Motokazu Fujiwara

Regulation of indoleamine 2,3-dioxygenase activity in the small intestine and the epididymis of mice

Abstract Indoleamine 2,3-dioxygenase activity was found to be ubiquitously distributed in various tissues of mice, such as brain, lung, stomach, intestine, and epididymis. The highest enzyme activity was detected in the alimentary canal and the epididymis. Developmental and daily rhythmic changes of indoleamine 2,3-dioxygenase activity and the effects of various regulatory factors were studied with the supernatant fractions derived from the small intestine and the epididymis. The enzyme activity in these two tissues was absent during the first 2 weeks (the weaning period). From the third week, there was a rapid increase in activities and a maximum was reached when the mice were 8 to 10 weeks of age (adolescence). The enzyme activity in the small intestine then gradually diminished to zero level at 30 weeks of age (prime) or later, while that in the epididymis remained at the high level throughout 69 weeks of age (senescence). The enzyme activity of the small intestine from mice fed during the hours 9:00–13:00 showed daily rhythmic changes; high in the daytime and low at night. Under night feeding (21:00–1:00), the enzyme activity was high at night and low in the daytime. The epididymal enzyme activity showed no daily fluctuations by either feeding schedule. With regard to the developmental and daily rhythmic changes, indoleamine 2,3-dioxygenase activity in the small intestine was similar to that of hepatic tryptophan 2,3-dioxygenase. However, in contrast to the hepatic tryptophan 2,3-dioxygenase activity, indoleamine 2,3-dioxygenase activity in the small intestine and the epididymis was not affected by adrenalectomy or intraperitoneal administration of adrenal steroid or tryptophan.

Diurnal variation of dopamine content in the rat pineal gland.

Abstract Levels of norepinephrine and dopamine in the rat pineal gland were determined by a radioenzymatic assay with modifications to separate the reaction products. Catecholamines were converted to 3-O-methylated derivatives in the presence of catechol-O-methyltransferase (EC 2.1.1.1) and S-adenosyl-L-[methyl- 3 H]-methionine. Following solvent extraction of the labelled normetanephrine and 3-methoxytyramine, the amines were separated by high-performance liquid chromatography. Contents of both catecholamines in the pineal gland varied with a 24-hr rhythm. The content of norepinephrine was maximal at about 6 A.M. (lights on from 8 A.M. to 8 P.M.) and declined gradually thereafter. In contrast to the level of norepinephrine, the dopamine level was highest at about 0 A.M. and fell rapidly to reach a trough after the lights were turned on. These observations suggest that the diurnal variation of norepinephrine is generated by changes in the contents of dopamine in sympathetic nerve terminals innervating the pineal.

Protocathechuate 3,4-dioxygenase. IV. Preparations and properties of APO- and reconstituted enzymes

Abstract Protocathechuate 3,4-dioxygenase is an enzyme containing the ferric form of nonheme iron as the sole cofactor. The iron could be removed from the enzyme by prolonged anaerobic dialysis against a buffer solution containing both o -phenanthroline and Na 2 S 2 O 4 . The apoenzyme thus obtained was fully reactivated by incubation with Fe 2+ under anaerobic conditions. The reactivation rate was greatly accelerated by the presence of reducing agents such as Na 2 S 2 O 4 . The visible absorption spectrum characteristic of the native enzyme disappeared upon removal of the iron and reappared upon reactiviation. The native enzyme and apoenzyme gave almost identical s values (18 S) on ultracentrifugaton and both also bound the same maximal amount of sustrate (8–10 moles per mole of enzyme). However, the dissociation constant for the apoenzyme was found to be approx. 20-fold higher than that for the native enzyme.

Regulation of the activity of histamine-N-methyltransferase from guinea pig skin by biogenic amines

Histamine-N-methyltransferase, a major histamine-degrading enzyme in the skin, was purified from guinea pig skin about 150-fold. The enzymological characteristics including pH optimum, Km values for substrates, and molecular weight were almost consistent with those reported in the brain. Regulatory mechanism of the enzyme activity by biogenic amines was investigated using the purified specimen. Serotonin, tryptamine, and 5-methoxytryptamine intensely inhibited the activity while tryptophan, melatonin, N-acetylserotonin, tryptophol, and 5-hydroxyindole acetic acid had no significant effects. Dopamine, tyramine, 3-methyltyramine, and phenylethylamine also inhibited the activity while no particular effects were obtained by adrenaline, noradrenaline, tyrosine, and DOPA. Spermidine and cadaverine caused significant but weaker inhibition. These amines acted competitively with respect to histamine, although varying manners were observed with respect to S-adenosyl-L-methionine. From these results, it was concluded that the enzyme activity was inhibited by such compounds in which a certain chemical structure, CH2-CH2-NH2 group neighboring the hydrophobic group, was contained. A possible mechanism of inhibition by the amines is postulated, and possible roles of such compounds in the inflammation by impairing the histamine metabolism is discussed.

Alterations in alpha adrenergic receptors in human cerebral arteries after subarachnoid hemorrhage.

The nature of alpha adrenergic receptors in human cerebral arteries was characterized and alteration of these receptors after subarachnoid hemorrhage (SAH) was examined using a radioligand binding assay. The specific 3H-prazosin binding to human cerebral arteries was saturable and of high affinity (KD = 4.1 nM) with a Bmax of 92 fmol/mg protein. Specific 3H-yohimbine binding to these tissues was also saturable and of high affinity (KD = 23 nM) with a Bmax 250 fmol/mg protein. IC50 values of adrenergic agents for 3H-prazosin binding were as follows: prazosin, 1.2 X 10(-10) M; phentolamine, 1.3 x 10(-6) M; yohimbine, 1.2 x 10(-5); norepinephrine, 4.9 x 10(-4) M; epinephrine greater than 1 x 10(-3) M. IC50 values of adrenergic agents for 3H-yohimbine binding were as follows: phentolamine, 1.7 x 10(-7) M; yohimbine, 4.2 x 10(-7) M; prazosin, 1.9 x 10(-5) M; epinephrine, 4.4 x 10(-5) M; norepinephrine, 7.9 x 10(-4) M. KD and Bmax of 3H-prazosin and 3H-yohimbine binding after SAH were compared with findings in the non-SAH group. KD and Bmax of 3H-prazosin binding of SAH group were 6 +/- 3 nM and 90 +/- 10 fmol/mg protein, respectively (N = 3). KD and Bmax of 3H-yohimbine binding of SAH group were 42 +/- 6 nM and 460 +/- 30 fmol/mg protein, respectively (N = 5). On the other hand, KD and Bmax of 3H-prazosin binding in the non-SAH group were 4 +/- 1 nM and 90 +/- 20 fmol/mg protein, respectively (N = 5), KD and Bmax of 3H-yohimbine binding of non-SAH group were 20 +/- 5 nM and 260 +/- 30 fmol/mg protein, respectively (N = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of beta adrenergic receptors in human cerebral arteries and alteration of the receptors after subarachnoid hemorrhage.

The nature of beta adrenergic receptors in human cerebral arteries was characterized and alteration of these receptors after subarachnoid hemorrhage was examined using a radioligand binding assay. The specific 3H-dihydroalprenolol, a beta adrenergic antagonist, binding to human cerebral arteries was saturable and of high affinity (KD = 12.3 nM) with a Bmax of 790 fmol/mg protein. Ki values and Hill coefficients of adrenergic agents for 3H-dihydroalprenolol were as follows; propranolol, 4.1 X 10(-8)M, 1.01; isoproterenol, 1.7 X 10(-6)M, 0.80; epinephrine, 8.3 X 10(-6)M, 0.48; norepinephrine, 2.3 X 10(-5)M, 0.45; metoprolol, 6.8 X 10(-8)M and 7.9 X 10(-6)M, 0.62; butoxamine, 2.2 X 10(-8)M and 2.1 X 10(-6)M, 0.43. The analysis of inhibition of specific 3H-dihydroalprenolol binding by these adrenergic agents suggests that human cerebral arteries contain a high density of beta adrenergic receptors and that the receptors are classified into two types, namely beta 1 and beta 2 adrenergic receptors. The calculated beta 1/beta 2 ratio from Hofstee plots was approximately 4/6. KD and Bmax of 3H-dihydroalprenolol binding to the cerebral arteries after subarachnoid hemorrhage were compared with those of control group. KD and Bmax of 3H-dihydroalprenolol binding of subarachnoid hemorrhage group were 13.9 nM and 1140 fmol/mg protein, respectively. The calculated beta 1/beta 2 ratio was approximately 6/4. These data suggest that the density of total beta adrenergic receptors increased without any significant change in the affinity after subarachnoid hemorrhage and that the increase of beta 1 adrenergic receptors was dominant.

Presynaptic and postsynaptic alpha 2-adrenergic receptors in human cerebral arteries and their alteration after subarachnoid hemorrhage.

The nature of alpha-adrenergic receptors in human cerebral arteries was characterized, and alteration of these receptors after subarachnoid hemorrhage was examined using a radioligand binding assay. Norepinephrine content of control arteries was also analyzed and compared with that of arteries after subarachnoid hemorrhage. Norepinephrine content in human cerebral arteries in cases of subarachnoid hemorrhage was about 5% of the control group. Specific binding of [3H]yohimbine, a selective alpha 2-antagonist, to cerebral arteries of the control group indicated two classes of binding sites: high-affinity sites with KD of 0.5 nM and Bmax of 18 fmol/mg protein and low-affinity sites with KD of 29 nM and Bmax of 248 fmol/mg protein. In cerebral arteries obtained from the subarachnoid hemorrhage group, [3H]yohimbine binding sites were of a single class with KD of 53 nM and Bmax of 456 fmol/mg protein. These results suggest that sympathetic denervation and subsequent alterations in alpha 2-adrenergic receptors occurred after subarachnoid hemorrhage in human cerebral arteries. These changes in sympathetic innervation to cerebral arteries were considered to be one of the antecedents of delayed vasospasm after subarachnoid hemorrhage.

2,5-dihydro-L-phenylalanine: A competitive inhibitor of indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase*

Summary 2,5-Dihydro-L-phenylalanine, an inhibitor of tryptophan 5-monooxygenase, was shown to be a potent inhibitor of indoleamine 2,3-dioxygenase (K i = 0.23 m M ) and tryptophan 2,3-dioxygenase (K i = 0.70 m M ), whereas tryptophan side chain oxidase from Pseudomonas was not inhibited by this compound.

Determination of Serotonin Turnover in the Rat Brain Using 6-Fluorotryptophan

Abstract: After intraperitoneal injection of rats with 6‐fluorotryptophan (6‐FT), brain 5‐hydroxytryptamine (5‐HT) levels decreased exponentially over 1 h. Depletion was dose‐dependent and maximum depletion was observed at 200 mg/kg. 6‐FT (200 mg/kg) did not significantly alter the content of 5‐hydroxyindoleacetic acid. Turnover rates of 5‐HT obtained by the 6‐FT and other methods were fairly consistent. 6‐FT had little effect on the content of noradrenaline and dopamine. These data suggest that 6‐FT completely inhibits tryptophan hydroxylase, in vivo, without affecting the release of 5‐HT from 5‐HT neurons and with little effect on the activities of tyrosine hydroxylase. Therefore, 6‐FT is a good pharmacological tool for studying the turnover rate of 5‐HT in the brain.

Effects of calmodulin antagonists and calmodulin on phospholipid base-exchange activities in rabbit platelets

Effects of various calmodulin antagonists and calmodulin on the incorporation of serine, ethanolamine and choline into the corresponding phospholipids, such as phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine by Ca2+-stimulated base-exchange reactions in rabbit platelet membranes were studied. Under a Ca2+-EGTA buffer system, the incorporation of three bases were stimulated by Ca2+ in a biphasic manner. Minimum requirement of free Ca2+ for the reactions was found to be around 0.5 microM and maximal incorporation took place at high Ca2+ concentrations (3-5 mM). Various calmodulin antagonists such as chlorpromazine, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, not only activated the three reactions but also greatly enhanced their sensitivity to Ca2+ (K0.5, 0.1-0.3 microM). In the absence of Ca2+, however, the drugs did not show any effect on the reactions. The concentrations of the drugs required for half maximal stimulation were approx. 30-40 microM. Although platelet membranes contained endogenous calmodulin (0.3-0.6 microgram/mg of membrane protein), the addition of exogenous calmodulin inhibited choline exchange activity but had no or little effect on serine or ethanolamine exchange activity. The results suggest that in the presence of low Ca2+ concentrations, these drugs markedly stimulate base-exchange activities, and choline exchange activity may be regulated by calmodulin.