Tadao Okegawa

CYTOLOGICAL LOCALIZATION OF NORADRENALINE, MONOAMINE OXIDASE AND ACETYLCHOLINESTERASE IN SALIVARY GLANDS OF DOG

The cytological localization of noradrenaline, monoamine oxidase and acetylcholinesterase was examined in the salivary glands of the dog. The noradrenaline-fluorescent nerve fibers surrounded the secretory acini of the submaxillary, sublingual and parotid glands, but the density in the sublingual gland was much less than in the other two glands. Noradrenaline fluorescence was not seen in the cytoplasms of the acinar cells nor in the vicinity of the excretory ducts. Intense fluorescence was present outside the smooth muscle layer in various sized arteries. The distribution of monoamine oxidase activity was different from that of noradrenaline fluoresence. All the cells of the secretory acini and the excretory ducts were evenly stained. The distribution of fibers showing acetylcholinesterase activity was different from that of noradrenaline-fluorescent fibers only in that the former fibers were present around the excretory ducts as well. The composition of secretory cells in the submaxillary and sublingual glands is different in dogs and rats. However, the cytological localization of noradrenaline, monoamine oxidase and acetylcholinesterase activities in the salivary glands of the dog was essentially similar to those of the rat. It was concluded, therefore, that the autonomic nerve supply to the salivary glands does not correlate with the type of secretory cells.

Arginine-vasopressin fragment 4–9 stimulates the acetylcholine release in hippocampus of freely-moving rats

We examined the effects of arginine-vasopressin (AVP) C-terminal fragment 4-9, which facilitates learning and memory, on the extracellular acetylcholine (ACh) release in hippocampus of freely-moving rats using the microdialysis technique. Following administration of AVP4-9, p-Glu-Asn-Cys[Cys]-Pro-Arg-Gly-NH2, through the dialysis probe into the hippocampus, ACh levels in dialysates from the hippocampus increased markedly in dose and time dependent manner at 2-2.5 and 2.5-3 hr. AVP1-9, the parent peptide, has a similar enhancing effect on ACh release as AVP4-9. Stimulated ACh release by AVP4-9 was significantly inhibited by V1-selective receptor antagonist ([1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-(O-methyl)-tyrosine]AVP), but not by V2-selective antagonist ([1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-D-Ile, 4-Ile]AVP). From these observations, it is demonstrated that AVP4-9 stimulates the ACh release in rat hippocampus via mediating V1-like vasopressin receptors.

Effect of a thromboxane A2 synthetase inhibitor (OKY-046.HCl) on airway hyperresponsiveness in guinea pigs.

We studied the effect of (E)-3-[p-(1H-imidazol-1-ylmethyl)phenyl]-2-propenoic acid hydrochloride monohydrate (OKY-046.HCl), a specific thromboxane (TX) A2 synthetase inhibitor, on airway hyperresponsiveness of guinea pigs. OKY-046.HCl (30-100 mg/kg, intraduodenally (i.d.) or orally (p.o.)) suppressed dose dependently the airway hyperresponsiveness to acetylcholine (ACh) induced by formyl-methionyl-leucyl-phenylalanine (FMLP), platelet activating factor (PAF) and repetitive antigen. OKY-046.HCl (100 mg/kg) also inhibited the increase in TXB2 in bronchoalveolar lavage fluid (BALF) induced by FMLP, PAF and antigen. Aspirin 10 or 30 mg/kg i.d. or p.o.) suppressed the airway hyperresponsiveness induced by FMLP and PAF but not by antigen. Azelastine (10 mg/kg i.d.) was ineffective on PAF- and antigen-induced airway hyperresponsiveness. TXA2 mimetic drugs caused airway hyperresponsiveness that was not inhibited by OKY-046.HCl (30 mg/kg i.v.). Furthermore, OKY-046.HCl showed no effect on propranolol- and physostigmine-induced airway hyperresponsiveness which did not accompany TXB2 generation in BALF. The number of eosinophils in BALF increased after FMLP exposure, an effect which was not inhibited by OKY-046.HCl. These results suggest that OKY-046.HCl inhibits airway hyperresponsiveness by suppressing TXA2 generation. We suggest that OKY-046.HCl will be a new antiasthmatic drug.

Effects of ONO-3708, an antagonist of the thromboxane A2/prostaglandin endoperoxide receptor, on platelet aggregation and thrombosis

The beneficial effects of an antagonist of the thromboxane A2/prostaglandin endoperoxide receptor, 7-[2 alpha,4 alpha-(dimethylmethano)-6 beta-(2-cyclopentyl-2 beta- hydroxyacetamido)-1 alpha-cyclohexyl]-5(Z)-heptenoic acid (ONO-3708) on thrombosis were examined. ONO-3708 at 0.1-3 microM inhibited the human platelet aggregation induced by thromboxane A2, prostaglandin H2, collagen, ADP (secondary phase) and epinephrine (secondary phase) without affecting prostanoid synthesis and the content of cyclic AMP in platelets. The in vivo effects, on coronary thrombosis in this case, were examined in two canine models. ONO-3708, 3 to 300 micrograms/kg i.v., prevented dose dependently the coronary thrombosis induced by partial obstruction of the coronary artery. ONO-3708, 3 micrograms/kg per min i.v., significantly prevented electrically stimulated coronary thrombosis without affecting systemic blood pressure and heart rate. These results indicate that the thromboxane A2/prostaglandin endoperoxide receptor could play an important role in the pathogenesis of thrombosis and that ONO-3708 may have therapeutic advantages in preventing thrombosis.

Effect of a prostacyclin analog OP-2507 on acute ischemic cerebral edema in cats

We evaluated the inhibitory activity of a novel prostacyclin analog, OP-2507 (15-cis-(4-n-propylcyclohexyl)-16,17,18,19,20-pentanor-9-deo xy-6,9 alpha- nitriloprostaglandin F1 methyl ester) on the brain edema induced by occlusion of the middle cerebral artery in cats. Middle cerebral artery occlusion for 4h caused a decrease of regional cerebral blood flow. The specific gravity of the cerebral cortex measured 4h after the middle cerebral artery occlusion as an index of cerebral edema showed a significant reduction. Intravenous infusion of OP-2507 at infusion rates of 10 and 50 ng/kg per min was started 30 min before the middle cerebral artery occlusion and was continued for 4.5 h. While OP-2507 did not affect the blood pressure, heart rate and regional cerebral blood flow before and after the middle cerebral artery occlusion, the reduction of the specific gravity of cerebral cortex was significantly prevented by OP-2507 treatment at both doses. Prostacyclin prevented the reduction of the specific gravity only at the higher dose of 50 ng/kg per min. The present results indicate the potential usefulness of OP-2507 in acute ischemic cerebral disorders.

Effects of ONO-3708, an antagonist of the thromboxane A2/prostaglandin endoperoxide receptor, on blood vessels

The pharmacological properties of a novel thromboxane A2/prostaglandin endoperoxide receptor antagonist, ONO-3708, on blood vessels were examined in vitro and in vivo. ONO-3708, 10 microM, inhibited the rabbit aorta contractions induced by thromboxane A2, prostaglandin H2, 11,9-epoxymethano-prostaglandin H2 (U-46619) or prostaglandin F2 alpha without affecting the contractions induced by angiotensin II, serotonin or norepinephrine. ONO-3708, at a concentration of 1 to 100 nM, appeared to be a competitive inhibitor of the contractile responses of the canine basilar artery to 9,11-epithio-11,12-methano-thromboxane A2 (STA2), U-46619 and PGF2 alpha, and a non-competitive inhibitor of the contractile responses to 15-hydroperoxy-eicosatetraenoic acid (15-HPETE). In in vivo studies, ONO-3708 (10 and 100 micrograms/kg per min i.v.) ameliorated the decrease in diameter of the basilar artery induced by the i.v. infusion of STA2 (0.1 microgram/kg per min) in cats. Furthermore, infusion of ONO-3708 (10 and 30 micrograms/kg per min i.v.) prevented the cerebral vasospasm in an experimental subarachnoid hemorrhage model in dogs. These results indicate that ONO-3708 is a potent antagonist of the thromboxane A2/prostaglandin endoperoxide receptor in vitro and in vivo and may be of therapeutic use in preventing cerebral vasospasm.

Platelet-Activating-Factor(PAF)吸入誘発モルモット気道過敏性に対する特異的Thromboxane A2合成酵素阻害剤(E)-3-〔p-(1H-imidazol-1-ylmethyl)phenyl〕-2-propenoic acid hydrochloride monohydrate (OKY-C)46・HCl)の抑制作用

We studied the inhibitory effects of OKY-046.HCl on PAF-induced airway hyperresponsiveness (AHR) in guinea pigs. 1) Inhalation of PAF (1 or 10 micrograms/ml) caused AHR to acetylcholine (ACh) aerosol and increased TXB2 generation in broncho-alveolar lavage fluid (BALF) at 30 min and 60 min, but the AHR and the TXB2 generation disappeared at 2 hr. OKY-046.HCl (100 mg/kg, intraduodenally) inhibited the AHR, which was accompanied by its inhibition of the TXB2 generation. However, no changes of 6-keto-PGF1 alpha in BALF were found. 2) There were no changes in the number of leukocytes; the activities of alkaline phosphatase, N-acetyl-beta-D-glucosaminidase, and lactate dehydrogenase; and the LTC4/D4/E4 in BALF. 3) In bronchus-lung preparations, PAF (1 microgram/min) also caused the AHR and increased TXB2 generation. OKY-046.HCl (100 micrograms/min) inhibited the AHR and TXB2 generation. 4) PAF (1 microgram/ml) evoked TXB2 generation in BALF from normal guinea pigs. OKY-046.HCl (10(-4)M) inhibited its increase. 5) Stable TXA2 (STA2, 1 ng/ml) inhalation also caused AHR to ACh at 30 min. STA2 (0.45 ng/min) also caused the AHR in bronchus-lung preparations. These results suggest that OKY-046.HCl can inhibit PAF-induced AHR by suppressing the generation of TXA2. We also supposed that TXA2 is released from lung parenchyma, airway epithelium and cell components in BALF.モルモットのPAF吸入誘発気道過敏性モデルにおけるthromboxane(TX)A2合成酵素阻害剤OKY-046・HClの抑制作用について検討した．その結果(1)PAF(1および10μg/ml)吸入30および60分後にacetylcholine(ACh)に対する気道反応は有意に充進し，気管支肺胞洗浄液(BALF)中のTXB2濃度も有意に上昇した，OKY-046・HCl(100mg/kg，十二指腸内投与)は気道反応の亢進とTXB2の産生を有意に抑制した．アスピリン(10mg/kg，十二指腸内投与)も気道反応の亢進を抑制した。BALF中の6-keto-prostaglandin(PG)F1αの濃度はPAF吸入により有意な影響を受けなかった．(2)PAF(10μg/ml)吸入15分後に末梢血中血小板数の減少が認められたが，血中TXB2濃度には変化が認められなかった．また吸入15分後にBALF中タンパク濃度および好中球数比の上昇が認められたが，末梢血中白血球数さらにBALF中の白血球数，細胞分画比，leukotriene(LT)C4/D4/E4濃度，alkaline phosphatase活性，N-acetyl-β-D-glucosaminidase活性およびlactate dehydrogenase活性にはPAF吸入60分後まで変化は認められなかった．(3)摘出気管支-肺灌流標本においてPAF(1μg/min)によりAChに対する気道反応の充進と灌流液中のTXB2産生の増加が有意に認められ，OKY-046・HCl(100μg/min)はこれらの反応を著明に抑制した．(4)正常モルモットのBALF中にPAF(1μg/ml)を添加するとTXB2産生は著明に増加し，OKY-046・HClは10-4Mでその増加を有意に抑制した．(5)stable TXA2(STA2，1ng/ml)吸入30分後にAChに対する気道反応の著明な充進を認めたが，2時間後には消失した．また摘出気管支-肺灌流標本においてもSTA2(0.45ng/min)によりAChに対する気道反応は充進した．以上より，PAF吸入誘発気道過敏性は，主として肺実質，上皮細胞および気管支-肺胞内細胞成分から産生されるTXA2によって発症し，OKY-046・HClはこのTXA2の産生を抑制することにより，この気道過敏性の発症を防ぐものと考えられ，OKY-046・HClは抗喘息薬として期待できる．