Tetsuhiro Hisayama

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.

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).

Effects of cyclic AMP and protein kinase on calcium uptake in a microsomal fraction from guinea pig taenia caecum

A microsomal fraction was isolated from guinea pig taenia caecum by differential centrifugation. Activities of ouabain-sensitive (Na+, K+)-ATPase, 5-nucleotidase and NADPH-cytochrome c reductase were enriched in the microsomal fraction. On the other hand, less cytochrome c oxidase and monoamine oxidase were contained in this fraction. These results suggest that the microsomal fraction used in this study was derived from both sarcolemma and sarcoplasmic reticulum. Ca2+ uptake by this fraction was strictly dependent on the presence of ATP and was facilitated by oxalate. An ATP-regenerating system was required for the determination of Ca2+ uptake, when a lower concentration of ATP (e.g. 0.25 mM) was used. Phosphorylation of the microsomal fraction was doubled when these membranes were incubated in the presence of cyclic AMP plus cyclic AMP-dependent protein kinase (protein kinase). When the microsomal fraction was pretreated with cyclic AMP plus protein kinase, Ca2+ uptake was stimulated. The increases in microsomal phosphorylation and Ca2+ uptake were significantly correlated (P less than 0.01). This stimulation of Ca2+ uptake by microsomal phosphorylation was observed only in the presence of protein kinase, oxalate, and low Ca2+ and Mg2+ concentrations. The results suggest that stimulation of Ca2+ uptake may be the mechanism by which cyclic AMP is involved in beta-adrenergic relaxation of smooth muscle.

Epithelium selectively controls hypersensitization of the response of smooth muscle to leukotriene D4 by endogenous prostanoid(s) in guinea-pig trachea

SummaryThe effect of epithelium removal on the sensitivity of smooth muscle to carbachol and leukotriene D4 (LTD4) was investigated in guinea-pig isolated tracheal preparations untreated and treated with a cyclooxygenase inhibitor, flurbiprofen (1 μol/1). The pD2 value of carbachol was not changed by epithelium removal or by flurbiprofen-treatment, alone or in combination. On the other hand, the pD2 of LTD4 was higher in the tracheal strips without epithelium than in the intact preparations. In preparations devoid of epithelium, the pD2 value of LTD4 was decreased by treatment with flurbiprofen, suggesting that the sensitivity of the smooth muscle (namely, epithelium-free preparations) to LTD4 is enhanced by intramurally produced excitatory prostaglandin(s) [PG(s)]. However, in intact preparations with epithelium, no such sensitized response to LTD4 was observed. After flurbiprofen-treatment, in the continued presence of prostaglandin F2α (PGF2α, 200 nmol/l), the sensitivity to LTD4 was partially recovered in the epithelium-free preparations, but not in the intact ones. These results suggest that epithelium diminishes the sensitizing effect of intramural excitatory PG(s) on responsiveness of tracheal smooth muscle to LTD4, possible via a non-prostanoid substance(s).

Pharmacological evidence for the possible coexistence of multiple receptor sites for mammalian tachykinins in rabbit iris sphincter smooth muscle.

SummaryContractile responses to neurokinin α and neurokinin β were characterized and compared with those to substance P (a SP-P agonist) and eledoisin (a SP-E agonist) in isolated rabbit iris sphincter. Neurokinin a and neurokinin β as well as substance P and eledoisin produced atropine- and tetrodotoxin-resistant contractions of the iris sphincter in nanomolar concentrations, and the rank order of sensitivity was eledoisin > substance P = neurokinin α = neurokinin β. After prolonged cold-storage of the preparations, responses to capsaicin, a releaser of tachykinins from sensory nerve endings, were nearly absent, but responses of considerable magnitude to carbachol and the tachykinins persisted. On wash-out of the tachykinins, responses faded at characteristic rates (neurokinin a > eledoisin > neurokinin β ≫ substance P). From the Schild analyses, [abetd-Arg1, abetd-Pro2, abetd-Trp7,9, Leu11]-substance P, a potent substance P antagonist, competitively antagonized the response to substance P, had no significant effect on the response to neurokinin β, and antagonized the response to neurokinin α and eledoisin in a more complex manner. Taken together, these results suggest that there coexist multiple receptor sites for mammalian tachykinins in rabbit iris sphincter smooth muscle.

Potentiating effect of 3-isobutyryl-2-isopropylpyrazolo-[1,5a]-pyridine (KC-404), a new cerebral vasodilator with anti-platelet activity, on prostacyclin-induced increase of cyclic AMP content in platelets of rat

1. The effect of KC-404, a new antiplatelet and cerebrovasodilating drug, on prostacyclin (PGI2)-induced accumulation of cyclic AMP was investigated using washed platelets of rat. 2. PGI2 enhanced the cyclic AMP content in a concentration-dependent manner. 3. KC-404 at a relatively high concentrations (above 4.34 microM) significantly increased the maximum cyclic AMP accumulation induced by PGI2, without change in its sensitivity. 4. It is concluded that KC-404 potentiates a mechanism of action of PGI2, and a possible relevance of KC-404 for the treatment of cerebrovascular disorders with a tendency of accompanying thromboembolism is suggested.

Nitro compounds (isosorbide dinitrate, 5-isosorbide mononitrate and glyceryl trinitrate) on the femoral vein and femoral artery

In organ bath studies, the selectivity of isolated femoral vein and artery of rabbit to isosorbide dinitrate (ISDN), 5-isosorbide mononitrate (ISMN), major metabolite of ISDN, and glyceryl trinitrate (GTN) was compared. The femoral vein and artery contracted by norepinephrine were relaxed by all the nitro compounds dose-dependently. Potency order was GTN greater than ISDN greater than ISMN. The maximum inhibitory responses to the nitrocompounds and their pIC50 values (negative logarithms of doses to induce the 50% response) were greater in the femoral vein than in the femoral artery. For ISMN a 3 times greater sensitivity of femoral vein than of femoral artery was found.