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Exposure to chronic hypoxic conditions causes various gastric diseases, including gastric ulcers. It has been suggested that gastric smooth muscle contraction is associated with aerobic metabolism. However, there are no reports on the association between gastric smooth muscle contraction and aerobic metabolism, and we have investigated this association in the present study. High K+- and carbachol (CCh)-induced muscle contractions involved increasing O2 consumption. Aeration with N2 (hypoxia) and NaCN significantly decreased high K+- and CCh-induced muscle contraction and O2 consumption. In addition, hypoxia and NaCN significantly decreased creatine phosphate (PCr) contents in the presence of high K+. Moreover, decrease in CCh-induced contraction and O2 consumption was greater than that of high K+. Our results suggest that hypoxia and NaCN inhibit high K+- and CCh-induced contractions in gastric fundus smooth muscles by decreasing O2 consumption and intracellular PCr content. However, the inhibition of CCh-induced muscle contraction was greater than that of high K+-induced muscle contraction.

The mechanism of imidazole-induced contraction on the bovine tracheal smooth muscle was investigated. Imidazole induced muscle contraction in a concentration-dependent manner on bovine, porcine and guinea-pig tracheas, but not in rat or mouse. In bovine tracheas, imidazole was cumulatively applied and induced muscle tension and increasesd intracellular Ca2+ level in a concentration -dependent manner. Imidazole, even at 300 µM, the concentration at which maximum contractile response occurs, did not significantly increase in cAMP content relative to control. Atropine inhibited imidazole-induced contraction at a concentration- dependent manner and pretreatment of hemicholinium-3 almost abolished imidazole-induced contraction. Conversely, pretreatment of tripelennamine, indomethacin or tetrodotoxin did not affect imidazole-induced contraction. Acetylcholine or eserine induced contraction in bovine, porcine, guinea pig, rat and mice trachea in a concentration-dependent manner. However, there was little difference in the rank order of maximum contraction of these agents. Imidazole-induced contraction was greater in bovine trachea compared to the other species tested. Further, cAMP did not appear to play a role in imidazole-induced contraction, suggesting other mechanisms, such as the release of endogenous acetylcholine.

Chlorogenic acid (CGA) is a polyphenol found in coffee and medicinal herbs such as Lonicera japonica. In this study, the effect of CGA-induced relaxation on carbachol (CCh)-induced contraction of mouse urinary bladder was investigated. CGA (30–300 μg/ml) inhibited CCh- or U46619-induced contraction in a concentration-dependent manner. SQ22536 (adenylyl cyclase inhibitor) recovered CGA-induced relaxation of CCh-induced contraction; however, ODQ (guanylyl cyclase inhibitor) did not have the same effect. In addition, 3-isobutyl-1-methylxanthine (IBMX) enhanced CGA-induced relaxation; however, forskolin or sodium nitroprusside did not have the same effect. Moreover, Ro 20–1724, a selective phosphodiesterase (PDE) 4 inhibitor, enhanced CGA-induced relaxation, but vardenafil, a selective PDE5 inhibitor, did not have the same effect. In the presence of CCh, CGA increased cyclic adenosine monophosphate (cAMP) level, whereas SQ22536 inhibited the increase of cAMP levels. Moreover, higher cAMP levels were obtained with CGA plus IBMX treatment than the total cAMP levels obtained with separate CGA and IBMX treatments. In conclusion, these results suggest that CGA inhibited CCh-induced contraction of mouse urinary bladder by partly increasing cAMP levels via adenylyl cyclase activation.

The present study was designed to clarify phosphodiesterase 9 (PDE9) expression in bovine tracheal smooth muscle tissue, and to elucidate that PDE9 may contribute to the regulation of airway relaxation. PDE9 mRNA expression was detected in bovine tracheal smooth muscle. Sodium nitroprusside (an NO donor) and BAY 73-6691 (a selective PDE9 inhibitor) reduced high K+- and carbachol-induced contraction. BAY 73-6691 relaxed tracheal tissue on the same level with vardenafil (a selective PDE5 inhibitor). These results support our hypothesis that PDE9 plays functional role in the tracheal smooth muscle relaxation. PDE9 inhibitors are expected to be a novel target of the add-on treatment of airway hyperresponsiveness.

Recent studies have shown that phloridzin, an inhibitor of sodium-glucose cotransporter (SGLT), strongly decreases high K+-induced contraction in phasic muscle, such as tenia coli, but slightly affects tonic muscle, such as trachea . In this study, we examined the inhibitory mechanism of phloridzin on high K+-induced muscle contraction in rat ileum, a phasic muscle. Phloridzin inhibited the high K+-induced contraction in the ileum and the aorta, and the relaxing effect of phloridzin at 1 mM in the ileum was approximately five-fold more potent than that in the aorta. The expression of SGLT1 mRNA in the ileum was higher than that of the aorta. Phloridzin significantly inhibited NADH/NAD ratio and phosphocreatine (PCr) content in the ileum; however, application of pyruvate recovered the inhibition of contraction and PCr content, but had no effect on ratio of NADH/NAD. High K+ increased 2-(N (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) uptake in ileal smooth muscle cells, and phloridzin inhibited the increase in a concentration-dependent manner. These results suggest that phloridzin inhibits high K+-induced contraction because of the inhibition of energy metabolism via the inhibition of SGLT1.

Eyes are supplied O2 through the cornea and vessels of the retina and iris, which are tissues characterized by aerobic metabolism. Meanwhile, there are no reports on the association between iris sphincter contraction and aerobic metabolism. In this paper, we studied the aforementioned association. Eyes from adult pigs of either sex were obtained from a local abattoir. A muscle strip was connected to a transducer to isometrically record the tension. O2 consumption was measured using a Clark-type polarograph connected to a biological oxygen monitor. Creatine phosphate (PCr) and adenosine triphosphate (ATP) contents were measured in the muscle strips by high-performance liquid chromatography (HPLC). Iris sphincter muscles were measured in resting, contractile or hypoxic phases. Contraction was induced by hyperosmotic 65 mM KCl (H-65K+) or carbachol (CCh), and hypoxia was induced by aeration with N2 instead of O2 or by addition of sodium cyanide (NaCN). H-65K+- and CCh-induced muscle contraction, involved increasing O2 consumption. Hypoxia and NaCN significantly decreased H-65K+- and CCh-induced muscle contraction and/or O2 consumption and PCr contents. Our results suggest that the contractile behavior in porcine iris sphincter highly depends on mitogen oxidative metabolism.

The effects of various selective phosphodiesterase (PDE) inhibitors on carbachol (CCh)-induced contraction in the bovine abomasum were investigated. Various selective PDE inhibitors, vinpocetine (type 1), erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, type 2), milrinone (type 3), Ro20-1724 (type 4), vardenafil (type 5), BRL-50481 (type 7) and BAY73-6691 (type 9), inhibited CCh-induced contractions in a concentration-dependent manner. Among the PDE inhibitors, Ro20-1724 and vardenafil induced more relaxation than the other inhibitors based on the data for the IC50 or maximum relaxation. In smooth muscle of the bovine abomasum, we showed the expression of PDE4B, 4C, 4D and 5 by RT-PCR analysis. In the presence of CCh, Ro20-1724 increased the cAMP content, but not the cGMP content. By contrast, vardenafil increased the cGMP content, but not the cAMP content. These results suggest that Ro20-1724-induced relaxation was correlated with cAMP and that vardenafil-induced relaxation was correlated with cGMP in the bovine abomasum. In conclusion, PDE4 and PDE5 are the enzymes involved in regulation of the relaxation associated with cAMP and cGMP, respectively, in the bovine abomasum.

The effects of papaverine on carbachol (CCh) -and high K+- induced contraction in the bovine abomasum were investigated. Papaverine inhibited CCh (1 µM) -and KCl (65 mM) -induced contractions in a concentration-dependent manner. Forskolin or sodium nitroprusside inhibited CCh-induced contractions in a concentration-dependent manner in association with increases in the cAMP or cGMP contents, whereas papaverine increased cGMP contents only at 30 µM. Changes in the extracellular Ca2+ from 1.5 mM to 7.5 mM reduced verapamil-induced relaxation in high K+-depolarized muscles, but papaverine-induced relaxation did not change. Futhermore, papaverine (30 µM) and NaCN (300 µM) decreased the creatine phosphate contents. These results suggest that the relaxing effects of papaverine on the bovine abomasum are mainly due to the inhibition of aerobic energy metabolism.

This study examined the inhibitory effects of papaverine on twitches directly elicited by electrical stimulation of the mouse diaphragm. Papaverine (3–100 µM) inhibited twitches in a dose-dependent manner. Papaverine increased the cyclic adenosine monophosphate (cAMP) but not cyclic guanosine monophosphate (cGMP) content. IBMX, Db-cAMP and 8-br-cGMP did not affect twitches, whereas verapamil and NaCN inhibited twitches. Increases in extracellular Ca2+ removed the twitch inhibition caused by verapamil but not that caused by papaverine. Papaverine (30 and 100 µM) and NaCN (1 mM) decreased creatine phosphate and ATP contents. These results suggest that the relaxing effects of papaverine on mouse diaphragm are mainly due to inhibition of aerobic energy metabolism.

A high-K(+), Na(+)-deficient (isosmotic 154 mM K(+) and 0 mM Na(+); I-154 K(+)) solution induced contraction followed by gradual relaxation of the smooth muscles of the porcine trachea, while hyperosmotic addition of 65 mM KCl (H-65 K(+)) induced a large sustained contraction. The I-154 K(+) solution also induced a sustained increase in [Ca(2+)](i) level. Decreases in muscle tension and increases in cellular water content were both prevented by the addition of sucrose or NaCl in the porcine trachea. An additional application of phloridzin inhibited recoveries of I-154 K(+) solution by addition of NaCl in the porcine trachea. Addition of pyruvate or oxaloacetate recovered the I-154 K(+) solution-induced relaxation in the porcine trachea. Although application of I-154 K(+) solution did not affect PCr and ATP contents in the bovine trachea, the solution induced a gradual decrease of PCr content in the porcine trachea. Moreover, application of pyruvate or oxaloacetate recovered the I-154 K(+) solution-induced decreases of PCr content in the porcine trachea. Phloridzin inhibited H-65 K(+)-induced contraction in porcine, but not in bovine trachea. In conclusion, the I-154 K(+) solution-induced relaxation in the porcine trachea is probably due to swelling of cells and inhibition of glucose utilization. Moreover, the inhibition of glucose utilization in I-154 K(+) medium in porcine trachea is different from that of bovine trachea.

This study examined the mechanism of vasorelaxation induced by dimethyl sulfoxide (DMSO) in endothelium-intact and -denuded rat aorta. DMSO (0.1-3%) inhibited phenylephrine (PE, 1 μmol/l)-induced contraction in a dose-dependent manner. However, this relaxation was lower in the absence of the endothelium. Increase in DMSO-induced relaxation in the presence of the endothelium was attenuated by preincubation in L-NG-nitroarginine methyl ester (L-NAME, 100 μmol/l) and by the removal of the endothelium. In the aorta with endothelium, DMSO (3%) and CCh (3 μmol/l) increased cGMP contents, significantly and L-NAME (100 μmol/l) inhibited the DMSO-induced increases of cGMP. In fura 2-loaded endothelium-denuded aorta, cumulative application of DMSO (1-3%) inhibited PE-induced muscle tension; however, this application did not affect the [Ca2+]i level. In PE-precontracted endothelium-denuded aorta, relaxation responses to fasudil were significantly less in the presence of DMSO compared to the control. These results suggest that DMSO causes relaxation by increasing the cGMP content in correlation with the release of NO from endothelial cells and by decreasing the Ca2+ sensitivity of contractile elements partly via inhibiting Rho-kinase in rat aorta.

Effects of various selective phosphodiesterase (PDE) inhibitors on muscle contractility and cyclic nucleotide contents in guinea pig taenia coli were investigated. Forskolin and sodium nitroprusside inhibited carbachol (CCh)-induced contraction in a concentration-dependent manner. Various selective PDE inhibitors, vinpocetine (type 1), erythro -9-(2-hydroxy-3-nonyl)adenine (EHNA, type 2), milrinone (type 3), Ro20-1724(type 4) and zaprinast (type 5) inhibited CCh-induced contraction in a concentration-dependent manner, but the inhibition of milrinone was noticeably smaller than that of the other PDE inhibitors. The rank order of potency was zaprinast > vinpocetine > EHNA > Ro20-1724 > milrinone. In the presence of CCh (0.3 microM), vinpocetine and Ro20-1724 both increased cAMP content, but not cGMP. By contrast, EHNA and zaprinast both increased cGMP content, but not cAMP. Pretreatment with ODQ (30 microM), a soluble guanylyl cyclase inhibitor, decreased the inhibition of CCh-induced contraction by EHNA or zaprinast. Pretreatment with SQ22536 (100 microM), an adenylyl cyclase inhibitor, decreased the inhibition of CCh-induced contraction by vinpocetine or Ro20-1724. In conclusion, it was indicated that vinpocetine- or Ro20-1724-induced relaxation was correlated with cAMP but EHNA- or zaprinast- induced relaxation was correlated with cGMP.

The effects of the type V phosphodiesterase (PDE V) inhibitors, MBCQ, zaprinast and dipyridamole, on the relationship between relaxation and cyclic nucleotide content were investigated in rat ileal smooth muscle. Each of MBCQ (0.01-10 microM), zaprinast (0.1-100 microM) and dipyridamole (0.1-100 microM) inhibited carbachol (CCh; 10 microM)-induced contractions in a concentration-dependent manner. When compared with the concentrations of these agents producing 50% relaxation (IC50) of CCh-induced contraction, MBCQ was 14-20 fold more potent than the other agents. The inhibitory potency of these agents against high K+ (65 mM KCl)-induced contractions were similar to that for CCh. MBCQ (1, 10 microM) did not significantly increase the cGMP content above control levels in the presence of CCh (10 microM). Both Zaprinast (1-100 microM) and dipyridamole (1-100 microM) increased the cGMP content of smooth muscle preparations in a concentration-dependent manner. There was a positive correlation between the inhibition of the CCh-induced contraction and the increase in cGMP content elicited by zaprinast and dipyridamole (zaprinast; r=0.72, P0.05, dipyridamole; r=0.92, P0.05). However, MBCQ at a concentration which induced a medium-sized relaxation did not significantly increase the cGMP content. Neither MBCQ, zaprinast nor dipyridamole significantly increased the cAMP content of the preparations above control. In summary, it is suggested that the inhibition of CCh-induced contractions by zaprinast and dipyridamole involves increases in cGMP content via inhibition of PDE V. However the inhibition of CCh-induced contraction by MBCQ may not involve cyclic nucleotides in rat ileal smooth muscle.

In the present paper, we examined a mechanism of the papaverine induced relaxation in the smooth muscle of non-pregnant rat uterus. The hyperosmotic 65 mM KCl (H-65K+)-or oxytocin-induced contraction in the uterus was inhibited by an addition of papaverine in a concentration-dependent manner. Papaverine did not increase both cAMP and cGMP contents in the uterus in the presence of H-65K+ or oxytocin. In fura 2 loaded muscles, papaverine did not affect an increase of [Ca2+]i level by high K+ or oxytocin. In permeabilized muscles, papaverine had no effect on the Ca2+-induced contraction. H-65K+ and oxytocin increased the rate of oxygen consumption 1.8 and 1.5 times higher than that in the resting condition, respectively. The increase of oxygen consumption in the H-65K+ or oxytocin was significantly inhibited by papaverine (1-100 microM). These results suggested that papaverine inhibits smooth muscle contraction mainly by inhibition of mitochondrial respiration in rat uterus as well as guinea pig ileum, which shows a highly spontaneous activity and a highly metabolic dependency of a contraction.

Effects of phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), on muscle tension and cytosolic Ca2+ ([Ca2+]i) level was investigated in rat anococcygeus muscle in comparison with other smooth muscles. 1) DPB (10(-6) M) induced a large contraction and an elevation of [Ca2+]i level in rat aorta and small and rhythmic changes in tension and [Ca2+]i level in guinea pig ileum. However, DPB did not change either of the parameters in rat anococcygeus muscle. 2) DPB caused tension development without changing the [Ca2+]i level elevated by high K+, ionomycin or beta-escin in the anococcygeus muscle. 3) In the beta-escin permeabilized muscles of guinea pig ileum and urinary bladder, rabbit mesenteric artery and rat anococcygeus muscle, DPB enhanced the Ca(2+)-developed tension. Moreover, the enhancement was inhibited by H-7 (3 x 10(-5) M). 4) DPB did not cause muscle tension to develop in the muscle of rat aorta, guinea pig ileum and rat anococcygeus muscle, pretreated with phorbol 12-myristate 13-acetate for 24 hr. In conclusion, DPB showed different contractile effects on the aorta, ileum and anococcygeus muscle, respectively. The initiation of muscle tension by DPB probably requires [Ca2+]i and the DPB-induced enhancement may be due to a Ca2+ sensitization of contractile elements in the anococcygeus muscle. Therefore, the difference between the DPB-induced response of the anococcygeus muscle and those of the other muscles seems to be due to a different Ca2+ movement caused by DPB. Moreover, it is suggested that DPB develops muscle tension by increasing [Ca2+]i and enhances it through the mediation of protein kinase C in the anococcygeus muscle as well as the other smooth muscles.

The mechanism of the inhibitory effect of Li + on contraction was examined in guinea pig ileal longitudinal smooth muscle. Li + -substitution (68.4 mM) reversed contractions induced by high K + (45.4 mM), carbachol (1 μM) and histamine (1 μM) without changing the cytosolic Ca 2+ level. Li + also had no effect on the increase in 45 Ca 2+ uptake stimulated by high K + . High K + transiently increased myosin light chain (MLC) phosphorylation, reaching a peak at 6-9 sec. Li + -substitution inhibited the high K + -induced MLC phosphorylation. In permeabilized ileal strips, contraction induced by 1 μM Ca 2+ was inhibited by 10 mM Li + . The inhibitory effect was antagonized by increasing the concentration of Ca 2+ or calmodulin. In the permeabilized muscle in which MLC was previously thiophosphorylated with 1 mM ATPγS and 3 μM Ca 2+ , ATP induced contraction in Ca 2+ free buffer. Li + added during this contraction did not show an inhibitory effect. In contrast, when 30 mM Li + was added during the thiophosphorylation, the contraction induced by the subsequent addition of ATP was inhibited. Li + (30 mM) changed neither the rate of relaxation induced by removing external Ca 2+ in permeabilized muscle nor the rate of dephosphorylation of MLC induced by crude phosphatase extracted from the ileum. Li + (15 mM), on the other hand, inhibited the rate of phosphorylation of MLC caused by crude MLC kinase extracted from the ileum. Li + did not inhibit the calmodulin activity as measured with the (Ca 2+ +Mg 2+ )-ATPase activity of the erythrocyte membrane. These results suggest that the inhibitory effect of Li + on contractions is attributable to the inhibition of MLC kinase in guinea pig ileum

A contractile property of phenylephrine (PE), alpha 1 agonist, on rat anococcygeus muscle was compared with that on rat aorta by simultaneously measuring changes in intracellular Ca2+ ([Ca2+]i) level and muscle tension. (1) PE (0.1-30 microM) and high K+ induced a sustained increases in [Ca2+]i level and muscle tension of both the muscles. (2) An application of verapamil (10 microM) and EGTA (4 mM) decreased the PE- or high K(+)-increased tension and [Ca2+]i level in both the muscle, respectively. (3) A cumulative application of PE or high K+ to anococcygeus muscle and aorta exhibited a positive relationship between [Ca2+]i and developed tension. The developed tension by PE was greater than that by high K+ at the same level of [Ca2+]i only in the aorta. A difference of regression slopes in the relationship between [Ca2+]i level and muscle tension under PE- and high K(+)-treatments in aorta was significant, but that in anococcygeus muscle was not. (4) An application of PE to anococcygeus muscle in Ca2+ free medium elicited a small transient contractile tension and increase in [Ca2+]i level, but that to aorta showed a large and transient increase in both the parameters. (5) Phorbol ester, DPB (1 microM), did not affect muscle tension or [Ca2+]i level in anococcygeus muscle, but DPB induced greater increases in aorta. (6) An application of PE (10 microM) with GTP produced a left shift in the pCa-tension curve in the beta-escin-permeabilized fiber of the anococcygeus muscle. In summary, it is suggested that the sustained contraction induced by PE in anococcygeus muscle is involved with the increases in [Ca2+]i which is due to Ca2+ influx mediated by alpha 1 receptor, but scarcely to Ca2+ release from the intracellular storage, and that an increase in Ca2+ sensitivity to PE is found only in the permeabilized anococcygeus muscle. The Ca(2+)-independent contractile mechanism in PE response as seen in aorta is probably to be absent in anococcygeus muscle. Moreover, it seems that the effect of the drug acting protein kinase C on anococcygeus muscle is extremely lesser than that on aorta.

The effect of CV-3988, a platelet activating factor antagonist, in the treatment of endotoxic shock was evaluated from the changes in plasma 6-keto-PGF1 alpha and thromboxane B2 concentrations. The animals consisted of 10 beagle dogs anesthetized with pentobarbital sodium and divided into a treatment group (n = 5) and a control group (n = 5). Endotoxic shock was experimentally induced in both groups by intravenous administration of endotoxin (lipopolysaccharide, 3 mg/kg). The treatment group was intravenously given 10 mg/kg of CV-3988 for 10 min from immediately after endotoxin. Mean aortic pressure, cardiac output and urine volume were remarkably decreased after the administration of endotoxin to both groups. These parameters were higher after the administration of CV-3988, in the treatment group than in the control group. Furthermore, the increase in plasma 6-keto-PGF1 alpha and thromboxane B2 concentrations was significantly inhibited. These results suggest the effectiveness of CV-3988 in the treatment of endotoxic shock.

The therapeutic effect of ulinastatin (25,000 U/kg, i.v.) on endotoxin-induced shock was compared with that of methylprednisolone (30 mg/kg, i.v.) in 17 anesthetized dogs. Both of these drugs had almost the same tendency to improve the hemodynamics, arachidonate cascade metabolites and pulmonary surface activity. There was little difference between the effectiveness of ulinastatin and that of methylprednisolone. It was newly confirmed that the release of 6-keto-PGF1 alpha, thromboxane B2 and leukotriene B4, arachidonate cascade metabolites and chemical mediators associated with endotoxin-induced shock, were significantly (p < 0.01 and p < 0.05) decreased by ulinastatin in the same way as methylprednisolone. These results suggest that ulinastatin is as useful as methylprednisolone for the treatment of endotoxin-induced shock.

The effects of various selective phosphodiesterase (PDE) inhibitors on muscle contractility and cyclic nucleotide content in the porcine coronary artery were investigated. Various selective PDE inhibitors, vinpocetine (type 1), erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, type 2), milrinone (type 3), rolipram (type 4), Ro20-1724 (type 4), and zaprinast (type 5), inhibited U46619-induced contractions in a concentration-dependent manner. The rank order of potency for the porcine coronary artery was rolipram > Ro20-1724 >milrinone > vinpocetine > zaprinast > EHNA, which was different from that of both the porcine carotid artery and aorta. Rolipram inhibited the U46619-induced muscle tension with a decreased [Ca(2+)](i) level, but inhibited the high K(+)-induced contraction without a change in [Ca(2+)](i) level. Rolipram increased cAMP but not cGMP content. Iberiotoxin restored the inhibition of muscle tension and the [Ca(2+)](i) levels induced by rolipram. U46619 and caffeine induced a transient increase in the [Ca(2+)](i) levels in a Ca(2+)-free solution, but rolipram only inhibited the U46619-induced Ca(2+) transient. In conclusion, rolipram is the most potent inhibitor in the porcine coronary artery, but not in the carotid artery and aorta. Moreover it is suggested that the mechanism by which rolipram causes relaxation is due to a decrease in the [Ca(2+)](i) levels and of the Ca(2+) sensitivity of the contractile elements to cAMP.

The effects of various selective phosphodiesterase (PDE) inhibitors on muscle contractility and cyclic nucleotide contents in porcine iris sphincter were investigated. Forskolin and sodium nitroprusside inhibited carbachol (CCh)-induced contraction in a concentration-dependent manner. Various selective PDE inhibitors, vinpocetine (type 1), erythro -9-(2-hydroxy-3-nonyl)adenine (EHNA, type 2), milrinone (type 3), Ro20-1724 (type 4) and zaprinast (type 5), also inhibited CCh-induced contraction in a concentration-dependent manner. The rank order of potency of IC50 was zaprinast > Ro20-1724 > EHNA ≥ milrinone > vinpocetine. In the presence of CCh (0.3 μM), vinpocetine, milrinone and Ro20-1724 increased cAMP, but not cGMP, contents. In contrast, zaprinast and EHNA both increased cGMP, but not cAMP, contents. This indicates that vinpocetine-, milrinone- and Ro20-1724-induced relaxation is correlated with cAMP, while EHNA- and zaprinast- induced relaxation is correlated with cGMP in porcine iris sphincter.

Kamizaki, J., Shimizu, K., Nakajyo, S. and Urakawa, N. A change in inhibitory mechanism of Na+deficiency on high K+-induced contraction in rat uterus with the progress of pregnancy. Jpn. J. Smooth Muscle Res.In the present paper, effects of high K+/Na+deficient solution on the rat uterus with the progress of the pregnancy were examined on the mechanical response and wet weight. The progress of pregnancy was divided in four stages, early stage (Day 4 and 5 of pregnancy), middle stage (Day 8-10), late stage (Day 14 and 15) and end stage (Day 21). In the early stage muscle, an isosmotically substituted high K+/Na+ deficient (iso-154K+) solution induced a large contraction followed by a decrease in tension level, though a hyperosmotic KCl addition induced a large sustained contraction. Similar results were shown in muscles of the middle and late stages, However, a maximum tension level and a subsequent inhibition by the iso-154K+ solution in the end stage muscle were smaller than those of other stages. On the other hand, iso-154K+solution remarkably increased the relative cellular water content in the early or middle stage muscle, but moderately in the late or end stage one. In the muscle of all stages, a hyperosmotic addition of sucrose reversed the inhibition of muscle tension and the cell swelling by the iso-154K+ solution. Moreover, a substitution of more impermeable anion (C2H5COO-) for Cl-in the iso-154K+solution decreased the inhibition of the contraction in the early stage muscle, but increased it in the end stage one. Thesubstitution for Cl-with more permeable anion (NO3-or I-) produced a greater inhibition of contraction in all stage muscles. Moreover, a substitution of pyruvate or oxalacetate forglucose in iso-154K+solution did not affect the decreased tension in the early stage muscle, however, reversed the decreased tension in those after the middle stage. From these results, the decrease of the developed tension in the iso-154K+solution in the early stage muscle is probably due to cell swelling and those after the middle stages is probably caused by both the cell swelling and the inhibition of glucose utilization by Na+deficiency in the medium. The data suggest that the mechanism of the decrease in tension is probably transferred from cell swelling to inhibition of glucose utilization with the progress of pregnancy.

The mechanism by which ibudilast induces vasodilation was examined in isolated endothelium-denuded rat aorta. Ibudilast inhibited the contractions induced by phenylephrine (PE) and high K+ with decrease of [Ca2+]i level in a concentration-dependent manner, to the same degree. 3-Isobutyl-1-methylxanthine (IBMX) inhibited PE-induced contraction and [Ca2+]i level in a concentration-dependent manner, but it inhibited high K+-induced contraction without decrease of [Ca2+]i level. In comparison with IBMX, the increases of cAMP and cGMP contents in ibudilast were much smaller than that of muscle tension. Ibudilast did not inhibit 12-deoxyphorbol 13-isobutyrate (DPB)-induced contraction in the presence of verapamil. Treatment with 30 µM ibudilast inhibited the extracellularly added Ca2+-induced muscle tension and increases in [Ca2+]i level during high K+ depolarization. These results suggested that ibudilast inhibited PE- and high K+-induced muscle contractions mainly by the inhibition of [Ca2+]i level in endothelium-denuded rat aorta. Keywords:: ibudilast, smooth muscle, cAMP, cGMP, rat aorta

In this study, we examined the inhibitory mechanism of monensin on high K+-induced contraction in guinea-pig urinary bladder. The relaxant effect of monensin (0.001 – 10 µM) was more potent than those of NaCN (100 µM – 1 mM) and forskolin (3 – 10 µM). Monensin (0.1 µM), NaCN (300 µM), or forskolin (10 µM) inhibited high K+-induced contraction without decreasing [Ca2+]i level. Monensin and NaCN remarkably decreased creatine phosphate and ATP contents. Monensin and NaCN inhibited high K+-induced increases in flavoprotein fluorescence, which is involved in mitochondrial respiration. Forskolin increased cAMP content but monensin did not. Monensin increased Na+ content at 10 µM but not at 0.1 µM that induced maximum relaxation. In the α-toxin-permeabilized muscle, forskolin significantly inhibited the Ca2+-induced contraction, but monensin did not affect it. These results suggest that the relaxation mechanism of monensin in smooth muscle of urinary bladder may be an inhibition of oxidative metabolism. Keywords:: monensin, urinary bladder, phosphocreatine, Na+ content

In guinea pig urinary bladder, the hyperosmotic 65 mM KC1 (H-65K+)- or carbachol (CCh)-induced contraction was inhibited by an addition of papaverine in a concentration-dependent manner. The cAMP content of the muscle in the presence of H-65K+ or CCh was increased by papaverine only at the higher concentration of 100 μM, but cGMP content was not affected by papaverine. Forskolin, compared with papaverine, increased cAMP content in a concentration-dependent manner, and nitroprusside did not significantly increase cGMP content. In a fura 2 loaded muscle, papaverine did not affect an increase of [Ca2+]i level by high K+ or CCh. The increase of oxidized flavoprotein (FPox) fluorescence and muscle contraction in the presence of H-65K+ or CCh was decreased by papaverine (l-00 μM), and the increase of pyridine nucleotide (PNred) fluorescence was not affected by papaverine. In summary, it was concluded that papaverine induced relaxation by inhibiting mitochondrial respiration in guinea pig urinary bladder as well as ileum. Moreover, it is proposed that the mechanism of papaverine-induced relaxation in the smooth muscle, which shows predominantly a metabolic dependency on its contraction, is an inhibition of mitochondrial respiration.

The effect of TCV-309, a newly developed platelet activating factor (PAF) antagonist, on the wet/dry weight ratio of the lung (index of pulmonary edema) and the pulmonary surface activity (index of pulmonary compliance) was evaluated in comparison with that of CV-3988 (PAF-antagonist). Administration of TCV-309 (1 mg/kg) or CV-3988 (10 mg/kg) significantly reduced the wet/dry weight ratio which was increased by endotoxin administration (3 mg/kg). It also augmented the pulmonary surface activity. Administration of either TCV-309 or CV-3988 alleviated the histologic lesions caused by endotoxic shock. These results suggest that lung injury during endotoxic shock can be controlled by TCV-309 as by CV-3988.

The therapeutic effect of dibutyryl cyclic AMP (DBcAMP) in endotoxic shock was evaluated, using 11 dogs with experimentally-induced endotoxic shock (5 in DBcAMP group and 6 in control group) under general anesthesia. The DBcAMP group was treated by single intravenous injection of DBcAMP (10 mg/kg) at 15 min before inoculation with endotoxin (3 mg/kg). After the inoculation of endotoxin, this group was given drip infusion of DBcAMP at a rate of 0.1 mg/kg/min over 180 min. Hemodynamic parameters and chemical mediators were measured until 360 min after endotoxin inoculation. The cardiac output and urinary volume, which were decreased in the control group, were significantly inhibited to decrease in the DBcAMP group (p < 0.01). The increases in 6-keto-PGF1 alpha and thromboxane B2, chemical mediators released in endotoxic shock, were significantly inhibited (P < 0.05 and p < 0.01, respectively). These results suggested that DBcAMP is useful for the treatment of endotoxic shock.

The inhibitory effect of tiamulin, an antibiotic produced by Pleurotus mutilis, on contractile and electrical responses in isolated thoracic aorta and cardiac muscle of guinea-pigs was studied. In the thoracic aorta, tiamulin with an IC50 of 9·7 × 10−6 m inhibited sustained contractions induced by isosmotically added 60 Mm KCl. The inhibitory effect of tiamulin on a Ca2+-induced contraction in a depolarized muscle was competitively antagonized by raising external Ca2+ concentration. Bay K 8644 (10−7 m) antagonized tiamulin's inhibition of the Ca2+ -induced contraction. Tiamulin (2×10−5 m) decreased the elevated cytoplasmic Ca2+ level measured by the fura 2 AM method in the depolarized muscle. In high K+-isoprenaline-treated left atria, tiamulin (2 × 10−5–2 × 10−4 m) produced negative inotropic effects. On the other hand in the membrane action potential of papillary muscles, tiamulin (2 × 10−6–2 × 10−4 m) produced decreases in action potential and durations and 2 × 10−4 m tiamulin depressed the slow response action potential in depolarized muscles. Tiamulin produced prolongations of the PR interval in ECG, negative chrono- and inotropic effects, and an increase in perfusion flow in guinea-pig isolated and perfused hearts. These effects of tiamulin on the aorta or cardiac muscle were similar to those of verapamil and nifedipine. These results suggest that both the inhibitory action of tiamulin on the high K+-induced contraction in the aorta and the negative inotropic effect of tiamulin on the cardiac muscle are due to an inhibition of Ca2+ entry through the voltage-dependent Ca2+ channels of cells of both these muscles.

The mechanism of endothelium-dependent relaxation induced by pirarubicin, (2″R)-4′-O-tetrahydropyranyladriamycin, THP, or carbachol was investigated in the rat isolated aorta. The relaxant effect of THP (1·5 × 10−6-4·5 × 10−5 m) or carbachol (10−8-10−4 m) on the aorta with endothelium was decreased by lowering Ca2+ in the medium. The relaxation induced by THP was not inhibited by pretreatment with verapamil (10−6–10−5 m), and that induced by carbachol was only partially inhibited. However, on replacement of all but 20 Mm Na+ with either Li+ or choline, the THP- or carbachol-induced relaxation was inhibited. Furthermore, the relaxing effect of THP or carbachol was inhibited by pretreatment with amiloride (10−4-3 × 10−4 m), with ouabain (10−4-10−3 m), or with K+-depletion. These results suggest that the THP- or carbachol-induced relaxation depending on endothelium was affected by modifying the calcium ion concentration, and that a Na+–Ca2+ exchange process is involved.

The mechanism of relaxation produced by pirarubicin [(2″R)-4′-O-tetrahydropyranyladriamycin, THP] has been studied in rat isolated aorta. THP (1·5 × 10−6–4·5 × 10−5 m) markedly relaxed contractions induced by noradrenaline (10−7 m) in the aorta with endothelium, but not in that without endothelium. The relaxation induced by 1·5 × 10−5 m THP was inhibited by methylene blue (5 × 10−6 m), hydroquinone (10−4 m), phenidone (5 × 10−5 m), haemoglobin (10−6 m) and p-bromophenacyl bromide (5×10−5 m), but not by indomethacin (2·5 ×10−5 m). The relaxation induced by THP (1·5 × 10−7 − 4·5 × 10−5 m) was inhibited by NG-nitro-l-arginine (10−5 m), but enhanced by superoxide dismutase (10 units mL−1) or by L-arginine (10−2 m). However, the THP-induced relaxation was not inhibited by various receptor antagonists such as atropine (10−6 m), cimetidine (10−5 m), diphenhydramine (3 × 10−6 m) and [D-Pro4, D-Trp7,9,10]-substance P(4–11) (1·5 × 10−6 m). In fifteen anthracycline analogues, THP and 13-dihydropirarubicin (both with a tetrahydropyranyl group) produced endothelium-dependent relaxations. These results suggest that the THP-induced relaxation which is probably mediated by endothelium-derived relaxing factor (EDRF) was not produced by an activation of muscarine, histamine H1 or H2, or substance P receptor, and further that the tetrahydropyranyl group must play an important role in the THP-induced relaxation.

The effects of several organic Ca antagonists and putative calmodulin antagonists on the release of [ 3 H]noradrenaline from rabbit aorta were examined. A 65.4 mM K solution and electrical stimulation induced a Ca-dependent increase in the 3 H-efflux from the adventitial layer (containing nerve terminals) but not from the media-intimal layer. Verapamil, D600, N-(6-aminohexyl)-5-chloro-l-naphthalenesulphonamide (W-7) and N 2 -dansyl-L-arginine-4-t-butylpiperidine amide (TI 233), in the concentration of 10 −5 M, did not inhibit the increase in 3 H-efflux. A high concentration (2 × 10 −4 M) of verapamil induced a Ca-independent increase in the 3 H-efflux from the adventitia but not from the media-intimal layer. D600, diltiazem, W-7 and TI233, in the concentration of 2 × 10 −4 M, also increased the 3 H-efflux; 65.4 mM K, added in the presence of 2 × 10 −4 M verapamil or W-7, produced no further increase in 3 H-efflux. The 3 H-efflux evoked by electrical stimulation was only partially inhibited by 2 × 10 −4 M verapamil. It is suggested that the evoked release of noradrenaline in rabbit aorta is relatively insensitive to organic Ca antagonists and calmodulin antagonists, and that high concentrations of these inhibitors themselves release noradrenaline by a mechanism independent of external Ca.

The effect of caffeine on contractile activity and Ca movement was investigated in guinuEa pig taenia coll. Caffeine at a concentration of 7 mM produced a contraction for approx. 7 min and increased 45Ca influx and efflux without a change in Ca content in the tissue. The effect of caffeine on Ca movement was the same as that of carbachol in the early phase of the contractile response. The caffeine-contraction was not potentiated by an addition of Ca up to four times that of normal concentration, however, the carbachol-contraction was potentiated. When the muscle was soaked in a Ca-free solution, it showed “Ca-free contraction” and a loss of the tissue Ca content. When caffeine was added immediately after the “Ca-free contraction” was relaxed, caffeine produced a transient contraction and increased the loss of the tissue Ca content. Under identical circumstances, carbachol did not induce a contraction or affect the Ca content of the tissue. In conclusion, caffeine was demonstrated to increase Ca exchangeability in smooth muscle cell as did carbachol, however, the results suggested the possibility that caffeine releases a bound Ca in the cell for contraction whereas carbachol induces a contraction by an increase in a permeability of the cell membrane to Ca ions.

1. The effects of procaine on muscle tension and 45Ca2+ movements were investigated in vascular smooth muscle of the rabbit aorta and intestinal smooth muscle of the taenia isolated from guinea-pig caecum. 2. Procaine (10 mM) induced a contraction in the taenia but had little effect on the resting tension in the aorta. 3. Procaine, 0.5-10 mM, relaxed the sustained contractions induced by 65.4 mM KCl and 10(-6) M noradrenaline in the aorta, and by 45.4 mM KCl, 10(-6) M carbachol and 10(-6) M histamine in the taenia. The inhibitory effect of procaine on the high K+-induced contractions was antagonized by external Ca2+ but not by the Ca2+ channel activators, Bay K 8644 and CGP 28,392. 4. 45Ca2+ uptake was increased by high K+ or noradrenaline in the aorta and by high K+ or carbachol in the taenia. The increments were inhibited by procaine at the concentrations needed to inhibit the muscle contractions. 5. In a Ca2+-free solution, noradrenaline and caffeine induced a transient contraction in the aorta, whereas a second application of each stimulant was almost ineffective. Addition of 1-10 mM procaine shortly before the first application of the stimulant inhibited the contraction. After washing the muscle with a Ca2+-free solution without procaine, the second application of the stimulant induced a greater contraction than that in control muscle without procaine pretreatment. 6. Noradrenaline and caffeine released 45Ca2+ from a cellular site in the aorta. Procaine inhibited the effects of these stimulants. 7. It was concluded that procaine may inhibit both the opening of Ca2+ channels and the release of Ca2 + from cellular stores and the former but not the latter effect may be attributable to a local anaesthetic action.

Ueda, F., Kishimoto, T., Karaki, H. and Urakawa, N. High K-induced Contraction in Rabbit and Monkey Tracheal Smooth Muscle. Jap.J. Smooth Muscle Res., 1983, 19 (6), 541-549. - High K-induced contractions in rabbit and monkey tracheal smooth muscle were characterized. A substituted 60mM K, 94.1mM Na solution produced a sustained contraction in both preparations. The 60 mM K-induced contraction in rabbit trachea was inhibited by Ca removal, verapamil, glucose-removal and hypoxia but not by sodium nitroprusside. The 60mM K-induced contraction in monkey trachea was inhibited by Ca removal, verapamil, sodium nitroprusside and hypoxia but not by glucose-removal. In rabbit trachea a substituted 154.1mMK, Na deficient solution induced a rapid rise in tension followed by a gradual decrease. Wet weight of rabbit trachea increased in the 154.1mM K solution. Both the decrease in the developed tension and the increase in the wet weight were prevented by the hyperosmotic addition of sucrose (50-100mM). The decrease in the developed tenison was also partially antagonized by the addition of pyruvate or oxalacetate. In monkey trachea, the sustained contraction induced by the 154.1mM K solution showed very slow decrease following the increase in the wet weight of the tissue. The decrease in muscle tension was prevented by the addition of hyperosmotic sucrose. These results suggest that, in rabbit tracheal smooth muscle, the decrease in the developed tension in isosmotically substituted high K, N a deficient solution may be attributable mainly to the swelling of muscle cells and partly to the inhibition of glucose utilization resulting from Na deficiency. In contrast, the contraction in monkey trachea seems to be inhibited, although slightly, by the swelling of the cells.

Nitroglycerine induced biphasic relaxation in the rat aorta, previously contracted by noradrenaline; a rapid decrease in tension was followed by a gradual increase reaching a steady level below the control contractile tension. No initial transient relaxation was induced by nitroglycerine in high K-stimulated muscle. The initial transient relaxation, but not the sustained relaxation, was dependent on the concentration of external K; maximum relaxation was observed in the presence of 2.7 mM K solution and only a slight relaxation was observed in 0 mM or 10.8 mM K solution. The initial transient relaxation was also inhibited by ouabain or low Na solution. On an appropriate increase in the concentration of external K, noradrenaline-induced contraction was transiently relaxed. Previous application of nitroglycerine potentiated this K-induced relaxation. Pretreatment of the muscle with methylene blue, an inhibitor of guanylate cyclase, inhibited both the initial transient and the sustained relaxations induced by nitroglycerine, but not the K-induced transient relaxation. It is suggested that the nitroglycerine-induced initial transient relaxation, but not the sustained relaxation, may be due to a stimulation of an electrogenic Na pump. Both relaxation phases may be mediated by cyclic GMP.

The differential effects of papaverine (Pap) and rotenone (Rot) were studied on the highly respiration-dependent contracture of guinea pig taenia coli in 40 mM potassium (40-K) medium, on isolated DT diaphorase activity and on mitochondrial respiration. The inhibition of guinea pig taenia coli to the 40-K induced tension by Rot (5 x 10(-7)M) was fully reversed by the addition of a water soluble vitamin K3 (VK3) derivative or menadione sodium bisulfite (MSB). A low concentration (10(-7)--10(-6)M) of Pap which had no effect on the 40-K induced tension inhibited the VK3 restored tension from the Rot suppression, corresponding to a Pap inhibition of the isolated DT diaphorase. Inhibition of the effective concentration of Pap to the 40-K induced tension development was never reversed by addition of VK3 or MSB. In taenia coli, both MSB and VK3 established a bypass of the Rot sensitive site on the mitochondrial respiratory chain by means of the DT diaphorase system. The difference in washout-efficacy between Pap and Rot on the inhibition of 40-K induced tension was ascribed to a difference in their mitochondrial binding properties.

1. 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.

In an attempt to elucidate the regulation mechanism of respiration in the smooth muscle cell, we investigated the roles of nicotinamide-adenine dinucleotide (NAD), adenosine diphosphate (ADP) and Ca++ in the muscle respiration using the tissues and subcellular fractions from guinea pig taenia coli. The tension in the strips of taenia coli increased with a concomitant increase in O2 consumption in high-K medium (40 mM K) containing 2.5 mM Ca. 10-3 M amytal and 10-5 M ouabain decreased the high-K induced tension and O2 consumption of the muscle. 10-4 M 2, 4-dinitrophenol (DNP) relieved the decreased respiration induced by ouabain, but not that with amytal. From these data it is suggested that NADH-linked respiration plays an important role in the respiration of the muscle. Ca++ in concentrations ranging from 0.5 to 2.5 mM in the high-K medium resulted in an increase in tension and in O2 consumption progressively. In spectrophotometric observations of subcellular fractions of the taenia coli, ADP increased in absorbance change at 340 mµ. Such occurred in mitochondrial fractions and was initiated by the addition of NADH. Therefore it is deduced that the increase in ADP level of the cytoplasm is primarily due to a contraction triggered by Ca++ thus stimulating respiration. On the other hand, at 0.1 mM of Ca++ concentration, the muscle strip increased O2 consumption without tension development in high-K medium. In the spectrophotometric observations, Ca++ and Sr++ increased the absorbance change in the homogenate and in the mitochondrial fraction. Hence, it seems that one part of the Ca++ entering into the smooth muscle treated with the high-K increased O2 consumption in mitochondria independent of an increase in muscle tension. From these results it is concluded that NADH-linked respiration plays an important role in the smooth muscle respiration in high-K medium and that ADP and Ca++ also play a role in regulating respiration.

A transport mechanism of methylmercury to egg albumen was investigated in laying Japanese quail which had received a single intravenous injection of 203Hg-methylmercuric chloride. Whole body distribution of 203Hg was analyzed by means of autoradiography. It was notable that the uptake of 203Hg was higher in the oviduct than in any other tissue checked. Further, the 203Hg was detectable in egg albumen which had been synthesized and secreted in the magnumean portion of the oviduct. From the observations mentioned above, it is suggested that methylmercury in the vascular system was taken up cumulatively by the oviduct and deposited in egg albumen by the secretory way. On the tissue level of the benzene-extractable form of 203Hg compounds (RHg) quantitative analysis was made in the respective organs. RHg decreased from the blood and liver with the lapse of time. The renal level of RHg showed a gradual increase which was followed by a rapid decrease. In contrast to the tissue tested above, a magnumean uptake of RHg increased gradually and reached the highest level of the four tissue after 24 hours. The RHg content was analyzed in the egg shell, albumen and yolk. It was the largest in the albumen and second largest in the yolk, but negligible in the shell. In 7 eggs laid over a period of 8 days after the injection, the total RHg content was approximately 44% of the dose of the radiocompound administered. The largest amount of RHg was found in the albumen of an egg the third in the order of oviposition after the injection. The fecal and urinary content of 203Hg was approximately 23% of the dose during the same period of time. These quantitative data suggest that the major part of methylmercury in the body is transported into egg albumen via the secretory mechanism of the magnumean portion of the oviduct. Starch gel electrophoresis of the soluble cytoplasmic fraction of the magnumean homogenate and of egg albumen revealed that 203Hg was associated with protein bands which had migrated near the front. The protein bands were considered to be ovalbumin. It was confirmed from an examination in vitro that 203Hg-methylmercuric chloride bound to a purified hen's ovalbumin preparation. These results mentioned above suggest that methylmercury is readily bound to ovalbumin which was newly synthesized in large quantity in the magnumean portion of the oviduct.

The effects of vanadate (Na3VO4) on the contractions induced by high concentrations of K, histamine and norepinephrine in isolated guinea-pig aorta were investigated. Vanadate (10(-3) M) relaxed the high K contraction only when the external K concentration was above 85.4 mM. Vanadate showed no inhibitory action on the contraction induced by either Na-containing 45.4 mM K solution or Na-poor 45.4 mM K solution. Ouabain (2 X 10(-5) M) had no inhibitory effects on the contractions induced by these high K solutions. Vanadate failed to relax the contraction induced by histamine or norepinephrine in normal (5.4 mM) medium, while it inhibited the contraction in the presence of 142.2 mM K. Ouabain also did not inhibit the histamine- or norepinephrine-induced contraction in either normal or high K solution. These results suggest that the inhibitory actions of vanadate are not due to an inhibition of Na pump and that these effects depend on the presence of a high concentration of external K ions.

1. Maltoxin isolated from the malt rootlet of Hordeum distichon, L., growing in Japan was identified as candicine (p-hydroxy-β-phenylethyltrimethylammonium base) in the form of chloride and picrate.2. Amount of candicine in two kinds of “malt sprouts” made from H. distichon, L., growing in Japan and H. hexastichon, L., being native to Japan was estimated by bioassay on the frog muscle after separation of the base by paper chromatography.

The mode of action of tetrodotoxin on the frog muscle fiber membrane has been analyzed with the aid of intracellular microelectrodes. Tetrodotoxin of 10–7 concentration made the applied cathodal current ineffective in producing action potential, whereas the resting potential and resting membrane resistance underwent little or no change. With 10–8 tetrodotoxin the muscle fibers responded with the small action potentials at high critical depolarizations. These results can be explained on the basis of the membrane being stabilized by inactivation of the sodium-carrying mechanism. Although delayed rectification was not observed in normal muscle fibers, it became apparent in the fibers rendered inexcitable by tetrodotoxin. This finding, together with other evidence in the existing literature, supports an applicability of the sodium theory to the frog muscle fibers.

Palytoxin (PTX), a highly toxic and sugar-containing substance isolated from Palythoa tuberculosa, caused K+ release from rabbit red blood cells. Cardiac glycosides, such as ouabain, convallatoxin, cymarin, digoxin and digitoxin, inhibited the PTX-induced K+ release. Their corresponding aglycones did not inhibit the K+ release, but antagonized the inhibitory effect of the glycosides. All these cardiotonic steroids equally inhibited the activity of (Na+ + K+-ATPase prepared from hog cerebral cortex. These results suggest that the sugar moiety of the cardiac glycosides is important for the inhibitory effect on the K+ release induced by PTX and that the inhibition is not related to their inhibitory potency on the (Na+ + K+)-ATPase activity.

Palytoxin (PTX) caused K+ release from rabbit erythrocytes at a concentration as low as 10−11 M. The K+ release due to PTX at a concentration below 10−9 M was dependent on Ca2+ in medium. The effect of Ca2+ was substituted fully by Sr2+ and partially by Ba2+. W-7 (2 × 10−4 M), a known inhibitor of calmodulin, markedly inhibited the rate of K+ release due to PTX. W-5 (2 × 10−4 M), an analog of W-7 with lower affinity to calmodulin than W-7, showed weaker inhibition. Other calmodulin antagonists, such as prenylamine, chlorpromazine and compound 48 80 , also inhibited the PTX-induced K+ release. These results suggest that the K+ release induced by PTX involves the process(es) mediated by intracellular Ca2+ and calmodulin.

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 × 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 × 10−5 M) or ouabain (1−2 × 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+ and -K+ pump is inhibited by ouabain, and that monensin inhibits aerobic energy metabolism of vascular smooth muscle.

Palytoxin (PTX), at extremely low concentrations (0.01-1 nM), caused K+ release from rabbit erythrocytes. Among the various chemical compounds tested, cardiac glycosides potently inhibited the PTX-induced K+ release. The order of inhibitory potency (IC50) was cymarin (0.42 microM) greater than convallatoxin (0.9 microM) greater than ouabain (2.3 microM) greater than digitoxin (88 microM) greater than digoxin (90 microM). Their corresponding aglycones, even at 10 microM, did not inhibit the K+ release, but competitively antagonized the inhibitory effect of the glycosides. All these cardiotonic steroids inhibited the activity of (Na+ + K+)-ATPase prepared from hog cerebral cortex in narrow concentration ranges (IC50 = 0.15-2.4 microM), suggesting that the inhibition of K+ release is not related to their inhibitory potency on the (Na+ + K+)-ATPase activity, and the sugar moiety of cardiac glycosides is involved in the inhibition. On the other hand PTX, at higher concentrations (greater than 0.1 microM), inhibited the (Na+ + K+)-ATPase activity. However, this inhibitory effect of PTX was not antagonized by ouabain. It is suggested that, compared with ouabain, PTX has additional binding site(s) on the (Na+ + K+)-ATPase.

The properties of contractions induced by ouabain and by K depletion were studied in comparison with those induced by other stimulants in guinea pig taenia coli. Ouabain 7.5 X 10(-6) M and K depletion produced biphasic contractions with similar time courses. Both contractions were slightly inhibited by low (0.25 mM) and high (7.5 mM) Ca concentrations, while the high K contraction was strongly inhibited by low Ca and potentiated by high Ca. Contractions induced by ouabain and K depletion were highly sensitive to lowering of the temperature and abolished at 15 degrees C, although contractions induced by carbachol 1.1 X 10(-7) M and histamine 5 X 10(-7) M were still observed below 15 degrees C. In Cl deficient solution substituted by SO4 or propionate, contractions induced by ouabain and K depletion were potentiated, while contractions induced by high K, carbachol and histamine were suppressed. These results indicate a striking similarity between contractions induced by ouabain and K depletion suggesting a common mechanism for both contractions.

Effects of atropine (ATR, 0.5 mg/kg, i.v.), proglumide (PGM, 100 mg/kg, i.v.)and metiamide (MTM, 0.4 mg/kg, i.v.) on gastric acid secretion induced by vagal stimulation, carbamylcholine (CCh, 5μg/kg, i.v.), tetragastrin (TG, 5μg/kg, i.v.) and histamine (Hist, 10μg/kg, i.v.) injections were examined by means of continuous pH recording of proventricular effluent in urethan anesthetized chickens. Birds were operated for fitting an esophageal cannula and gastric fistel, artificial respiration, bilateral vagotomy and instillation of physiological saline solution. Electrical stimulation (0.1msec duration, 30volts, 50 Hz, for 40 to 60 sec) was applied to Trunks communis n. vagi at the proximal region of the proventriculus where the proventricular rami (Rr. gastrici glandulares) branched. The vagal stimulation and the administration of CCh produced a biphasic profile of pH fall response consisting of an early transient and a secondary lasting phases. The transient response was larger in the degree of pH fall than the lasting one, and was inhibited by ATR but not by PGM and MTM. The lasting response was reduced by all of the three inhibitors. PGM and MTM inhibited pH fall responses induced by TG and Hist, respectively. These results show that the vagally stimulated acid secretion consists of an early transient response which is major component and a secondary lasting response Which is minor component. It is suggested that the transient response is directly mediated by cholinergic transmission and the lasting response is possibly mediated by endogenous gastrin and histamine in the pathways sensitive to PGM and MTM.