Tadashi Kishimoto

A quantitative relationship between cellular Na accumulation and relaxation produced by ouabain in the depolarized smooth muscle of guinea-pig taenia coli

Summary1.An investigation was made of the effect of oubain on the membrane potential, tension development,45Ca uptake and intracellular Na and K contents of smooth muscle of the guinea-pig taenia coli depolarized by high-K solution. The results were compared with the effects of 2,4-dinitrophenol (DNP) and verapamil.2.All drugs produced concentration-dependent relaxations of high-K induced contractures without affecting the depolarization of the membrane. After removal of Na from the medium, the contracture was inhibited by DNP or verapamil but not by ouabain. The inhibitory effects of ouabain and verapamil were antagonized by raising the concentration of external Ca ([Ca]0), but the relaxation produced by DNP was independent of [Ca]0.3.Estimates of cellular Na were made by bathing the tissues in cold Li-solution. After an initial rapid loss of Na due to an exchange of extracellular Na with Li, the tissue Na reached a steady state. The residual after 1 h was regarded as cellular Na.4.During relaxation induced by ouabain, the depolarized muscle gained cellular Na. The increase in cellular Na was dependent upon the concentration of ouabain. A smaller increase was noted with DNP, while verapamil had no effect on cellular Na.5.The relaxation produced by ouabain was related to the intracellular Na content of the tissues. Also, the magnitude of the contraction produced by adding 1 mM Ca to Ca-free K-depolarized tissues was inversely correlated with the logarithm of the intracellular Na content, and an increase in the Na from 7.3–26.6 mmole/kg wet wt. halved the size of the Ca contracture.6.45Ca uptake, which was measured by a modified “La-method”, was increased in high-K solution. This increase was inhibited by pretreatments with ouabain, DNP and verapamil.7.It is suggested that the action of ouabain is closely related to an accumulation of cellular Na, which may cause the relaxation mainly by inhibiting Ca influx in response to depolarization with high-K. The data also suggest that the mechanism of relaxation induced by DNP or verapamil is different from that induced by ouabain.

Effects of the Na ionophore monensin on the contractile response and the movements of monovalent cations in the vascular smooth muscle of rabbit aorta

SummaryThe effects of monensin, a Na ionophore, on the muscle contraction and the movements of monovalent cations were investigated in rabbit aorta. Experiments were conducted in the presence of phentolamine (10−6 M) to avoid the vasoactive effect of monensin due to the release of endogenous catecholamine. Both monensin (2×10−5 M) and ouabain (2×10−5 M), added separately, produced a small and slowly developing contraction, whereas simultaneous application of these agents produced more rapid and greater contraction. Verapamil (10−6 M) decreased the contraction by 75%. Ouabain gradually increased cellular Na content. Monensin augmented the ouabain-induced Na increase. Further, the loss of cellular Na into Na deficient solution was enhanced by monensin. Low temperature (0.5°C) inhibited the monensin-induced increase in Na permeability. The relaxation of noradrenaline-contraction induced by a K-readmission was inhibited by ouabain but not by monensin. These results suggest that monensin increases Na movement down its electrochemical gradient, augments cellular Na accumulation when the Na pump is inhibited by ouabain, and induces muscle contraction, and that the contraction induced by monensin and ouabain is mainly due to an increased Ca influx through voltage sensitive Ca channels.

Inhibition of calcium channels by harmaline and other harmala alkaloids in vascular and intestinal smooth muscles

1 Effects of harmaline and other harmala alkaloids on the contractions induced in the vascular smooth muscle of rabbit aorta and intestinal smooth muscle of taenia isolated from guinea‐pig caecum were examined. 2 In rabbit isolated aorta, harmaline inhibited the sustained contraction induced by 65.4 mM K+ with an IC50 (concentration needed for 50% inhibition) of 4.6 × 10−5 M. This inhibitory effect on high K+‐induced contraction was antagonized by raising the concentration of external Ca2+ but not by Bay K 8644, a Ca2+ channel facilitator. Harmaline also inhibited the sustained contraction induced by noradrenaline (10−6 M) with an IC50 of 7.6 × 10−5 M. The inhibitory effects on noradrenaline‐induced contractions were not antagonized by raising the external Ca2+ concentrations or by Bay K 8644. 3 In guinea‐pig taenia, harmaline inhibited the 45.4 mM K+‐induced contraction with an IC50 of 6.8 × 10−5 M and the carbachol (10−6 M)‐induced contraction with an IC50 of 7.0 × 10−5 M. The inhibitory effects on both high K+‐ and carbachol‐induced contractions were antagonized by raising the external Ca2+ concentrations but not by Bay K 8644. 4 Harmaline, at the concentrations needed to inhibit the muscle contraction, inhibited the increase in 45Ca2+ uptake induced by high K+, noradrenaline and carbachol in aorta and taenia. 5 Harmaline did not change the cellular Na+ and ATP contents in resting and high K+ stimulated taenia. 6 Other harmala alkaloids also inhibited the contractions in these smooth muscles. The order of the inhibitory potency was 6‐methoxyharman = harmine > harmaline = 2‐methylharmine = harmane > 6‐methoxyharmalan > harmalol = harmol for the contractions induced by high K+ in aorta and taenia and by carbachol in taenia, and 2‐methylharmine > 6‐methoxyharman > 6‐methoxyharmalan = harmol = harmalol = harmane > harmine > harmaline for the contraction induced by noradrenaline in aorta. 7 These results suggest that harmaline inhibits the contractile response of rabbit aorta and guinea‐pig taenia by inhibiting different types of Ca2+ channel. The structure‐activity relationship indicates that the potency and selectivity of the inhibitory effects on these channels are varied by modification of the structure of this alkaloid.

The inhibitory effect of monensin on high K-induced contraction in guinea-pig taenia coli

This study deals with the effects of monensin, a Na ionophore, on contraction, cellular Na content, oxygen consumption and tissue ATP content of smooth muscle of the guinea-pig taenia coli depolarized by high K (62.7 mM) solution. The application of monensin (10(-8) -10(-6) M) had little effect on the phasic component of the K contraction while it decreased the tonic component. The inhibitory effect of monensin, unlike that of verapamil, was not antagonized by raising the concentration of external Ca. Under hypoxic conditions, the inhibitory action of monensin (10(-7) M) was greatly reduced. The removal of Na (choline or sucrose substitution) from the high K solution did not attenuate the inhibitory effect of hypoxia or monensin. Monensin (10(-7) M) produced only a small increase in cellular Na in the depolarized muscle. The K-induced increase in the rate of oxygen consumption was suppressed by monensin (10(-6) M). In high K solution, but not in normal solution, monensin (10(-6) M) decreased the tissue ATP content. These results suggest that the relaxing action of monensin is mainly due to the inhibition of aerobic energy metabolism of the smooth muscle of guinea-pig taenia coli.

Effect of TEI-9874, an inhibitor of immunoglobulin E production, on allergen-induced asthmatic model in rats.

As TEI-9874, 2-(4-(6-cyclohexyloxy-2-naphtyloxy)phenylacetamide)ben zoic acid reduces allergen-specific immunoglobulin E (IgE) production by human peripheral blood mononuclear cells in vitro, we evaluated its potency on an allergen-induced asthmatic model in Brown-Norway rats. Inhaled ovalbumin induced the immediate-phase asthmatic response, the late-phase asthmatic response, the infiltration of leukocytes into bronchoalveolar lavage fluid, and an increase of serum anti-ovalbumin IgE. These parameters were suppressed by the treatment with TEI-9874 (3, 10, and 30 mg/kg p.o.). The ovalbumin-induced airway hyperresponsiveness was prevented by TEI-9874 (30 mg/kg p.o.). Furthermore, the suppression of the immediate-phase asthmatic response and the late-phase asthmatic response by TEI-9874 was almost completely extinguished by the exogenous administration of rat anti-ovalbumin antiserum. These results indicate that the efficacy of TEI-9874 on the asthmatic response is mainly mediated by the suppression of allergen-specific IgE production and TEI-9874 appears to be a good candidate as therapy for IgE-mediated allergic asthma.

Differences in Responses to TC-81 Among Various Arteries in Dogs

Summary: The vasodilation induced by TC-81 was investigated in vitro and in vivo. In helical strips of dog arteries precontracted with 65.4 mM KCl, TC-81, nicardipine, nifedipine, diltiazem, and papaverine produced concentration-dependent relaxation. The potencies of TC-81 were in the following order: basilar > coronary > femoral > renal > mesenteric arteries. The other drugs also showed a similar property of greater response in basilar and coronary arteries than in renal and mesenteric arteries. However, the difference in reactivity to TC-81 was greater. On the other hand, TC-81, nicardipine, and papaverine dose-dependently increased the blood flow in vertebral, coronary, and femoral arteries in anesthetized dogs. However, the blood flow in renal and mesenteric arteries was not changed or decreased. The decreases in vessel resistance induced by TC-81 were in the following order: vertebral > coronary > femoral > renal = mesenteric arteries, agreeing with the experimental results in vitro. These results suggest that Ca2+ antagonists have the pharmacological property of vasodilating cerebral and heart blood vessels more selectively than other arteries, and this property of TC-81 might be an advantage compared with other drugs.

Action of the Na+ ionophore monensin on vascular smooth muscle of guinea-pig aorta

The effects of the Na+ ionophore monensin on contractile responses were investigated in guinea-pig aorta in normal and high K+ solutions. In normal K+ (5.4 mM) solution, monensin (2 X 10(-5) M) produced a rapid increase in tension followed by slow relaxation. This contraction was markedly inhibited by phentolamine (10(-5) M) or prazosin (10(-6) M) and was accompanied by an increase in tritium efflux from tissue preloaded with [3H]norepinephrine. In the presence of phentolamine, monensin (1-2 X 10(-5) M) or ouabain (1-2 X 10(-5) M) caused only a small and slowly developing contraction. Simultaneous application of these agents caused a more rapid and greater contraction. Either monensin or ouabain gradually increased cellular Na+ and decreased cellular K+ content. When monensin was applied simultaneously with ouabain, there was a rapid increase in cellular Na+ and loss of cellular K+. In high K+ (65.4 mM) solution, monensin (10(-6) M) slightly reduced the increased tension level but when external glucose was omitted monensin markedly inhibited the contraction. A significant decrease in tissue ATP content was observed only when monensin was applied in glucose-free solution. Similarly, hypoxia (N2 bubbling) markedly inhibited the high K+ contraction and decreased the tissue ATP content only in the absence of glucose. These results suggest that monensin produces a neurogenic contraction due to the release of endogenous catecholamines and also produces a myogenic contraction by a decrease in transmembrane Na+ and K+ gradients when the Na+-K+ pump is inhibited by ouabain, and that monensin inhibits aerobic energy metabolism of vascular smooth muscle.

The inhibitory effect of lithium on high-K induced contraction in guinea-pig taenia coli

Summary1.This study deals with the effects of lithium (Li) on the membrane potential, tension development, intracellular Na, K and Li contents and 45Ca uptake of smooth muscle of the guinea-pig taenia coli depolarized by high-K (62.7 mM) solution.2.Replacement of Na with Li in high-K solution resulted in a concentration-dependent inhibition of muscle contraction without affecting membrane depolarization. The effect of Li-substitution was also dependent on the duration of exposure to Li and was antagonized by raising the concentration of external Ca ([Ca]0). Replacement of Na with tris (hydroxymethyl) aminomethane (Tris), sucrose or choline did not show such an inhibitory effect.3.To estimate the intracellular Na content by removing extracellular bound Na, muscle strips were washed with cold Li solution and the amount of Na remaining in the tissue was measured. During washout with the cold Li-solution, a part of the tissue Na was rapidly lost followed by a rapid Li uptake while the remaining Na was lost very slowly as a single exponential process. A similar change in tissue Li level was observed in Li-loaded tissue during washout with cold Na-solution. The residual Na and Li after a 1 h washout with the cold solutions were regarded as intracellular Na and Li, respectively.4.Replacement of a part of the extracellular Na in high-K solution with either Li or sucrose led to a slight decrease in intracellular Na. In the case of Li substitution, Li gradually accumulated in the muscle cells. The increase in intracellular Li was dependent on the concentration of external Li.5.The relaxation produced by Li was correlated with the intracellular Li content of the tissues. Both the Li-induced relaxation and the increased intracellular Li were fully reversible with similar time courses following removal of extracellular Li.6.45Ca uptake of the muscle measured by a modified “La-method” increased in high-K solution. This increase was inhibited by a pretreatment with Li.7.It is suggested that the action of Li is closely related to an accumulation of intracellular Li, which may produce muscle relaxation mainly by inhibiting the depolarization-induced Ca influx.

Relationship between tissue content of TC-81 and relaxation of rat aorta

This study dealt with the relationship between the relaxant action of TC-81, a new Ca2+ antagonist, and its distribution in rat aorta depolarized by high K+ (65.4 mM). The inhibitory effect by TC-81 on K(+)-induced contraction and 45Ca2+ uptake was strongly time-dependent. TC-81 and nicardipine, each 10(-9) M, produced gradual relaxation of high-K(+)-induced contraction. The tissue contents of TC-81 and nicardipine increased with time courses that reflected decreasing tension. Both the maximum tissue content of TC-81 and the maximum relaxation were significantly greater than those for nicardipine. Also, the relaxations produced by TC-81 and nicardipine were correlated with the logarithm of the content of each drug in muscle tissues. However, the dissociation of TC-81 from the tissues was slower than that of nicardipine. The data suggest that the action of TC-81 is closely related to a gradual distribution of drug into muscle tissue, resulting in a slow onset of action. Also, the saturation time of TC-81 was longer than that of nicardipine, thereby accounting for the greater relaxation with TC-81.