Seigo Fujimoto

The antisense homology box: A new motif within proteins that encodes biologically active peptides

Amphiphilic peptides approximately fifteen amino acids in length and their corresponding antisense peptides exist within protein molecules. These regions (termed antisense homology boxes) are separated by approximately fifty amino acids. Because many sense–antisense peptide pairs have been reported to recognize and bind to each other, antisense homology boxes may be involved in folding, chaperoning and oligomer formation of proteins. The antisense homology box–derived peptide CALSVDRYRAVASW, a fragment of human endothelin A receptor, proved to be a specific inhibitor of endothelin peptide (ET–1) in a smooth muscle relaxation assay. The peptide was able to block endotoxin–induced shock in rats as well. Our finding of endothelin receptor inhibitor among antisense homology box–derived peptides indicates that searching proteins for this new motif may be useful in finding biologically active peptides.

Mechanisms of hydrogen peroxide-induced relaxation in rabbit mesenteric small artery.

The effects of hydrogen peroxide were studied on isolated rabbit mesenteric small artery; rabbit superior mesenteric artery and mouse aorta were also studied as reference tissues. For mesenteric small artery, hydrogen peroxide (1 to 100 microM) relaxed a norepinephrine-stimulated artery in a concentration-dependent manner. The relaxation was not significantly affected by removal of the endothelium and was less pronounced in arteries contracted with high-KCl solution plus norepinephrine than in those contracted with norepinephrine alone. The relaxation response to hydrogen peroxide was increased by isobutylmethylxanthine and zaprinast, inhibited by diclofenac, methylene blue and dithiothreitol and unaffected by atropine, tetraethylammonium, superoxide dismutase, deferoxamine, dimethyl sulfoxide or the Rp stereoisomer of adenosine cyclic monophosphothioate. Hydrogen peroxide shifted concentration-contractile response curves for norepinephrine to the right and downwards. Norepinephrine and caffeine elicited a transient, phasic contraction of the mesenteric small artery exposed for 0.5, 1 and 2 min to a Ca2+-free solution. Hydrogen peroxide inhibited the norepinephrine-induced contraction, and to a lesser extent the caffeine-induced contraction, and verapamil did not alter the contraction to norepinephrine. These pharmacological properties of hydrogen peroxide were similar to those of 8-bromo cGMP; 8-bromo cGMP inhibited more potently the norepinephrine-induced than the KCl-induced contraction and the contraction elicited by norepinephrine in Ca2+-free solution. The present results suggest that hydrogen peroxide induces endothelium-independent relaxation of the rabbit mesenteric small artery precontracted with norepinephrine. The effects of hydrogen peroxide may be at least in part mediated by cGMP and cyclooxygenase products in the vascular smooth muscles now used.

Diminished β-adrenoceptor-mediated relaxation of arteries from spontaneously hypertensive rats before and during development of hypertension

beta-Adrenoceptor agonists and other drugs were studied for their relaxant effects on femoral and mesenteric arterial strips from spontaneously hypertensive rats (SHR). The potency and efficacy of isoproterenol (ISO) in these arteries were decreased in SHR before and during the development of hypertension as compared with age-matched Wistar Kyoto rats (WKY). Reserpine and 6-hydroxydopamine inhibited the development of hypertension but did not alter the reduced ISO-induced relaxation of the arteries. These arteries from prehypertensive SHR (PHSHR) were less sensitive to salbutamol and cyclic AMP and cyclic GMP derivatives than arteries from age-matched WKY. The relaxation response to nitroprusside was less in the femoral but not in the mesenteric arteries from PHSHR than in arteries from age-matched WKY. The relaxation response to papaverine was not diminished in the PHSHR arteries. It was found that the SHR arteries had a reduced responsiveness to the beta-adrenoceptor agonists before the initiation of hypertension and that the diminished relaxation was not specific to the beta-agonists, although there was no generalized defect in vasorelaxation in PHSHR.

Mechanisms underlying the hydrogen peroxide-induced, endothelium-independent relaxation of the norepinephrine-contraction in guinea-pig aorta

The mechanisms underlying the hydrogen peroxide-induced relaxation of the norepinephrine-contraction were studied by measuring isometric force, myosin light chain (MLC(20)) phosphorylation and cyclic GMP in endothelium-denuded muscle from the guinea-pig aorta. Norepinephrine (5.2+/-1.3 microM) produced a phasic, followed by a tonic contraction. Hydrogen peroxide (10 and 100 microM), glyceryl trinitrate (30 and 300 nM) and 8-bromo cyclic GMP (30 and 100 microM) did not change the basal tone, but reduced the norepinephrine-induced contraction. Phosphorylation of MLC(20) (percentage of phosphorylated to total MLC(20)) was increased 1 min (5.9+/-1.0% vs. 35.9+/-4.9%) and, to a lesser extent, 20 min (3.7+/-1.7% vs. 13.9+/-1.6%) after the addition of norepinephrine. Hydrogen peroxide (100 microM) did not modify basal MLC(20) phosphorylation, but reduced the increase in MLC(20) phosphorylation induced by 1-min exposure to norepinephrine (20.9+/-4.1%). Its effect was abolished by catalase. When the tissue was incubated for 20 min with norepinephrine in the presence of hydrogen peroxide, norepinephrine-induced MLC(20) phosphorylation was not changed (13.6+/-1.5%), as compared to that in the absence of hydrogen peroxide. Hydrogen peroxide relaxed norepinephrine-stimulated aortas in a concentration-dependent fashion with EC(50) values of 5.9+/-0.2 microM. The relaxation was inhibited by soluble guanylate cyclase inhibitors and increased by an inhibitor of cyclic GMP-selective phosphodiesterase. In aorta precontracted with norepinephrine, hydrogen peroxide (100 microM) relaxed the tissue by 89+/-11% and almost doubled tissue concentrations of cyclic GMP, whereas sodium nitroprusside (1 microM) relaxed the tissue by 100% and increased cyclic GMP concentrations 30-fold. It is suggested that the inhibitory effects of hydrogen peroxide on the norepinephrine-induced phasic and sustained contractions are explained by a decrease in MLC(20) phosphorylation and by an alteration in MLC(20) phosphorylation-independent mechanisms, respectively. The effects of hydrogen peroxide were in part mediated by cyclic GMP.

Role of nitric oxide and nitric oxide-independent relaxing factor in contraction and relaxation of rabbit blood vessels.

It has been shown that spontaneous release of nitric oxide (NO) from the vascular endothelium attenuates contractile responses of vascular smooth muscles to norepinephrine, and that acetylcholine-induced relaxation is mediated by the evoked release of NO and endothelium-derived hyperpolarizing factor. Since the involvement of these substances (or factors) in mechanical responses is heterogeneous among blood vessels, we have investigated the role of these substances in agonist-induced contraction and relaxation in 6 rabbit blood vessels. Vascular reactivity for the contractile response to norepinephrine was potentiated after removal of endothelium and by 100 microM N(G)-nitro-L-arginine (L-NA) but not by 80 nM-0.4 microM clotrimazole. This potentiation was most marked in the mesenteric artery among the blood vessels tested, suggesting that the basal release of NO reduced the contractile response of the vascular smooth muscle to norepinephrine in this artery. Acetylcholine-induced relaxation was abolished by removal of the endothelium and was attenuated by L-NA (1-100 microM) in all blood vessels. The attenuation by 100 microM L-NA was most obvious in aorta and vein and least in mesenteric resistance artery in which the acetylcholine-induced, L-NA-resistant relaxation was inhibited by 80 nM-0.4 microM clotrimazole. These results suggested that there is a regional difference in the degree of involvement of NO in acetylcholine-induced relaxation. In mesenteric resistance artery, the NO-independent, clotrimazole-sensitive factor, possibly hyperpolarizing factor may also contribute to the response to acetylcholine at high concentrations.

K(+) channel blockers and cytochrome P450 inhibitors on acetylcholine-induced, endothelium-dependent relaxation in rabbit mesenteric artery.

Acetylcholine caused an endothelium-dependent relaxation in isolated rabbit mesenteric small artery in the presence of nitro L-arginine and indomethacin. The acetylcholine-induced relaxation was attenuated by high K(+) solution, suggesting that the response is mediated by a membrane potential-sensitive mechanism, presumably an endothelium-derived hyperpolarizing factor. The acetylcholine-induced relaxation was also inhibited with tetraethylammonium, 4-aminopyridine and charybdotoxin, but not with Ba(2+), apamin, iberiotoxin nor glibenclamide. The relaxation was abolished by a combination of apamin and charybdotoxin, but iberiotoxin could not replace charybdotoxin in this combination. The responses to charybdotoxin and 4-aminopyridine were synergistic but neither apamin nor iberiotoxin increased the effect of 4-aminopyridine. Clotrimazole and proadifen inhibited the acetylcholine-induced relaxation, but these drugs also inhibited the cromakalim-induced relaxation, while protoporphyrin IX inhibited the acetylcholine- but not cromakalim-induced relaxation. 17-Octadecynoic acid and 1-aminobenzotriazole did not affect the response to acetylcholine. Four regioisomers of epoxyeicosatrienoic acids did not relax endothelium-denuded artery. A gap junction inhibitor 18alpha-glycyrrhetinic acid attenuated the relaxation to acetylcholine. It is suggested that in rabbit mesenteric artery, the acetylcholine-induced, nitric oxide- and prostacyclin-independent relaxation is mainly mediated by 4-aminopyridine- and charybdotoxin-sensitive K(+) channels and that the relaxation is not mediated through cytochrome P450 enzyme metabolites. The contribution of heterocellular gap junctional communication to the relaxation is discussed.

Kinin release from kininogens by calpains

During the investigation of inhibitory activity of kininogens toward calpains [EC 3.4.22.17], we found that lysyl-bradykinin was liberated from both high molecular weight (HMW) and low molecular weight (LMW) kininogens by the action of the calpains. The kinin liberation occurred in a limited range of calpain to kininogen molar ratios of 0.5:1 to 8:1, and in that condition calpains were simultaneously inhibited 20 to 80% by kininogens. The maximum level of kinin release from HMW and LMW kininogens by calpain I was about 25% and that by calpain II was 20%. These results suggest that in case of inflammation the kininogens play two physiologically distinct roles by interaction with calpains: to release lysyl-bradykinin and to inhibit proteinase activity of calpains derived from the damaged tissues.

Malfunction of arterial sarcoplasmic reticulum leading to faster and greater contraction induced by high-potassium depolarization in young spontaneously hypertensive rats

The contribution of sarcoplasmic reticulum was studied with regard to the increase in arterial contraction induced by a high-potassium depolarization in spontaneously hypertensive rats (SHR). The 20 mmol/l potassium-induced contraction of femoral arteries was faster and greater in 6-week-old SHR than in age-matched normotensive Wistar-Kyoto (WKY) rats. Relaxation after washing the arteries with a Krebs solution was slower in SHR than in WKY rats. When the sarcoplasmic reticulum of SHR arteries had been depleted of calcium by caffeine in a calcium-free solution, the rate of high-potassium-induced contraction of the calcium-depleted SHR arteries was slowed, the same result as that with non-calcium-depleted WKY arteries. In ryanodine-treated arteries, the rate and magnitude of high-potassium-induced contraction were enhanced slightly in SHR and greatly in WKY rats, resulting in no final difference between SHR and WKY rats. Ryanodine slowed the relaxation rate in WKY rats but not in SHR. These results suggest that the diminution in ability of sarcoplasmic reticulum to sequester calcium may be responsible for the faster rate and greater magnitude of high-potassium-induced contraction with the slower relaxation in SHR arteries. We postulated that genetic malfunction of sarcoplasmic reticulum causes the increased contraction of arterial smooth muscle leading to the enhanced vasoconstriction and elevated blood pressure in SHR.

Beta1- and beta2-adrenoceptor-mediated relaxation responses in peripheral arteries from spontaneously hypertensive rats at pre-hypertensive and early hypertensive stages

Beta-adrenoceptor-mediated relaxation of isolated arteries from spontaneously hypertensive rats (SHR) was studied. The relaxation responses of the femoral arteries of pre-hypertensive SHR (PHSHR) to isoprenaline (ISO) and dibutyryl cyclic AMP were less than those of the age-matched tissues of rats of the Wistar-Kyoto strain (WKY). The responses of the mesenteric arteries of PHSHR to ISO and noradrenaline (NA) but not fenoterol (FEN) and forskolin were diminished when compared to those from the WKY tissues. The diminished relaxation response to ISO was observed in the SHR arteries with and without endothelial cells as compared to similar arteries from WKY. Relative potency ratios of ISO:FEN:NA and the Schild plot data for atenolol and butoxamine suggested that there were only beta 1-adrenoceptors and predominantly beta 2-adrenoceptors mediating relaxation in the femoral and mesenteric arteries, respectively. There was no difference in beta-adrenoceptor subtypes between the SHR and WKY arteries. The evidence in this study suggests that beta 1-adrenoceptor-mediated relaxation was diminished in the femoral and mesenteric arteries of PHSHR.

Vasorelaxant effect of olprinone, an inhibitor of phosphodiesterase 3, on mesenteric small artery and vein of rabbits

The effects of olprinone, a cardiotonic agent that inhibits cyclic GMP (cGMP)-inhibited phosphodiesterase, was studied on isolated rabbit mesenteric small artery and vein. In the presence of indomethacin and propranolol, olprinone at concentrations of 10 nM to 10 microM and 1 microM to 100 microM relaxed norepinephrine-stimulated mesenteric artery and vein in a concentration-dependent manner, respectively. The relaxation was not endothelium-dependent in the artery. Removal of the endothelium, however, increased marginally the response of the vein to olprinone. Olprinone-induced relaxation was less pronounced in arteries contracted with high KCl solution + norepinephrine than in those contracted with norepinephrine alone. Nicardipine inhibited this attenuating effect of high KCl solution on the olprinone-induced relaxation. Olprinone (1 microM) enhanced the relaxation of artery and vein in response to a cAMP-increasing agent, 6-(3-dimethylaminopropionyl) forskolin (NKH477), but not to a cGMP- increasing agent, glyceryl trinitrate. Norepinephrine (10 microM) and caffeine (5 mM) elicited a transient, phasic contraction of the artery in Ca2+-free solution. Both olprinone and NKH477 attenuated more potently the norepinephrine-induced contraction than the caffeine-induced contraction. When norepinephrine (10 microM) and caffeine (5 mM) were successively applied in Ca2+-free solution, the contractile effect of caffeine was diminished compared to that in artery which had not been pretreated with norepinephrine. When the contraction in response to norepinephrine was partially attenuated by 1 microM olprinone, the following contraction evoked by caffeine was enlarged. It is concluded that olprinone relaxes the small artery more strongly than the vein via its direct action on smooth muscles. It is suggested that olprinone attenuates norepinephrine-induced contraction through inhibition of receptor-operated transmembrane Ca2+ influx and Ca2+ release from intracellular storage sites.