Motohiko Ueda

A novel non‐peptide endothelin antagonist isolated from bayberry, Myrica cerifera

A potent non‐peptide ET receptor antagonist, myrireron caffeoyl ester (50–235), was isolated from the bayberry, Myrica cerifera. This compound selectively antagonized specific binding of [125I]ET‐1, but not of [125I]ET‐3, to rat cardiac membranes, ET‐1‐induced increase in the intracellular free calcium concentration in Swiss 3T3 fibroblasts, and ET‐1‐induced contraction of rat aortic strips. Thus, 50–235 is the first non‐peptide ETA receptor antagonist. This compound can be useful for studying the physiological role of endothelin and exploring its role in various diseases.

Changes in extracellular concentration of amino acids in the hippocampus during cerebral ischemia in stroke-prone SHR, stroke-resistant SHR and normotensive rats

To investigate the role of glutamate release in cerebral ischemia, the amounts of amino acids (glutamate, taurine, alanine, glycine and glutamine) released in the hippocampal CA1 region of stroke-prone SHR (SHRSP), stroke-resistant SHR (SHRSR) and normotensive rats (WKY) were determined during and after cerebral ischemia by the microdialysis method under halothane anesthesia. Cerebral ischemia was produced by the occlusion of both common carotid arteries for 20 min. The basal amino acids release did not differ among the three strains of rats, but ischemic glutamate and taurine releases were more marked in SHRSP than in other strains. These results suggest that the massive glutamate release during cerebral ischemia of SHRSP might be related with severe neuronal cell injury.

Effect of pancreatic type phospholipase A2 on isolated porcine cerebral arteries via its specific binding sites

The addition of porcine pancreatic group I phospholipase A2 (PLA2‐I) produced a transient contraction followed by a relaxation in helical strips of porcine cerebral arteries. Its ED10 value (2.3 nM) was almost identical to the K 4 value(3.9 nM) calculated from the specific binding of 125I‐labeled porcine PLA2‐I in cultured porcine cerebral arterial smooth muscle cells. Type‐specific action of PLA2s and homologous desensitization strongly implicated the involvement of PLA2‐I‐specific sites in the response. The transient contraction was abolished by treatment with indomethacin as well as by the removal of endothelium, indicating the dependence of vasoconstrictor prostoglandins synthesized by PLA2‐I in endothelium. The PLA2‐I‐induced relaxation response was also observed in bovine and cat cerebral arteries, thus providing a new aspect of PLA2‐I as a vasoactive substance.

Vasodilatation induced by Pinacidil in dogs. Comparison with hydralazine and nifedipine

Summary: In helical strips of dog arteries precontracted with prostaglandin (PG) F2α, pinacidil and nifedipine produced a dose-related relaxation. The potencies of pinacidil were in the order of coronary and renal > mesenteric > basilar and middle cerebral arteries, whereas those of nifedipine were in the order of basilar and renal > mesenteric and coronary arteries. Pinacidil caused a greater relaxation in mesenteric veins than in the arteries. Hydralazine consistently relaxed the arteries only at 10−3M. In mesenteric artery strips exposed to Ca2+-free, high K+ media, contractions induced by Ca2+ were reduced by 10−8M nifedipine, but they were not influenced by 10−5M pinacidil or by 10−4M hydralazine. In the arteries exposed to Ca2+-free media and stimulated by PGF2α or norepinephrine, tonic contractions induced by Ca2+ were reduced moderately by 10−5M pinacidil but only slightly by 10−8M nifedipine. In Ca2+-free media, PGF2α-induced contractions were inhibited only by pinacidil. In isolated mesenteric vasculature, perfusion pressure was lowered by pinacidil and hydralazine. It may be concluded that pinacidil produces vasodilatation due to interference with the transmembrane influx of Ca2+ into smooth muscle evoked by receptor stimulation but not that due to inhibition in the Ca2+ influx associated with K+-induced membrane depolarization. Decreased release of Ca2+ from intracellularly stored sites or increased sequestration to the sites may also be involved. Pinacidil appears to dilate arteries and veins as well as resistance vessels, whereas hydralazine appears to act exclusively on resistance vessels.

A dicarba analog of β‐atrial natriuretic peptide (β‐ANP) inhibits guanosine 3′,5′‐cyclic monophosphate production induced by α‐ANP in cultured rat vascular smooth muscle cells

The synthesis and biological properties are described of [Asu7,23′]‐β‐ANP‐(7–28) (Asu, L‐α‐aminosuberic acid), a dicarba analog of β‐atrial natriuretic peptide (β‐ANP, an antiparallel dimer of human α‐ANP with the chains linked by 7–23′ and 7′–23 disulfide bonds). This Asu‐analog (referred to as analog III) displaced 125I‐α‐ANP specifically bound to cultured rat vascular smooth muscle cells (VSMC) with an apparent K i of 2.1 × 10−8 M, but did not stimulate formation of intracellular cGMP at 10−8–10−5 M. Analog III inhibited the α‐ANP‐stimulated cGMP production in VSMC competitively with a pA 2 value of 7.45 and behaved as an antagonist of α‐ANP in rat aorta smooth muscle relaxation. In addition, β‐ANP was also shown to inhibit the α‐ANP‐induced cGMP production in a dose‐dependent manner. The mechanism of action of β‐ANP is also discussed.

Comparison of vascular and platelet thromboxane A2/prostaglandin H2 receptors in the pig

We compared the properties of vascular and platelet thromboxane A2/prostaglandin H2 receptors in the pig. The binding profiles of U46619, several prostaglandins and thromboxane A2/prostaglandin H2 receptor antagonists to the aorta and platelet receptors were almost the same irrespective of whether the agonist ([3H]U46619) or the antagonist ([3H]SQ29,548) radioligand was used, except that the receptor density in the latter was 4 times higher than that in the aorta. The antagonists suppressed U46619-induced contraction of pig coronary artery and secondary aggregation of platelets at potencies comparable to their KI values in the binding experiments. On the other hand, the responses of the artery specimens to U46619 and the prostaglandins differed from those of the platelets. Thus, the binding sites in the vascular and platelet receptors seem to be the same or quite similar, but there seem to be different mechanism(s) leading the agonistic binding signal to final responses.

Characterization of [3H]U46619 binding in pig aorta smooth muscle membranes

The binding characteristics of [3H]U46619, a tritiated thromboxane A2/prostaglandin H2 mimetic, were studied in pig aorta smooth muscle membranes. Binding was fast, saturable, selective and reversible. The KD values determined by kinetics, equilibrium binding and drug competition methods were 68, 42 and 53 nM, respectively. The Bmax was 87.8 fmol/mg protein. Specific binding was competitively displaced by thromboxane A2/prostaglandin H2 antagonists. Binding was also displaced by prostaglandins D2, E2 and F2 alpha with IC50 values of 8, 21 and 12 microM, respectively. U46619 contracted the rat and pig aorta smooth muscle, but the response of the latter was slower than that of the former. The antagonists prevented the U46619-induced contraction of rat aorta with the same rank order as that for the inhibition of ligand binding in pig aorta smooth muscle membranes. These results provide evidence that the putative thromboxane A2/prostaglandin H2 receptor in vascular smooth muscle membranes can be detected by a ligand binding technique.

Actions of a novel thromboxane A2-receptor antagonist, S-145, on isolated monkey and cat arteries

The effects of a novel thromboxane (Tx) A2-receptor antagonist, S-145, were investigated mainly in helical strips of monkey and cat arteries. S-145 (3 × 10−10 to 3 × 10−9 M) attenuated the contraction induced by U46619 (2 × 10−10 to 10−7 M), which produced concentration-dependent contraction in monkey cerebral, coronary, and mesenteric arteries, and cat cerebral arteries. The attenuation in the different monkey arteries did not differ much, and it tended to be greater in cat cerebral arteries than in monkey at concentrations of <10−9 M S-145. S-145 also suppressed contractions in cat cerebral arteries induced by prostaglandin (PG) F2α, PGE2, and PGD2. However, S-145 did not affect the contractile responses to PGF2α in cat iris sphincter muscle and to PGE2 in guinea pig ileum. In cat mesenteric arteries, S-145 did not affect contractions induced by norepinephrine or K+, or relaxations induced by PGI2 or adenosine. The addition of S-145 (10−9-10−8 M) produced a transient contraction in cat cerebral arteries, and when S-145 (3 × 10−11 to 3 × 10−7 M) was cumulatively added, the contraction was not produced. The measurement of antagonistic potency of S-145 was not complicated by its agonistic effect, since the former potency was always determined after confirming absence of the latter effect. These results suggest that S-145 is a potent TxA2-receptor antagonist with partial agonistic activity in vascular smooth muscle. PGF2α, PGE2, and PGD2, however, at least in part, seemed to interact with the TxA2 receptor in vascular smooth muscle.

A new blood pressure measuring apparatus equipped with a microcomputer system for conscious rats

In order to measure the systolic blood pressure of conscious rats without thermal stress, a highly sensitive pulse sensor was developed using a light-emitting diode-photo diode system. The combination of this pulse sensor and microcomputer system led to the development of a six-channel automatic blood pressure measuring apparatus for rats. It can measure the systolic tail arterial pressure of conscious rats at 28-30 degrees C. The relationship between direct carotid arterial pressure (Y) and indirect tail arterial pressure (X) in conscious spontaneously hypertensive rats can be expressed as follows: Y = 0.95X + 19.9 (mm Hg, r = 0.988, 28 degrees C). The new apparatus was also used to confirm the acute hypotensive effects of hydralazine, nifedipine, and pindolol in spontaneously hypertensive rats and the subacute antihypertensive effect of trichlormethiazide in desoxycorticosterone/saline hypertensive rats.

Antagonistic actions of S-145 on vascular and platelet thromboxane A2 receptors

We studied the actions of a potent thromboxane A2/prostaglandin in H2 (TP) receptor antagonist, (+/-)-(5Z)-7-[3-endo-[(phenylsulfonyl)amino]bicyclo [2.2.1]hept-2-exo-yl]heptenoic acid (S-145) on vascular and platelet receptors in the pig. S-145 showed almost the same affinity for both receptors in ligand binding studies with [3H]U46619 or [3H]SQ29, 548. The binding affinity of S-145 was 5-8 times higher than that of SQ29,548, a well-characterized TP receptor antagonist. However, S-145 inhibited U46619-induced contractions of pig coronary arteries with an IC50 value 52.5 times lower than that of SQ29,548, and was approximately equipotent with SQ29,548 in inhibiting U46619-induced secondary aggregation of pig platelets. Detailed kinetic studies on [3H]S-145 binding revealed that the apparent discrepancy between the pharmacological potency of S-145 in platelet and vascular systems was not due to tissue selectivity, but its small association constants for both receptors.