reversed by ATP, GTP, ITP, UTP and CTP. However, ITP only, among all the triphospho-nucleotides tested, reversed the activity of adenylate cyclase previously activated by NaF plus MgC12. The activity of the enzyme reversed by ITP was not, however, re-enhanced by the presence of NaF in the assay medium. These results suggest the possiblity that F- induces a change in the membrane structure itself, and this change can be reversed by incubation with ITP. Consequently, adenylate cyclase may be conformed either to an activated or an unactivated state.

Adenylyl Cyclases↗
PubMed1975

Cellular levels of feedback regulator of adenylate cyclase and the effect of epinephrine and insulin.

We have obtained direct evidence that shows the cellular formation and subsequent release of a potent inhibitor (feedback regulator) of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by adipocytes, upon stimulation with epinephrine. The appearance of such a feedback regulator in adipocytes preceded its release into the medium. During a 30 min incubation, intracellular regulator levels rose rapidly and reached 39-61 units/g of adipocyte at 10 min. Release of inhibitor into the medium increased slowly and was 11-16 units/g of adipocyte at 10 min. Upon continued incubation, the cells at 30 min contained 30-41 units/g of ingibitor, slightly less than the content at 30 min; meanwhile, the medium content rose more than 3-fold. The inhibitor from both locations appeared to have the same characteristics, judging from the purification procedures and the biological activities on hormone-stimulated adenylate cyclase. Adenylate cyclase was inhibited by the feedback regulator in vitro when either epinephrine, corticotropin (ACTH), or glucagon was used as activator. The site of action of this inhibitor is therefore most likely beyond the specific hormone receptors. A new in vitro action of insulin has been found. Insulin, 50-500 microunits/ml, inhibited the formation and release of this factor from isolated rat or hamster adipocytes by 29-81% after these cells were stimulated by hormones that raise intracellular adenosine 3':5'-cyclic monophosphate. This factor enhaced the effect of insulin in lowering the adenosine 3':5'-cyclic monophosphate levels in fresh rat adipocytes. A reduced formation of such a factor may modify the metabolic events in adipocytes, and some as yet unexplained effects of insulin could therefore be linked to the metabolic effects of this factor.

Adenylyl Cyclases↗
PubMed1975

Inhibition of cyclic nucleotide phosphodiesterase activity by an endogenous factor.

Cyclic nucleotide phosphodiesterase activity of several tissues of rat is inhibited by an endogenous factor isolated from rat adipocytes following exposure of these cells to agents that raise intracellular cyclic AMP levels. The inhibitory action was demonstrated with varying cAMP concentrations from 0.1-400 muM. Enzyme from 10,000 X g supernatant of epididymal adipose tissue was inhibited approximately 2-3 fold more than the plasma membrane of adipocytes by a given concentration of the feedback regulator. Kinetic analysis of cAMP phosphodiesterase of plasma membrane showed that feedback regulator (8.8 U/ml) inhibited the Vmax 48%. The maximum inhibition of phosphodiesterase by feedback regulator (20 U/ml) was about 80%. The apparent Km for cAMP was increased. The ability of phosphodiesterase from several tissues of rat (10,000 X g supernatant) to hydrolyze cAMP and cGMP was tested. Feedback regulator inhibited cGMP hydrolysis in cardiac muscle and 5 other tissues 23-92% more than it inhibited the hydrolysis of cAMP. The physiological significance of this inhibitory effect can begin to be clarified when the feedback regulator is purified to homogeneity and characterized.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1975

Influence on adipocyte plasma membrane bound protein kinase by feedback regulator.

Protein kinase, phosphodiesterase and adenylate cyclase of plasma membrane of adipocytes and the effect of the feedback regulator (FR) on these three enzymes was measured and compared. The basal level ratio of adenylate cyclase to phosphodiesterase to protein kinase was 1:1.9:3.0. Epinephrine and/or FR alters this ratio. FR stimulated protein kinase activity up to 3 fold in the presence of a wide range of enzyme concentrations, 5-50 mug membrane protein/tube. The concentration of FR effective for stimulation of membrane protein kinase was much greater than that needed for inhibition of adenylate cyclase and phosphodiesterases. The inhibition by FR on adenylate cyclase was the most potent effect among the 3 enzymes. 1 U (or 2 U/ml) of FR inhibited 50% of the adenylate cyclase activity in a defined system. The maximum effective concentration of FR for stimulation of membrane protein kinase was greater than 10 U/ml. Histone type 11A was the best substrate for protein phosphorylation so far observed. The FR stimulatory effect was observed at all substrate concentrations used ranging from 1-5 mg/ml. A NaF concentration curve shows that 15 mM NaF gave maximum phosphorylation. The stimulatory effect of FR was observed both in the presence and absence of NaF. Protein kinase of adipocyte plasma membrane was mainly cAMP-independent. The effect of FR (20 U/ml) in stimulation of protein phosphorylation was much greater than that of cAMP (1 X 10(-6) M). The cAMP and FR effects seemed to be additive. Preincubation of plasma membrane with FR in the absence of ATP resulted in no decrease but slight increase in protein kinase activity. A shift in protein kinase, phosphodiesterase and adenylate cyclase ratios by FR suggests the regulatory role of FR in cAMP metabolism in adipocytes.

3',5'-Cyclic-AMP Phosphodiesterases↗