Hiroo Itoh

Effects of Cilostazol, a Selective cAMP Phosphodiesterase Inhibitor on the Contraction of Vascular Smooth Muscle

The effects of cilostazol (OPC-13013, 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-quin olinone) on cyclic nucleotide metabolism and Ca2+-induced contraction of intact and skinned rabbit arterial smooth muscles were investigated. The concentrations of cilostazol producing 50% inhibition of cyclic adenosine monophosphate phosphodiesterase and Ca2+-dependent cyclic nucleotide phosphodiesterase were 0.4 microM and above 100 microM, respectively. This compound has no significant effect on adenylate cyclase in concentrations of up to 100 microM. Addition of cilostazol increased significantly the cAMP content without significant effect on cyclic guanosine monophosphate level of rabbit thoracic aorta in the presence of forskolin. Moreover, the ED50 value of cilostazol in relaxation of rabbit mesenteric arterial strips was decreased selectively by addition of 0.01 microM forskolin, which alone at this concentration has no effect on vascular contraction. Cilostazol of up to 30 microM did not suppress the Ca2+-induced contraction of the chemically skinned rabbit mesenteric artery. Therefore, cilostazol may produce the relaxation of intact vascular smooth muscle by its inhibition of cyclic adenosine monophosphate hydrolysis.

Isoenzymes of cyclic nucleotide phosphodiesterase in the human aorta : characterization and the effects of E4021

In extracts of the human aorta, five isoenzymes of cyclic nucleotide phosphodiesterase, namely, phosphodiesterase I, phosphodiesterase II, phosphodiesterase III, phosphodiesterase IV and phosphodiesterase V, were identified exclusively in the cytosolic fraction, and no phosphodiesterase activity was detected in the particulate fraction. Phosphodiesterase V and phosphodiesterase I were the major cGMP-hydrolyzing enzymes in the human aorta. A novel vasorelaxant, sodium 1-[6-chloro-4-(3,4-methylenedioxybenzyl)aminoquinazolin-2-yl ]piperidine-4- carboxylate sesquihydrate (E4021), relaxed prostaglandin F2 alpha-precontracted strips of human pulmonary artery with an ED50 value of 0.5 microM. E4021 potently and highly selectively inhibited the activity of phosphodiesterase V from human aorta with a Ki value of 2.4 nM. These results suggest that there is a unique distribution of phosphodiesterase isoenzymes in the human aorta and that inhibitors of phosphodiesterase V might be useful as a new type of vasodilator in the treatment of clinical disorders.

Vitamin A acid-induced activation of Ca2+-activated, phospholipid-dependent protein kinase from rabbit retina.

Ca2+-activated, phospholipid-dependent protein kinase from rabbit retina was partially purified. Vitamin A acid (retinoic acid) stimulated this protein kinase in the presence of Ca2+, while other metabolites of vitamin A such as retinol or retinal were less effective. The order of the extent of phosphorylation of the various substrate proteins by this protein kinase was identical in the presence of vitamin A acid or phosphatidylserine. The major spots of the 32P labeled peptide from histone H1 phosphorylated in the presence of vitamin A acid by this protein kinase did not differ from those obtained from histone H1 phosphorylated in the presence of phosphatidylserine. Retinol caused a further enhancement of the enzymatic activity, whereas the addition of retinal inhibited the activation by vitamin A acid. Thus, vitamin A and its metabolites may play an important role in the regulation of Ca2+-activated, phospholipid-dependent protein kinase activity in the retina.

Ca2+-dependent and Ca2+-independent vasorelaxation induced by cardiotonic phosphodiesterase inhibitors

Cardiotonic agents that belong to a group of phosphodiesterase inhibitors--vesnarinone, amrinone, enoximone, pimobendan, MS-857 and E-1020--inhibited the 35 mM KCl- and 10(-7) M norepinephrine-induced contractions of helical strips from rat thoracic aorta in a concentration-dependent manner. In the absence of extracellular Ca2+, 10(-7) M phorbol 12,13-dibutyrate caused a sustained contraction of the muscle strip without an increase in intracellular Ca2+ level ([Ca2+]i). The phorbol 12,13-dibutyrate-induced contractions in Ca2+(-)free buffer were also inhibited by the cardiotonic phosphodiesterase inhibitors. A cyclic GMP-inhibited cyclic nucleotide phosphodiesterase was partially purified from rat aorta and the activity of the phosphodiesterase was inhibited by the cardiotonic agents. The inhibitory effect of these agents on the KCl-, norepinephrine-and phorbol 12,13-dibutyrate-induced contractions showed good correlations to the concentrations of the agents producing 50% inhibition (IC50) of cyclic GMP-inhibited cyclic nucleotide phosphodiesterase. Vesnarinone inhibited the norepinephrine-induced contraction with a decrease in [Ca2+]i, but inhibited the phorbol 12,13-dibutyrate-induced contraction in Ca(2+)-free buffer without significant changes in [Ca2+]i. Dibutyryl cyclic AMP and NKH477 also inhibited the phorbol 12,13-dibutyrate-induced contraction in Ca(2+)-free buffer without significant changes in [Ca2+]i. The six cardiotonic phosphodiesterase inhibitors increased the cyclic AMP contents of the muscle strips. These results suggest that the inhibitory actions of these cardiotonic phosphodiesterase inhibitors on cyclic GMP-inhibited cyclic nucleotide phosphodiesterase may cause vasorelaxation through a decrease in [Ca2+]i and an inhibitory effect on a Ca(2+)-independent contractile process (or a decrease in Ca(2+)-sensitivity of contractile elements).

Contractile actions of endothelin-1 in isolated helical strips from rat pulmonary artery : potentiation of serotonin-induced contraction

Summary: Endothelin-1 (ET-1) caused a concentration-dependent contraction of helical strips from rat pulmonary arteries. Removal of endothelial cells did not change the response to ET-1. ET-1 (10-8M) induced a small contraction of the arterial strips in the absence of extracellular Ca2+ (31.0% of the contraction in the presence of extracellular Ca2 + ). Pretreatment of pulmonary arterial strips with 6 x 10-11M ET-1 potentiated the serotonin-induced contraction (10-7-3 x 10-6 M), but showed no significant effect on KCl-induced contraction. The ET-1-induced potentiation of the response to serotonin was prevented by nordihydroguaiaretic acid (3 x 10-6M), quinacrine (10-6M), and AA861 (10_6M), but not by in-domethacin (10-7M) and baicalein (10-6M). These results suggest that ET-1 may cause pulmonary hypertension through a direct vasoconstrictor action and potentiation of serotonin-induced contraction, which may involve the 5-lipoxygenase pathway.

Contraction of rat thoracic aorta strips by endothelin‐1 in the absence of extracellular Ca2+

1 Endothelin‐1 (ET‐1) caused a concentration‐dependent contraction of helical strips from rat thoracic aorta in the absence of extracellular Ca2+. The Ca2+‐depleted muscle strips, prepared by three repeated applications of 10−2 m caffeine or 10−6 m noradrenaline in Ca2+‐free buffer, were contracted by 10−8 m ET‐1 in the same manner as non‐treated strips. 2 In the absence of extracellular Ca2+, 10−7 m phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C, induced a small but sustained contraction of the rat thoracic aorta strips within 60 min. Preincubation of the strips with 10−7 m PMA for 60 min in Ca2+‐free buffer, did not affect the 10−8 m ET‐1‐induced contraction, but decreased the 5 × 10−8 m phorbol 12,13‐dibutyrate (PDB)‐, or the 10−7 m PMA‐induced contraction, and potentiated the contraction induced by 10−8 m urotensin II. Preincubation with 10−8 m ET‐1 (which induced maximum contraction) for 25 min in Ca2+‐free buffer did not change the subsequent contraction induced by PMA (10−7 m) or urotensin II (10−8 m) but gave a somewhat lower maximum tension than in non‐treated strips. 3 Calyculin‐A, a potent inhibitor of phosphatase, also induced a contraction of the Ca2+‐depleted muscle strips in Ca2+‐free buffer. Preincubation of the strips with ET‐1 (10−8 m) or PMA (10−7 m) decreased the calyculin‐A (3 × 10−8 m)‐induced contraction. 4 These results suggest that ET‐1 may induce phosphorylation of an unknown protein either without an increase in myoplasmic Ca2+ concentration or, alternatively, with mobilization of intracellular Ca2+ from noradrenaline‐ and caffeine‐insensitive Ca2+ sources, through a mechanism different from that of phorbol ester.

Comparison of the effects of various vasodilators on the rat portal vein and mesenteric artery

The effects of various vasodilators on isolated helical strips of rat portal vein and mesenteric artery were examined. Dilazep, ibudilast, nifedipine, verapamil and papaverine relaxed the KCl- and norepinephrine-induced contractions of the portal vein to a greater extent than they relaxed those of mesenteric artery. Dibutyryl cyclic AMP and NKH477 did not show any significant difference in terms of the concentrations that produced 50% inhibition (IC50) of the KCl-induced contraction of the portal vein and mesenteric artery. The relaxant effects of nitroprusside and isosorbide dinitrate on the KCl- and norepinephrine-induced contractions of the portal vein were less potent than their effects on contractions of the mesenteric artery. The agents that inhibited the contractions of the portal vein more potently than those of the mesenteric artery were associated with parallel shifts to the right of the concentration-response curves for CaCl2 in both preparations. These results suggest that drugs with a Ca2+ channel blocking action may be preferable to those that cause a decrease in portal pressure for treatment of portal hypertension.

Effects of the endothelin ETA receptor antagonist, TA-0201, on pulmonary arteries isolated from hypoxic rats

To investigate the roles of endothelin-1 in the pathogenesis of hypoxic pulmonary hypertension, we studied the effects of a selective endothelin ET(A) receptor antagonist, TA-0201 (N-(6-(2-(5-Bromopyrimidin-2-yloxy) ethoxy)-5-(4-methylphenyl) pyrimidin-4-yl)-4-(2-hydroxy-1,1-dimethylethyl) benzensulfonamide sodium salt sesquihydrate), on helical strips of pulmonary arteries isolated from hypoxia-induced pulmonary hypertensive rats as compared with those of normoxic rats. Endothelin-1-induced maximum contractions were significantly inhibited by exposure to hypoxia in the pulmonary arterial strips, but not in the mesenteric arterial strips. The hypoxia also induced right ventricular hypertrophy in rats. Addition of TA-0201 to the bath inhibited the endothelin-1-induced contraction of pulmonary arterial strips isolated from hypoxic rats more effectively than in those of normoxic rats. Oral administration of TA-0201 to hypoxic rats inhibited the hypoxia-induced right ventricular hypertrophy, and decreased the maximum contractile response to endothelin-1 in pulmonary arterial strips isolated from these rats. Those inhibitory effects induced by the oral administration of TA-0201 were not observed in the pulmonary arteries from normoxic rats or in the mesenteric arteries from both hypoxic and normoxic rats. These results suggest that endothelin-1 has important pathophysiological roles in hypoxia-induced pulmonary hypertension, and that TA-0201 may inhibit the endothelin-l-induced contraction through a change in the function of endothelin ET(A) receptor as well as competitive inhibition for endothelin ET(A) receptor.