Masayuki Ishikawa

Endothelium‐dependent inhibition of platelet aggregation

1 In cascade perfusion and superfusion experiments on rabbit tissues, when acetylcholine (ACh) was introduced into the circuit so as to perfuse the aorta under perfusion with noradrenaline (NA), the effluent relaxed the transverse aortic strip which had been denuded of endothelium. 2 The effluent from the perfused aorta which was capable of relaxing the transverse aortic strip also significantly inhibited platelet aggregation induced by arachidonic acid (AA) in a volume‐related manner. The inhibitory activity was decreased by the prolongation of transit time before addition of the effluent to platelet‐rich plasma. 3 Neither the inhibition of AA‐induced aggregation nor the relaxation of the transverse strip by the effluent could be observed after the removal of endothelium from the aorta, or after pretreatment of aorta with mepacrine or nordihydroguaiaretic acid (NDGA). 4 The AA‐induced platelet aggregation was unaffected by pretreatment of platelets with mepacrine or NDGA at the concentration tested. 5 Pretreatment of aorta with indomethacin failed to modify the relaxation of the transverse strip induced by the effluent. 6 These results strongly suggest that endothelium‐derived vascular relaxant factor (EDRF) possesses inhibitory activity on AA‐induced aggregation in addition to its vasodilator activity.

Synthesis and biological activity of 1α-hydroxyvitamin D3

Abstract Hydroboration of cholesta-1,5-diene-3β-ol followed by alkaline-peroxide oxidation resulted in the formation of 1α- and 2α-hydroxy derivatives of cholesterol in nearly equal amounts. 1α-Hydroxycholesterol was then transformed to 1α-hydroxyvitamin D 3 , via 1α-hydroxycholest-5,7-diene-3β-ol. 1α-Hydroxyvitamin D 3 was as active as 25-hydroxyvitamin D 3 in the stimulation of intestinal calcium transport and bone mineral mobilization in intact rats, and moreover was able to produce both response in anephric rats similar to 1α,25-dihydroxyvitamin D 3 , the active metabolite of vitamin D 3 , as reported originally by DeLucas group.

Calcium‐dependent contractile response of arterial smooth muscle to a jellyfish toxin (pCrTX: Carybdea rastonii)

1 The purpose of the present experiments was to investigate the pharmacological mechanisms of the vasoconstriction caused by the toxin (pCrTX) which had been partially purified from the tentacles of the jellyfish Carybdea rastonii (‘Andonkurage’). 2 pCrTX (0.1 to 10 μg ml−1) produced a tonic contraction of rabbit aortic strips, which was nearly abolished in Ca2+‐free medium and was significantly reduced by verapamil or diltiazem. 3 pCrTX stimulated 45Ca2+‐influx and this effect was markedly attenuated by verapamil. 4 pCrTX‐induced vasoconstriction was significantly attenuated by phentolamine, 6‐hydroxydopamine (6‐OHDA) and in low Na+‐medium, but not by bretylium, guanethidine, reserpinization or tetrodotoxin (TTX). 5 pCrTX continuously and significantly increased the 3H‐efflux from [3H]‐noradrenaline preloaded aortic strips and this effect was completely inhibited by pretreatment with 6‐OHDA and in Ca2+‐free medium, but not by phentolamine, bretylium, guanethidine or TTX. 6 A single exposure to pCrTX for 30 min greatly reduced the contractile responses to tyramine, nicotine and transmural electrical stimulation, but not those to noradrenaline or KCl. In addition, incorporation of [3H]‐noradrenaline was reduced. 7 Pretreatments with chlorphenylamine or indomethacin failed to modify the contractile response to pCrTX. 8 These results suggest that the pCrTX‐induced vasoconstriction is caused by a presynaptic action, releasing noradrenaline from the intramural adrenergic nerve terminals, and by a postsynaptic action, which consists at least in part of stimulation of the transmembrane calcium influx. Both pre‐ and postsynaptic actions depend on the external calcium concentration. The data further suggest that pCrTX damages the noradrenaline uptake and/or storage mechanisms without damaging postsynaptic contractile systems.

A new synthetic method of 1α-hydroxy-7-dehydrocholesterol

Abstract Cholesta - 1,4,6 - trien - 3 - one ( 1 ) was converted to 3β - hydroxycholesta - 1,5,7 - triene ( 3 ) via the deconjugation procedure using t-BuOK in DMSO followed by the subsequent reduction with Ca(BH 4 ) 2 . The compound ( 3 ) readily reacted with 4-phenyl-1,2,4-triazoline-3,5-dione to yield the corresponding 1,4-addition product ( 4 ). Epoxidation of 4 with m -chloroperbenzoic acid resulted in the formation of the 1α,2α-epoxide ( 5 ) and the 1β,2β-epoxide ( 6 ) in the ratio 2:3. Reduction of 5 with LAH under reflux in THF afforded the titled compound ( 7 ). The same reduction of 6 gave 2β-hydroxy- and 1β - hydroxy - 7 - dehydrocholesterol ( 8 and 9 ) in the ratio 8:1. The compound ( 4 ) can be obtained in 25% yield from 1 without any purification of the intermediate compounds; cholesta - 1,5,7 - trien - 3 - one ( 2 , a very unstable compound) and 3 . Since 1 is obtained readily from cholesterol in high yield, the present study provides a simple and efficient synthetic method of 1α-hydroxycholecalciferol and is reasonably expected to be applicable in the synthesis of 12,25-dihydroxycholecalciferol and the other metabolites of vitamin D 3 .

Photolysis of 2,6-dicyanopyridine 1-oxides

Abstract Irradiation of 2,6-dicyanopyridine 1-oxide (VIIa) in dichloromethane with > 290 mμ rays gave rise to 2,6-dicyanopyridine (VIIIa; 20%), 5-cyano-2-pyrrolecarbonyl cyanide (IXa) (20%), and an oxazepine (Xa) tentatively assigned as 2,4-dicyano-1,3-oxazepine (Aa; 35%). 4-Methyl-2,6-dicyanopyridine 1-oxide (VIIb) gave similar photo-products. The mechanism implied in these photochemical reactions has been discussed.

Platelet aggregation caused by a partially purified jellyfish toxin from Carybdea rastonii

A partially purified toxin (pCrTX) was obtained from the tentacles of the jellyfish, Carybdea rastonii. When pCrTX (3 X 10(-8) - 3 X 10(-7) g/ml) was added to citrated platelet-rich plasma, aggregation was produced in a concentration-dependent manner. Scanning electron microscopic examination revealed that both pCrTX and collagen produced aggregates of platelets possessing many pseudopods. The concentration which produced 50% aggregation for pCrTX was 1.8 X 10(-7) g/ml, as compared to 2.3 X 10(-6) g/ml for collagen. The pCrTX-induced aggregation was only slightly inhibited by indomethacin and quinacrine in concentrations sufficient to inhibit arachidonic acid- and collagen-induced aggregation. pCrTX was less active in washed platelets suspended in Ca2+ free medium, whereas the pCrTX-induced aggregation was significantly augmented in the presence of Ca2+. The augmentation of aggregation by Ca2+ was only slightly attenuated by pretreatment with 100 microM verapamil. pCrTX significantly increased the concentration of cytoplasmic free Ca2+ ([Ca2+]i) and depolarized the platelet membrane in concentrations that produced aggregation. The increase in [Ca2+]i caused by pCrTX was little affected by verapamil. The depolarization by pCrTX was unchanged in the presence or absence of Ca2+, or by sodium or potassium transport inhibitors. The movement of 22Na+ into platelets was significantly increased by pCrTX. This increase in the movement of 22N+ into platelets was unaffected by tetrodotoxin. On the other hand, pCrTX-induced aggregation, depolarization and the increase in [Ca2+]i were all significantly attenuated in low Na+ medium. These results suggest that pCrTX causes a massive depolarization by increasing cation permeability indiscriminately and this generalized depolarization permits an inward movement of calcium down an electrochemical gradient which, in turn triggers platelet aggregation.

α1‐Adrenoceptor antagonist activity of novel pyrimidine derivatives (SHI437 and IK29) in rabbit aorta and trigone of the bladder

1 In the rabbit isolated aorta and trigone of the bladder, noradrenaline, phenylephrine and clonidine elicited concentration‐dependent contractions, which may be caused through activation of postsynaptic α1‐adrenoceptors. 2 SHI437, IK29, prazosin and yohimbine competitively antagonized the contractile responses induced by noradrenaline in the aorta and trigone. The pA2 values of SHI437, IK29, prazosin and yohimbine were 7.35 ± 0.09, 7.47 ± 0.10, 8.55 ± 0.02 and 6.28 ± 0.05 in the aorta, and 8.07 ± 0.04, 8.30 ± 0.03, 8.22 + 0.04 and 6.46 ± 0.04 in the trigone, respectively. 3 SHI437, IK29, prazosin and yohimbine also possessed competitive α2‐adrenoceptor blocking properties, judging from their antagonism of the clonidine‐induced inhibitory effect on the twitch responses in the electrically stimulated vas deferens of the rat. The pA2 values of SHI437, IK29, prazosin and yohimbine were determined to be 4.76 ± 0.02, 4.74 ± 0.02, 5.06 ± 0.03 and 7.86 ± 0.04, respectively. 4 SHI437, IK29 and prazosin inhibited the contractile responses elicited by transmural electrical stimulation without affecting the evoked 3H‐overflow from the [3H]‐noradrenaline‐preloaded rabbit aorta. Yohimbine augmented the contractile responses and 3H‐overflow. 5 SHI437 and IK29 at a concentration sufficient to inhibit noradrenaline‐induced contraction failed to attenuate the contractile responses of aorta to KCl, 5‐hydroxytryptamine and prostaglandin F2α, and of the trigone to acetylcholine and histamine. 6 The present results suggest that SHI437 and IK29 are highly selective α1‐adrenoceptor antagonists, especially in the trigone of the bladder.