Issei Takayanagi

Inhibitory effects of a fullerene derivative, dimalonic acid C60, on nitric oxide-induced relaxation of rabbit aorta.

Dimalonic acid C60 (10(-5) M), a new fullerene derivative, produced an augmentation of phenylephrine-induced tone and reduced both the acetylcholine-induced maximum relaxation and the amplitude of substance P (10(-8) M)-induced relaxation in endothelium-containing thoracic aorta of rabbit; the acetylcholine- and substance P-induced relaxation was restored in the presence of superoxide dismutase (250 U/ml). Dimalonic acid C60 (10(-5) M) did not influence the phenylephrine-induced contractile response in the absence of endothelium, but the acetylcholine-induced relaxation was eliminated by removal of the endothelium. Superoxide anion generation, using hypoxanthine (1 mM)/xanthine oxidase (16 mU/ml), reduced the acetylcholine-induced relaxation and produced an augmentation of phenylephrine-induced tone in endothelium-containing strips; these effects were negated by the addition of superoxide dismutase (250 U/ml). A nitric oxide-generating agent, S-nitroso-N-acetylpenicillamine, caused relaxation of aorta without endothelium in a concentration-dependent manner, and the concentration-response curve was shifted to the right in the presence of dimalonic acid C60. This inhibitory effect of dimalonic acid C60 was also masked in the presence of superoxide dismutase. Sodium nitroprusside-induced relaxation was not affected by either dimalonic acid C60 or superoxide dismutase. These observations suggest that dimalonic acid C60 inhibits endothelium (nitric oxide)-dependent agonist-induced relaxation through the production of superoxide.

Endothelin-1 induced contraction of rat aorta: contributions made by Ca2+ influx and activation of contractile apparatus associated with no change in cytoplasmic Ca2+ level

SummaryThe modes by which Endothelin-1 (ET) induces Ca2+-influx and the relative functional importance of the different sources of Ca2+ for ET-induced contraction were studied using fura 2-loaded and unloaded rat aortic strips. ET caused an increase in the cytosolic free Ca2+ level ([Ca2+]i) followed by a tonic contraction in Ca2+-containing solution, and produced a transient elevation of [Ca2+]i followed by a small sustained contraction in Ca2+-free medium. ET also stimulated 45Ca influx into La2+-inaccessible fraction significantly. With the same change of [Ca2+]i, ET caused a larger tension than that induced by high K. ET-induced contraction and [Ca2+]i elevation were not significantly inhibited by 0.1–0.3 μM nicardipine which nearly abolished the contraction and [Ca+]i elevation produced by high K. During treatment of the strips with high K, addition of ET induced further increases in [Ca2+]i and muscle tension, and vice versa. In Ca2+-free medium, ET-induced contraction was influenced neither by ryanodine-treatment nor by high K-treatment, although the former attenuated and the latter potentiated the [Ca2+]i transient induced by ET. Further, the ET-induced sustained contraction under Ca2+-free conditions began to develop after the [Ca2+]i level returned to the baseline. Thus, it seems that the Ca2+ released from the ryanodine-sensitive and -insensitive Ca2+ stores by ET may provide only a minor or indirect contribution, if any, to the tension development. ET might cause a contraction mainly by stimulating Ca2+-influx through Ca2+ channel(s) other than voltage-dependent Ca2+ channels in character, and by increasing the sensitivity of the contractile filaments to Ca2+ or activating them Ca2+-independently.

Ryanodine: its possible mechanism of action in the caffeine-sensitive calcium store of smooth muscle

The caffeine-sensitive intracellular Ca store was characterized and the mechanism of action of ryanodine in the store was studied using K-depolarized guinea-pig taenia caecum. (1) After incubation of the preparation with CaCl2 (Ca loading), caffeine was applied in Ca-deprived medium, to produce a transient contraction and to monitor the amount of the stored Ca. As duration of Ca deprivation was prolonged, the amplitude of the caffeine-induced contraction was decreased. When ryanodine was applied during Ca deprivation, the rate of the decrease was remarkably accelerated. (2) The rate of rise of the contraction induced by external Ca ((Ca)o) was slowed by preceding depletion of the stored Ca by caffeine, compared with that observed in the Ca loaded preparation. However, in the presence of ryanodine, even if stored Ca was depleted by caffeine, the rate of rise of the (Ca)o-induced contraction remained at a higher level. (3) These results suggest that ryanodine stimulates a leak of the stored Ca, and that the contraction induced by the transmembrane influxed Ca could be modulated by the amount of Ca in, or leakiness of, the caffeine-sensitive Ca store.

Ryanodine reveals multiple contractile and relaxant mechanisms in vascular smooth muscle: simultaneous measurements of mechanical activity and of cytoplasmic free Ca2+ level with fura-2

1 The effects of ryanodine on changes in cytoplasmic Ca2+ level ([Ca2+]i) and muscle tension induced by maximum concentrations of phenylephrine (Phe; 1μm), prostaglandin F2α (PGF2α, 10 μm), caffeine (Caf, 30 mm) and isoprenaline (Iso, 1 μm) were examined in rat aortic strips using fura‐2. 2 In normal media, Phe and PGF2α produced a phasic contraction, followed by a tonic one. Caf elicited only a transient contraction. When the preparation was treated with 10 μm ryanodine, an increase in [Ca2+]i was induced accompanied by a nicardipine (1 μm)‐resistant contraction which was [Ca2+]o‐dependent. 3 In Ca2+‐free solution, the three stimulants elicited transient increases in [Ca2+]i. Transient contractions to Phe and Caf were accompanied by changes in [Ca2+]i. The transient increase in [Ca2+]i induced by PGF2α was not accompanied by a corresponding contraction. 4 Sustained contractions were induced by Phe and PGF2α in the absence of external Ca2+, while the increase in [Ca2+]i was reduced. A larger maximum contraction was induced by PGF2α than by Phe. 5 Ryanodine abolished both the Caf‐ and Phe‐induced [Ca2+]i transient increases and the corresponding contractions, but had no substantial effect on the PGF2α‐induced [Ca2+]i transient increase. Ryanodine had no influence on the sustained contractions induced by Phe and PGF2α. 6 Iso relaxed both sustained contractions almost completely, without any detectable change in [Ca2+]i. Treatment of the preparation with ryanodine had no effect on the concentration‐response curves for Iso in relaxing the 0.1 μm Phe‐ or 40 mm K+‐induced precontraction. 7 It is suggested that Phe and Caf mobilize Ca2+ from a ryanodine‐sensitive Ca2+ store and that PGF2α releases Ca2+ from a ryanodine‐insensitive Ca2+ store. The former contributes to the transient contraction through a Ca2+‐dependent process, while the latter seems not to be directly associated with the contraction. The sustained contraction under Ca2+‐free conditions might involve a Ca2+‐independent process or a change in the sensitivity of the contractile filaments to Ca2+. 8 In addition to lowering cytoplasmic Ca2+ concentration, it is suggested that Iso counteracts the apparently Ca2+‐independent process. The ryanodine‐sensitive Ca2+ store plays no substantial role in active relaxation by Iso, although it does play a major role in the maintenance of cytoplasmic Ca2+ in a quiescent muscle.

Mechanism of action of nicotine in isolated urinary bladder of guinea-pig.

1 Nicotine produced a transient contraction of isolated strips of guinea‐pig urinary bladder. The response to nicotine was antagonized by the nicotinic receptor antagonist, hexamethonium but was insensitive to tetrodotoxin. 2 The nicotine‐induced contraction was potentiated by the cholinesterase inhibitor, physostigmine, and was reduced to 50% and 70% by the muscarinic cholinoceptor antagonist, atropine and the sympathetic neurone blocking drug, guanethidine, respectively. Chemical denervation with 6‐hydroxydopamine abolished the inhibitory effect of guanethidine. Simultaneous treatment with atropine and guanethidine did not abolish the response to nicotine, but the degree of inhibition was comparable to that obtained with atropine alone. 3 The nicotine‐induced contraction was insensitive to bunazosin and yohimbine (α1‐ and α2‐adrenoceptor antagonists, respectively), and exogenously applied noradrenaline did not cause a contraction even in the presence of blockade of noradrenaline uptake mechanisms with desipramine and normetanephrine and of β‐adrenoceptors with propranolol, suggesting a non‐adrenergic nature of the sympathomimetic effect of nicotine in this tissue. 4 The nicotine‐induced contraction in the presence of atropine was abolished after desensitization of P2‐purinoceptors with α, β‐methylene adenosine 5′‐triphosphate, a slowly degradable ATP analogue selective for P2‐purinoceptors. By this desensitization, the response to ATP, but not to histamine, was also abolished. 5 A cyclo‐oxygenase inhibitor flurbiprofen partially inhibited the nicotine‐induced contraction. The degree of the inhibition was more pronounced in the presence of atropine than in its absence. Flurbiprofen antagonized the response to exogenously applied ATP in an unsurmountable manner, but not that to carbachol. 6 The present results suggest that nicotine might induce a contraction through an interaction with nicotinic receptors located on the terminals of, possibly, (i) parasympathetic cholinergic, (ii) sympathetic non‐adrenergic and (iii) non‐sympathetic purinergic nerves in guinea‐pig detrusor preparations, and that a portion of the contraction due to the purine nucleotide released is possibly potentiated by intramural prostaglandin(s). Parasympathetic cholinergic output might be modulated by an unknown excitatory substance released by nicotine from sympathetic nerve. 7 Nicotine reveals a latent excitatory effect of the sympathetic hypogastric nerve which innervates guinea‐pig detrusor.

Possible mechanisms of β-adrenoceptor-mediated relaxation induced by noradrenaline in guinea pig taenia caecum

The mechanisms of the beta-adrenoceptor-mediated relaxation induced by noradrenaline in guinea pig taenia caecum were investigated. Noradrenaline caused graded relaxation of this preparation. However, the concentration-response curves for noradrenaline were unaffected by propranolol (approximately 10(-5) M) or phentolamine (approximately 10(-5) M). The responses to noradrenaline were antagonized in a concentration-dependent manner by bupranolol, and Schild plots of the data revealed a pA2 value of 5.53. Also, bupranolol antagonized responses to isoprenaline, and Schild plots of the data revealed the pA2 value to be 8.53. Noradrenaline significantly increased the cyclic AMP level in this preparation. Bupranolol (10(-4) M) significantly decreased the cyclic AMP response elicited by noradrenaline, whereas propranolol (10(-5) M) produced no effect. These results suggest that the relaxant response to noradrenaline in guinea pig taenia caecum is mainly mediated by beta 3-adrenoceptors (or atypical beta-adrenoceptors) and that in guinea pig taenia caecum noradrenaline behaves as a beta 3-selective adrenoceptor agonist.

Eicosanoid‐induced Ca2+ release and sustained contraction in Ca2+‐free media are mediated by different signal transduction pathways in rat aorta

1 The effects of 12‐O‐tetradecanoyl 4β‐phorbol 13‐acetate (β‐TPA) on the inositol 1,4,5‐trisphosphate (IP3) production, Ca2+ release from the intracellular Ca2+ stores and sensitization of contractile apparatus, induced by prostaglandin F2α (PGF2α) and U46619, a thromboxane A2‐mimetic, were studied, using fura‐2‐loaded and ‐unloaded rat thoracic aortic strips. 2 Both eicosanoids had characteristic patterns of responses in Ca2+‐free, 2 mm EGTA‐containing solution (Ca2+‐free solution). They induced transient increases in intracellular Ca2+ concentration ([Ca2+]i) without corresponding transient contraction, but produced delayed, sustained contraction, where [Ca2+]i was returned to the basal level. 3 Treatment with β‐TPA for 60 min reduced the eicosanoids‐induced IP3 production, suggesting that the treatment inhibits PIP2 breakdown. 4 The treatment also attenuated [Ca2+]i transient induced by the eicosanoids, but not by caffeine (an IP3‐independent releaser of stored Ca2+), in fura‐2‐loaded preparations incubated in Ca2+‐free solution. 5 In contrast in the presence of β‐TPA, the sustained contractions evoked by the eicosanoids in Ca2+‐free solution were potentiated, suggesting that the sites of actions of β‐TPA and the eicosanoids may differ from each other. 6 PGF2α and U46619 utilize different and parallel signal transduction pathways to release Ca2+ by IP3 produced by PIP2 breakdown (β‐TPA‐sensitive), and to increase the sensitivity of contractile apparatus, in which protein kinase C may not be involved (β‐TPA‐insensitive).

Possible involvement of substance P immunoreactive nerves in the mediation of nicotine-induced contractile responses in isolated guinea pig bronchus.

Nicotine-induced contraction of the isolated guinea pig bronchial preparation was abolished by capsaicin and a substance P (SP) antagonist [( D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP). Nicotine increased the release of immunoreactive SP from the preparations. The nicotine-evoked release of immunoreactive SP from the bronchial preparation was reduced by hexamethonium but not by tetrodotoxin. The results indicate that the responses to nicotine of the guinea pig bronchial preparation were mediated through the release of SP-like material(s), and that the nicotine-induced response may be produced through a process independent of the sodium action potential. In conclusion, the most likely site of action of nicotine in the isolated guinea pig bronchial preparation is the nicotinic receptor of SP immunoreactive nerves.

Comparison of the muscarinic cholinoceptors in the rabbit ciliary body and the guinea-pig ileum

Interactions of several muscarinic drugs with their receptors were studied in the ciliary body smooth muscle of rabbits and the ileal longitudinal muscle of guinea-pigs, using pharmacological and biochemical procedures. The dissociation constants of carbachol, pilocarpine, atropine and pirenzepine estimated by these procedures indicate no heterogeneity of muscarinic receptors in either tissue; thus both are probably of the M2 type. However, the density of the receptors in the ciliary body is lower than in the ileum. Pilocarpine, with lower intrinsic efficacy, demonstrated a pronounced organ selectivity when compared to carbachol as it was a potent full agonist in the ileum and a competitive antagonist in the ciliary body. These results suggest the importance of both receptor density and threshold as determinants of agonist potency.

Characterization of postsynaptic alpha1-adrenoceptors in the rabbit iris dilator smooth muscle

SummaryInteractions of several alpha-adrenoceptor agonists and antagonists with their receptors were studied in rabbit and guinea pig iris dilator smooth muscle and rabbit aortic strips using pharmacological procedures.In rabbit iris dilators and aortic strips, noradrenaline acted as a full agonist, while oxymetazoline, clonidine and tizanidine acted as partial agonists. The dissociation constants of full and partial agonists in the dilators, calculated after irreversible blockade of a proportion of the active receptors with phenoxybenzamine, were similar to those in the aortic strips. Furthermore, the relative intrinsic efficacies of partial agonists were practically equal in the two tissues, suggesting that these drugs act on the same alpha-adrenoceptors. Since the alpha2-agonists clonidine and tizanidine had low affinity in the rabbit dilators, the alpha-adrenoceptors in this tissue appear to be of alpha1-type. These results were further supported by the fact that the pA2-value of prazosin, an alpha1-antagonist, was approximately 2 log units higher than that of yohimbine, an alpha2-antagonist.However, pA2-values of four quinazolines (prazosin, bunazosin, SM911 and SM2470) and two yohimbine alkaloids (yohimbine and corynanthine) were significantly lower in the rabbit dilator muscle than in rabbit aortic strips. Two imidazoline antagonists (phentolamine and tolazoline) and a phenethanolamine (labetalol) acted on the alpha1-adrenoceptors in the two tissues nonselectively. These results suggest that alpha1-adrenoceptors in the rabbit dilator muscle and aortic strips may not be identical and that both selective and nonselective antagonists which act on these receptor sites exist. The pA2-values of prazosin, SM911 and SM2470 against noradrenaline in guinea pig iris dilator smooth muscle were also significantly lower than those in the rabbit aortic strips, but practically equal to those in the rabbit iris dilators, suggesting that the alpha1-adrenoceptors in the dilators are the same in these two species.