Yuta Kobayashi

Endogenously Occurring β‐Carboline Induces Parkinsonism in Nonprimate Animals: A Possible Causative Protoxin in Idiopathic Parkinson's Disease

Abstract: To examine whether simple β‐carbolines induce parkinsonian‐like symptoms in vivo via N‐methylation, the simple β‐carbolines norharman (NH), 2‐mono‐N‐methylated norharmanium cation (2‐MeNH+), and 9‐mono‐N′‐methylnorharman (9‐MeNH) were systematically administered to C57BL/6 mice for 7 days. These substances induced bradykinesia with reduction of locomotion activity. NH or 2‐MeNH+ decreased dopamine (DA) contents to 50–70% of values in controls in the striatum and midbrain. 9‐MeNH potently decreased not only DA but also serotonin content in various regions. Immunohistochemical examination revealed that the numbers of tyrosine hydroxylase (TH)‐positive cells in the substantia nigra pars compacta of NH‐ and 9‐MeNH‐treated mice were diminished to 76 and 66% of values in control mice, respectively. The formation of a toxic metabolite, 2,9‐di‐N,N′‐methylated norharmanium cation (2,9‐Me2NH+), was 14 and eight times higher in the brain of mice receiving 9‐MeNH than that in NH‐ and 2‐MeNH+‐treated mice, respectively. In cultured mesencephalic cells from rat embryo, 2,9‐Me2NH+ selectively killed TH‐positive neurons only at a lower dose but was toxic to all neurons at higher doses. Thus, the excess formation of 2,9‐Me2NH+ would induce nonspecific neurotoxicity. These results indicated that 9‐indole nitrogen methylation should be the limiting step in the development of the toxicity. NH, a selective dopaminergic toxin precursor, is sequentially methylated to form 2,9‐Me2NH+, which could be an underlying factor in idiopathic Parkinsons disease.

Skin mast cell promotion of matrix remodeling in burn wound healing in mice: relevance of chymase

Abstract Inflammation, granulation, and collagen accumulation, which are observed in the wound healing process, occasionally lead to hypertrophic scarring. Several in vitro reports have suggested that skin mast cells (MCs) and their major protease, chymase, participate in the healing process as well as in fibrotic skin diseases. The present study examined the potential involvement of MCs and MC chymase in the healing of burns in mouse dorsal skin. The size of the burn wounds, density of the capillaries, collagen accumulation, MC number, and chymase activity were measured before and 1, 3, 7, and 14 days after burning. The healing process corresponded strongly with MC density and chymase activity in both acute and subacute phases. The maximum decrease in MC number and chymase activity occurred on day 3 when tissue loss due to necrosis was maximal. From day 7 to 14, the burn wounds retracted rapidly accompanied by increases in capillaries and collagen fibers, in correspondence with fast increments in MC numbers and chymase activity at the wound edges. The present results combined with previous in vitro results strongly support the contention that skin MC chymase plays a role in the normal wound healing process, and presumably in dermal fibrotic disorders.

Neuronal Nitric-Oxide Synthase Inhibition Facilitates Adrenergic Neurotransmission in Rat Mesenteric Resistance Arteries

The effects of nonselective nitric-oxide synthase (NOS) inhibitors [N-ω-nitro-l-arginine methyl ester (l-NAME) and N-ω-nitro-l-arginine (l-NNA)] and specific neuronal NOS (nNOS) inhibitor [vinyl-l-N-5-(1-imino-3-butenyl)-l-ornithine (l-VNIO)] on adrenergic nerve-mediated vasoconstriction were studied in rat perfused mesenteric vascular beds without endothelium. Perfusion of l-NAME, l-NNA, or l-VNIO markedly augmented vasoconstrictor responses to periarterial nerve stimulation (PNS; 2-8 Hz) without affecting vasoconstriction induced by exogenously injected norepinephrine (NE). Addition of l-arginine, a precursor for the synthesis of nitric oxide (NO), reversed the augmentation of the PNS response by l-NAME. The PNS (8 Hz)-evoked NE release in the perfusate was increased by l-NAME perfusion. In preparations treated with capsaicin [a depleter of calcitonin gene-related peptide (CGRP)-containing nerves], l-NAME did not augment vasoconstrictor responses to PNS or NE injection. Combined perfusion of CGRP(8-37) (a CGRP receptor antagonist) and l-NAME induced additive augmentation of the vasoconstrictor response to PNS but did not affect the response to NE injection. In preparations with active tone produced by methoxamine and in the presence of guanethidine, l-NAME perfusion did not affect the vasodilator response induced by PNS. Immunostaining of the mesenteric artery showed the presence of nNOS-like immunopositive nerve fibers, which were absent in arteries pretreated with capsaicin. These findings suggest that NO, which is released from perivascular capsaicin-sensitive nerves, presynaptically inhibits neurogenic NE release to modulate adrenergic neurotransmission.

Role of nitric oxide from the endothelium on the neurogenic contractile responses of rabbit pulmonary artery

The effects of L-NG-nitro arginine (L-NO2Arg), a stereospecific inhibitor of nitric oxide formation, on the responsiveness of rabbit pulmonary artery to transmural electrical stimulation were studied. The contractile response evoked by electrical stimulation at 4 Hz was abolished by tetrodotoxin (10(-7) M) and depressed to approximately 10% by bunazosin (10(-6) M), an alpha 1-antagonist. Pretreatment with L-NO2Arg (10(-5) M) significantly potentiated the response to electrical stimulation without changing the resting tension. D-NO2Arg (10(-5) M) did not show such a potentiating action. In endothelium-denuded arteries, L-NO2Arg did not potentiate the response to electrical stimulation. The effect of L-NO2Arg on endogenous noradrenaline release in response to electrical stimulation was also examined by HPLC with electrochemical detection; L-NO2Arg did not affect noradrenaline release. The contractions induced by exogenous noradrenaline (10(-6)-10(-5) M) were enhanced by L-NO2Arg, but not by D-NO2Arg. These results suggest that the vasoconstriction induced by sympathetic nerve stimulation in the rabbit pulmonary artery is modulated by endogenous nitric oxide or nitric oxide-like substances released from endothelial cells.

Congenic Rats For Hypertension: How Useful Are They For The Hunting Of Hypertension Genes?

1. Linkage studies have revealed quantitative trait loci (QTL) for blood pressure in the rat genome using genetic hypertensive rat models. To identify the genes responsible for hypertension, the construction of congenic rats is essential.

Anesthetic Effects of a Mixture of Medetomidine, Midazolam and Butorphanol in Two Strains of Mice

The combination of ketamine and xylazine is a widely used anesthetic for laboratory animals. However, due to an abuse problem in Japan, ketamine has been specified as a narcotic since 2007. Instead of using ketamine, Kawai et al. reported an injectable formula with an equivalent effect to the mixture of ketamine and xylazine [11]. The mixture of 0.3 mg/kg body weight (b.w.) medetomidine (Med.), 4.0 mg/kg b.w. midazoram (Mid.), and 5.0 mg/kg b.w. butorphanol (But.) produced an anesthetic duration of around 40 min in outbred ICR mice. However, the anesthetic effect of the mixture for inbred mice strains remains unknown. Therefore, we examined anesthetic effects of the mixture of Med., Mid., and But. in the BALB/c and C57BL/6J strains. After intraperitoneal injection into mice, right front paw, left hind paw, and tail pinch reflexes as well as corneal and righting reflexes were observed. Every 5 min, we scored each reflex category as 0 for reaction or 1 for no reaction. As long as the total score was at least 4 out of 5, we considered the mixture as putting a mouse in a surgical anesthetic state. The mixture produced an anesthetic duration of more than 45 min in both strains of mice. These results indicate that the mixture of Med., Mid., and But. can be a useful and effective anesthesia for the BALB/c and C57BL/6J strains of inbred mice as well as outbred ICR mice.

Methoxamine enhances the release of endogenous noradrenaline from rabbit ear artery: possible involvement of ATP

SummaryThe effect of methoxamine, an α1-adrenoceptor agonist, on the electrically-evoked release of endogenous noradrenaline was examined in the isolated rabbit ear artery. Noradrenaline was quantified by high performance liquid chromatography-electrochemical detection. The release of adenine nucleotides and nucleosides by methoxamine was examined using high performance liquid chromatography-fluorescence detection.The release of noradrenaline evoked by electrical field stimulation (EFS) at 4 Hz was reduced by tetrodotoxin 0.3 μmol/l and clonidine 1 μmol/l by approximately 80% and 50%, respectively. On the other hand, methoxamine at 10 but not 1 μmol/l enhanced the release of noradrenaline to approximately twice the control, and the enhancement was prevented by prazosin 1 μmol/l. The facilitatory action of methoxamine was also abolished after desensitization of P2-purinoceptors by α,β-methylene ATP 30 μmol/l as well as by the presumed P2-purinoceptor antagonist suramin given at 10 μmol/l. Exogenous ATP 10 μmol/l significantly enhanced the EFS-evoked release of noradrenaline, and the enhancement was abolished by α,β-methylene ATP and suramin. None of the drugs changed the spontaneous outflow of noradrenaline. These results indicate that endogenous ATP, acting at prejunctional purinoceptors, may participate in the facilitatory effect of methoxamine. Indeed, methoxamine 10 μmol/l significantly enhanced the spontaneous outflow of ATP and, less so, ADP. The methoxamine evoked release of ATP and ADP was antagonized by prazosin 1 μmol/l.It is concluded that methoxamine releases endogenous ATP from postjunctional sites which then, via prejunctional purinoceptors, facilitates action potential-evoked release of noradrenaline in rabbit ear artery.

CORONARY VASODILATORY RESPONSE TO A NOVEL PEPTIDE, ADRENOMEDULLIN 2

1. Adrenomedullin‐2 (AM2) is a novel peptide originally found in a fish and it is structurally related to mammalian AM or fish AM1. Cloning of AM2 cDNA in the mouse, rat and human has been successful. In the present study, the vasodilatory effect of synthetic human AM2 was analysed in isolated artery ring preparations of porcine.

Endothelium-dependent and -independent relaxation by dopamine in the rabbit pulmonary artery

1. The relaxing response of dopamine (DA) was studied in the rabbit pulmonary artery. DA caused concentration‐related relaxation in helically cut strips of the artery contracted with prostaglandin F2α in the presence of prazosin.

L‐NITROARGININE INCREASES BLOOD PRESSURE IN THE RAT

1. Effects of administration of NG‐nitro‐L‐arginine (NO2Arg), a guanidino nitroarginine derivative, for 1 week on blood pressure and some vascular responses of rats were studied.