Biomedical subjects
Publications and source records attributed to T Hata.
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1-Sarcosine, 8-isoleucine angiotensin II (Sar1-Ile8-AII) was infused intravenously in 5 normal volunteers and 66 subjects with various hypertensive, fluid and electrolyte disorders. Changes of blood pressure (BP), plasma renin activity (PRA) and plasma aldosterone concentration (PAC) were studied. In normal subjects, Sar1-Ile-AII showed pressor (agonistic) activity, which was related to both dosage and sodium intake. Hyporeninaemic hypertensive subjects (pirmary aldosteronism) showed pressor responses to a smaller dose of this compound than the dose employed in normal subjects. Hyporeninaemic hypertensive subjects and normal volunteers after 3 days of high sodium intake showed significant elevations of BP and PAC and reduction of PRA. Changes of BP, PAC and PRA in normoreninaemic subjects including those with Bartter's syndrome, renal tubular acidosis or liver cirrhosis with ascites showed reduction of BP and PAC and elevation of PRA. The results indicate that the compound has both agonistic and antagonistic activities for blood pressure; which of these is obtained apparently depends upon endogenous angiotensin II levels, as well as the dosage employed. The results in subjects with high and low PRA suggest that the compound has antagonist and agonist actions at 3 sites of angiotensin II action, i.e. peripheral vascular bed, renin release mechanism from juxta-glomerular apparatus and the zona glomerulosa of the adrenals.
Analgesic effects of a neuroleptica "Neurotropin" (NSP) were studied in mice while the analgesic effects of other agents were studied in SART-stress mice (specific stress caused by alterating rhythm in temperature) and the results compared with those in normal mice. NSP is an extract containing many conjugated polysaccarides isolated from the skin or tissues of rabbits which had been inoculated with the living cowpox virus. (a) NSP given alone to normal mice resulted in slight analgesic effects as observed with the application of the acetic acid-, phenylquinon-writhing method or the modified Randall-Selitto method. Little effect was seen when the D'Amour-Smith was used. (b) Synergism was evident when NSP and aminopyrine or NSP and morphine were given concomitantly and the acetic acid- or phenylquinon-writhing methods were applied. (c) Analgesic effects of morphine, levomepromazine, imidazole acetic acid and particularly NSP were greater in SART-stress mice than in normal mice. With the D'Amour-Smith method, only NSP had a greater effect in SART-stress mice than in normal mice.
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The possible role of histidine residues in the catalytic function of carboxypeptidase Y from bakers' yeast has been investigated using site-specific reagents. Among the reagents tested, benzyloxy-L-phenylalanylchloromethane (Z-PheCH2Cl) was the most powerful inhibitor of the enzyme. It irreversibly inactivated both the peptidase and esterase activities with an apparent second order rate constant of 3.8 M-minus 1 S-minus 1; the D isomer caused essentially no effect on either activity. Inhibition by L-Z-PheCH2Cl, the reaction retarded by certain competitive inhibitors of the enzyme. Using radioactive L-Z-PheCH2Cl, the reaction with the enzyme was shown to be essentially stoichiometric. Diisopropylphosphorofluoridate (iPr2PF)-inactivated enzyme failed to react with Z-PheCH2Cl, and conversely, the Z-PheCH2Cl-inhibited enzyme failed to react with radioactive iPr2PF. Amino acid analyses of the Z-PheCH2Cl-inactivated enzyme revealed the loss of essentially 1 residue, with a concomitant yield of a 0.62 residue of N-t-carboxymethylhistidine. Since carboxypeptidase Y has a reactive serine at its active center, we concluded from these results that the mechanism involves a charge-relay system in the hydrolysis of peptide and ester substrates, as in chymotrypsin. An -SH group of carboxypeptidase Y was not affected during the reaction with L-Z-PheCH2Cl. The generic name "serine carboxypeptidase" has been proposed for carboxypeptidase Y and for the iPr2PF-sensitive carboxypeptidases from plants, molds, and animal tissues, in order to distinguish them from "metal carboxypeptidase" to which carboxypeptidase A (EC 3.4.12.2) and B (EC 3.4.12.3) belong.
Kinetic parameters for carboxypeptidase Y [EC 3.4.12.1], characterized as a nonspecific enzyme, are given for the hydrolysis of a series of acylated peptides, acylated amino acid esters, and amides. We confirmed that the enzyme released COOH-terminal proline and beta-alanine at an appreciable rate, as well as neutral amino acids with aromatic and aliphatic side chains at a very high speed. The rates of hydrolysis of ester and amide substrates were compatible with those produced by chymotrypsin [EC 3.4.21.1]. Stereospecificity was also demonstrated by the failure to hydrolyze peptide, ester, amide, and anilide substrates containing a D-amino acid. The effects of pH, solvents, and salt concentrations on the kinetic parameters of hydrolysis of peptide and ester substrates are also described.
Reversible inhibition of the peptidase and esterase activities of CPase Y [EC 3.4.12.1] was investigated with substrate and product analogs known to be inhibitors or effectors of pancreatic carboxypeptidases A or B [EC 3.4.12.2 or 3]. L-Amino acids and NH2-blocked L-Amino acids showed competitive-type inhibition, whereas their D-enantiomers caused less inhibition than the L-enantiomers, and showed non-competitive or mixed-type inhibition. Some phenylalanine analogs, e.g. beta-phenylpropionic acid and t-cinnamic acid, were also reversible inhibitors of CPase Y. The type of these inhibitors and their K1 values were generally parallel for both the peptidase and esterase activities.
Cathepsin A [EC 3.4.2.-] of small molecular size (cathepsin A, S) has been purified about 800-fold from pig kidney by procedures including chromatographies on DEAE-Sephadex, SP-Sephadex, and Sephadex G-150. 1. The homogeneity of the purified enzyme was proved by ultracentrifugation and polyacrylamide gel electrophoresis. The molecular weight (100,000) and isoelectric point (pI=5.0) were estimated. 2. The enzyme was remarkably stabilized by sucrose and KCl, and was most stable at pH 5-5.5 in the presence of both stabilizers. The enzyme had not only peptidase activity but also esterase and amidase activity; it was optimally active at pH 5.2 for peptide hydrolysis and at pH 8 for the hydrolysis of esters and amides. 3. Diisopropyl fluorophosphate and iodoacetamide completely inhibited these three activities. 4. The enzyme hydrolyzed various benzoyl- and benzyloxycarbonyl-dipeptides with neutral, acidic, and basic amino acids, and proline in the C-terminal position. The carboxypeptidase nature of the enzyme was proved by its action on an oligopeptide. 5. Several enzymatic properties of cathepsin A, S were almost the same as thoas of cathepsin A of large molecular size (cathepsin A, L) and the crude homogenate.
Kinetic parameters of carboxypeptidase Y are given for the hydrolyses of ester, amide, and anilide substrates. The kcat/Km values were compatible with those of chymotrypsin [EC 3.4.21.1] with a few exceptions. One ionizable group with a pK of around 5.8 was suggested to be involved in the free enzyme in hydrolyzing all the substrates, including peptide substrates. In addition, hydroxylaminolysis and the kinetic isotope effects of deuterium oxide indicated, with some reservations, a reaction mechanism which proceeds via the formation of an acyl intermediate.
Exactly how the organic mechanism corresponds to variations of environmental temperature has not been clarified and for elucidation we reared mice and rats under the conditions of alterating rhythm in temperature (ART). Mice (or rats) were reared at a temperature of 24 degrees C and 8 degrees C (or -3 degrees C) every hour continuously from 10 a.m. to 5 p.m. and at 8 degrees C (or -3 C) from 5 p.m. to the following 10 a.m. Severe stress was evidenced in the animals and was termed specific ART-stress (SART-stress). In this type of stress, there was no apparent increase in body weight, respiration and heart rate were increased slightly and the QRS-time on the ECG was prolonged. ACh sensitivity of the isolated duodenum tested by the Magnus method in SART-stress mice was found to have declined considerably as compared to the normal. Histological observation of the SART-stress rats revealed that the wet weight of the spleen was lighter than that of the controls, while that of the lungs, heart, liver, stomach, kidneys and adrenal glands was close to that of control. Macroscopically, red brown spots of the lungs were visible. Ventricular hypertrophy, slight erosion and hyperemia were present in the interior of the stomach. On the galvanic skin response (GSR) test, electrical resistance of the skin of SART-stress rats was less than that of normal rats and the rate of increase of resistance caused by external stimulus was greater. Recovery time from change in GSR was shorter than in normal animals. Consequently, SART-stress appears to be a form of disease and sudden changes in temperature even in humans may contribute to this state.
In preliminary experiments to investigate the influence of masking the eyes of E1-mice on the rate of non-convulsions (RNC), it was suggested that the quantity of light as well as visibility itself played a role in inducing convulsions. The following experiments were thus carried out: (a) Both TSC (the threshold of number of shakings required for induction of convulsions) and RNC increased remarkably on the 5th day after enucleation of bilateral eyeballs from E1-mice, but 15 days later recovery to control level was observed. Moreover, TSC and RNC at 2 p.m. increased considerably compared with the control, however, the increase was rarely observed at 10 a.m.. (b) 2 groups of E1-mice reared intact under radiation of light plus different time schedules were shaken at 10 a.m. and 2 p.m., respectively. Consequently, TSC after radiation for 5 hours was less than that after radiation for 1 hour. When observed 9 hr after radiation, TSC could not be differentiated from TSC at 5 hr after the radiation. (c) E1-mice were reared under continuous lighting, continuous darkness or a normal lighting situation, after which TSC and RNC were examined. From our observations, the intensity of light appears to play a role in inducing convulsions. (d) When mice were shaken immediately after lighting, RNC decreased in inverse proportion to increase in the quantity of light. TSC was not influenced by the luminosity itself. One hour after lighting, however, the influence of lighting had disappeared. The intensity, quantity or irradiation time would not, however, influence the induction of E1-mice convulsions so much as the cognizance of light by the individual animals. Such a conception is derived from the "cognitive theory" in psychology.
We reported previously that by altering the rhythm of environmental temperature (ART) the physiological mechanism was changed to an abnormal state which we termed specific ART-stress (SART-stress). In the present report, the effects of neurotropin and other tranquilizers on this SART-stress state were investigated. Neurotropin is an extract containing many conjugated polysaccarides isolated from the skin or tissues of rabbits which had been inoculated with the living cowpox virus. Neurotropin prevented the decrease in body weight of both mice and rats caused by the SART-stress. However, there was no such prevention when an extract isolated from vaccinia virus-uninjured, healthy rabbit skin was given. Chlorpromazine (Cp), reserpine (Rp), diazepam (DZ), imipramine or diphenhydramine gave no protection against the decrease in body weight. Slight increase in number and variation of respirations in SART-stress animals was to some extent prevented by neurotropin. Prolongation of the QRS interval was restored to control with the administration of the above mentioned drugs. Decrease in ACh sensitivity of the isolated duodenum tested by the Magnus-method was prevented by neurotropin. In the galvanic skin response test in rats, decrease in electric resistance on the skin, increase in reactivity and shortening of reaction time caused by SART-stress was prevented to the greatest extent by neurotropin and also to a considerable degree by parenteral administration of DZ or beta-hydroxy gamma-aminobutyric acid (GABOB), the effective results of the drugs being: Neurotropin, DZ, GABOB in that order. Cp and Rp, major tranquilizers were found to be the least effective.
The circadian rhythms of acute toxicity of N-methyl D-aspartic acid, picrotoxin, pentetrazol, strychnine, chlorpromazine and Na-methylhexabital in dd-mice were investigated. The drugs were injected into mice on the hour at 2, 6, 10, 14, 18 or 22 in one day, after which the cumulative mortalities were calculated for 72 hours. Regarding central stimulants, the mortality of mice injected at 22 (o'clock) was lowest, and when injections were given at 2, 10 or 18 (o'clock), the mortality was higher than at any other time. On the other hand, regarding central depressants, the mortality was lowest at 10, and highest at 14 or at 18 o'clock. Thus, the administration time of central stimulants showing the lowest mortality was shifted about 12 hours in comparison with central depressants. As compared to the central depressants, the mortality rate as the result of central stimulants showed a great contrast when injected at 10 o'clock.
The exploratory movement recorder of Tokyo University of Education type was utilized to assess the exploratory motor activity of groups of 1, 2 and 3 mice of the dd-strain. As the animals were observed during 15 min periods to move spontaneously, psychotropic drugs were administered and the specific actions of these agents were determined. Each drug was given i.p. in a dose which proved non-effective in a single mouse. Observations were as follows: The ratio of exploratory motor activity of each pair of mice as compared to a single mouse was not always different from that of control animals. When a 3rd mouse was added to the pair, the motor activity either increased or decreased considerably. The rate of increase in motor activity was calculated 45 min after caffeine, pentetrazol, ephedrine and DL-amphetamine and was found to be less than in the control animals. After administration of chlordiazepoxide, diazepam, imipramine, chlorpromaxine or mescaline, the rate of increase in motor activity was considerably greater than in the controls. With dimorphoramine or hexobarbital, the rate was much the same as control. After beta-oxy gamma-aminobutyric acid, the rate was slightly increased and with diphenhydramine the rate was slightly less than control. Thus the exploratory motor activity of 3 mice as compared to that of 2, decreased considerably after administration of psychotropic stimulants, while after psychotropic depressants, an even greater increase was observed. We have already reported on the mutual relation of mice in fighting episodes and E1-mice convulsions. Those relations were, however, responses elicited by the physical stimulation. In contrast, the mutual relation in this paper is more of a natural nature. In view of observations in this paper, it is considered that CNS stimulants increase the mutual, emotional, checking behavior in groups of 3 animals while CNS depressants decrease the checking behavior. Such behavior is one example of the emotional conduct when a 3rd party is added to a 2 member group.
The metal content of carboxypeptidase Y was analyzed by the atomic absorption method. After exhaustive dialysis against an EDTA solution, the enzyme showed no loss of activity nor any significant content of metals (Zh,Mg,Ca,Cu,Mn,Ni,Fe, and Co). The activity was, however, rather sensitive to preincubation with various metals. The reactivity of a serine residue of the enzyme was also reevaluated. Diisopropyl fluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF) stoichiometrically and irreversively inhibited the enzyme. The rate of inactivation with DFP was much faster than that for typsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1.], while the rate with PMSF was one-fifteenth of that for chymotrypsin. The pH-dependence of the inactivation by DFP was similar to that of the enzymatic hydrolysis of acetylphenylalanine ethyl ester. The present results indicate that carboxypeptidase Y is free of metals and has a serine residue with a vital role in the catalytic process, though the functional role of this SH group remains to be clarified.
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