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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.

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.

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.

The effects of various selective phosphodiesterase (PDE) inhibitors on muscle contractility and cyclic nucleotide contents in the guinea pig gall bladder 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), and zaprinast (type 5), inhibited CCh-induced contractions in a concentration-dependent manner. The rank order of potency for the gall bladder was Ro20-1724vinpocetineEHNAmilrinonezaprinast, which was different from that of the trachea, taenia coli, and aorta. In the presence of CCh (0.3 muM), vinpocetine, milrinone, and Ro20-1724 each increased cAMP content, but not cGMP. By contrast, zaprinast increased cGMP content, but not cAMP, and EHNA increased both cAMP and cGMP contents. These results suggest that vinpocetine-, milrinone-, and Ro20-1724-induced relaxation was correlated with cAMP, zaprinast-induced relaxation was correlated with cGMP, and that EHNA-induced relaxation was correlated with cAMP and cGMP in the guinea pig gall bladder. In conclusion, the effect of PDE inhibitors in the guinea pig gall bladder was different from those in smooth muscles, such as the trachea, taenia coli, and aorta.

We have demonstrated that the relaxing mechanism of papaverine in phasic muscles such as ileum, urinary bladder, and uterus is different from tonic muscles such as aorta. In this study, we examined the inhibitory mechanism of papaverine on carbachol (CCh)-induced contraction in the bovine trachea. Papaverine inhibited muscle contraction and increase in [Ca2+]i level induced by CCh. Papaverine increased cAMP content but not cGMP content. Papaverine did not affect CCh-induced oxidized flavoproteins fluorescence or reduced pyridine nucleotides fluorescence. Papaverine (30 µM) remarkably inhibited muscle tension, but slightly decreased creatine phosphate and ATP contents. Iberiotoxin restored the inhibitions of muscle contraction and [Ca2+]i level induced by papaverine or dibutyryl-cAMP. These results suggested that the relaxing mechanism of papaverine in the bovine trachea is mainly due to increases of cAMP content by inhibiting phosphodiesterase and the mechanism is partially involved in the activation of BK channel by cAMP. Keywords:: papaverine, bovine, trachea, cAMP, iberiotoxin

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.

Papaverine (0.3-100 microM) more potently inhibited phenylephrine (1 microM)-induced contraction than 65 mM K+-induced contraction of the aorta, while it equally inhibited contractions induced by 65 mM K+ and carbachol (1 microM) in ileal smooth muscle. In phenylephrine-treated aorta, papaverine (1-10 microM) increased the cAMP and cGMP content. However, in carbachol-treated ileum, 30 microM papaverine partially increased the cAMP content while it maximally relaxed the preparation. In fura2-loaded aorta, papaverine (0.3-10 microM) inhibited both the contraction and the increase in intracellular Ca2+ level ([Ca2+]i) induced by phenylephrine in parallel. However, papaverine inhibited carbachol-induced contraction with only a small decrease in [Ca2+]i. Papaverine (1-30 microM) inhibited the carbachol-induced increase in oxidized flavoproteins, an indicator of increased mitochondrial oxidative phosphorylation, in ileal smooth muscle whereas it did not change the phenylephrine-induced increase in the aorta. These results suggest that papaverine inhibits smooth muscle contraction mainly by the accumulation of cAMP and/or cGMP due to the inhibition of phosphodiesterase in the aorta whereas, in ileal smooth muscle, papaverine inhibits smooth muscle contraction mainly by the inhibition of mitochondrial respiration.

Gymnema sylvestre (GS) is one of the Asclepiad strains that grows in South-east Asia. Their therapeutic effects for treating diabetes mellitus, rheumatic arthritis and gout have been well known for a long time. However, the problem is that GS suppresses sweetness and tastes bitter. For this study, we chose Gymnema inodorum (GI) instead of GS, since it has an advantage that it does not suppress sweetness nor is it bitter in taste. In this paper, effects of glucose availability of some saponin fractions (F-I to F-IV) extracted from GI leaves, which were obtained by high-performance liquid chromatography were studied on a high K(+)-induced contraction of guinea-pig intestinal smooth muscle, O2 consumption on guinea-pig ileum, glucose-evoked transmural potential difference (delta PD) of guinea-pig everted intestine and blood glucose level in glucose tolerance tests on rats. The extracts of GI leaves suppressed the intestinal smooth muscle contraction, decreased the O2 consumption, inhibited the glucose evoked-transmural potential, and prevented the blood glucose level. Our studies suggest that the component of GI inhibits the increase in the blood glucose level by interfering with the intestinal glucose absorption process.

Two substances identified as ((3 beta, 4 alpha, 16 beta, 21 beta, 22 alpha)-21-tigloxy-16, 22, 23, 28-tetrahydroxyolean-12-en-3-yl-beta D-glucopyranosiduronic acid) (GA1) and ((3 beta, 4 alpha, 16 beta, 21 beta, 22 alpha)-21-(2-methylbutyroxy)-16, 22, 23, 28-tetrahydroxyolean-12-en-3-yl-beta-D-glucopyranosiduronic acid) (GA2) identified among the gymnemic acids are triterpene glycosides extracted from Gymnema sylvestre leaves. We examined the effects of GA1 or GA2 on high K(+)-induced contraction in the guinea-pig longitudinal muscle. A sustained muscle contraction induced by hyperosmotically added 65.4 mM KCI (H-65K+) was suppressed by GA1 or GA2 (7.7 x 10(-5) M). Simultaneous measurements of reduced pyridine nucleotide (PNred) or oxidized flavin protein (FPox) by the fluorescence technique and of contractile force revealed that GA1 and GA2 reduced the increase of PNred fluorescence and contractile force induced by H-65K+, whereas FPox fluorescence induced by it further increased. Reduced muscle contraction induced by GA1 or GA2 was restored by 5.5 mM pyruvate. Simultaneous measurements of intracellular Ca2+ [Ca2+]1 level and contractile force indicated that [Ca2+]1 level, which increased by H-65K+, hardly changed with GA1 and GA2. In summary, both GA1 and GA2, which are among the gymnemic acids, suppressed high K(+)-induced contraction in the guinea-pig ileal longitudinal muscle. The difference between these two gymnemic acids was not significant. The inhibitory effect of GA1 and GA2 on smooth muscle were assumed to be a result of inhibiting glucose uptake, which is an energy source of the muscle, whereas the inhibitory mechanisms were probably not mediated by Ca2+.

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.

To elucidate the role of glycogen in the contraction of tracheal smooth muscle, we investigated the changes in the glycogen contents of the bovine trachea during contractions induced by high K(+) and hypoxia (achieved by bubbling N(2) instead of O(2)), either in a glucose-free condition or in the presence of iodoacetic acid (IAA), an inhibitor of glycolysis. Hyperosmotic addition of 65 mM KCl (H-65 K(+)) induced a sustained contraction. A glucose-free condition did not affect H-65 K(+)-induced contraction. However, hypoxia slightly inhibited the contraction, and glucose-free PSS with hypoxia or IAA remarkably inhibited the H-65 K(+)-induced contraction. H-65 K(+) induced a sustained increase in reduced pyridine nucleotide (PNred) fluorescence, representing glycolysis activity. Hypoxia alone slightly enhanced PNred fluorescence, and when combined with a glucose-free condition, it remarkably enhanced the H-65 K(+)-induced PNred fluorescence. IAA inhibited PNred fluorescence. In the presence of H-65 K(+), a glucose-free condition, hypoxia and the combination of glucose-free PSS and hypoxia decreased the glycogen contents. However, IAA had no effect on glycogen contents. Although hypoxia or glucose-free PSS did not affect PCr and ATP contents, the combination of hypoxia and glucose-free PSS or IAA induced a gradual decrease of PCr content. In conclusion, we suggest that endogenous glycogen was utilized to increase the activity of glycolysis for maintaining high K(+)-induced contraction of the bovine trachea in the glucose -free and/or hypoxic condition.

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.

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

A high-K+, Na+-deficient (I-154 K+) solution induced contraction followed by gradual relaxation of the smooth muscles of the bovine trachea, while hyperosmotic addition of 65 mM KCl induced a large sustained contraction. Exposure of the muscle to the I-154 K+ solution induced an increase in the ratio of cellular water content and a sustained increase in oxidized flavoprotein fluorescence or reduced pyridine nucleotide fluorescence. The I-154 K+ solution also induced a sustained increase in [Ca2+]i level. Decreases in developed tension and increases in cellular water content were both prevented by the addition of sucrose or NaCl but not pyruvate. Substitution of KI for KCl in the I-154 K+ solution produced a greater inhibition of contraction, while substitution with K-propionate produced no inhibition of contraction. Moreover, decreases in developed tension and increases in cellular water content were both prevented by addition of 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), but not 10 microM bumetanide or 1 mM acetazolamide. In conclusion, I-154 K+ solution induced-relaxation in the bovine trachea may be due to swelling of smooth muscle cells and the mechanism of swelling is probably involved in DIDS-sensitive anion movement.

The leaves of Gymnema inodorum (GI) have been known to be effective for some diseases including diabetes mellitus, rheumatic arthritis and gout. The crude saponin mixtures extracted from GI leaves inhibited glucose absorption in the isolated intestinal tract and suppressed the increased blood glucose in rats. In this study, we examined the relationship between chemical structure and pharmacological activity of the four components from GI leave extracts (GiA-1, GiA-2, GiA-5 and GiA-7). These components were the derivatives of (3beta,4alpha,16beta)-16,23,28-trihydroxyolean-12-en-3-yl-beta-D-glucopyranosiduroic acid. GiA-2, GiA-5 and GiA-7 that have suppressive effects on the high K+-induced contraction, an increase in deltaPD and the increased blood glucose level in the glucose tolerance test have -H at the 21st position and -CH2OH at 4beta of aglycon. On the other hand, GiA-1 that does not have any effects on the three parameters mentioned above has -H at the 21st position and -CH3 at 4beta of aglycon. In conclusion, it is suggested that the inhibitory effect of triterpenoids in Gymnema leaves on glucose absorption from the intestinal tract relies on -CH2OH at 4beta.

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.

Extracts containing gymnemic acids, which were extracted from the leaves of Gymnema sylvestre (GS) as nine fractions, were evaluated for their effects on a high K(+)-induced contraction of guinea-pig ileal longitudinal muscles, on glucose transport mediated by the difference of glucose-evoked transmural potential difference (delta PD) in the inverted intestine of guinea-pig and rat, and on blood glucose in rat. Among nine fractions obtained by high performance liquid chromatography from the extract, f-2 and f-4 strongly suppressed the high K(+)-induced contraction of the ileal muscle, f-3 and f-5 did so moderately, and f-8 and f-9 did so weakly, whereas the other fractions did not affect it. The degree of suppression of high K(+)-induced contraction by f-2 at 74% was almost the same as that of f-4 at 67%, at concentrations of 0.1 mg/ml. The suppressed contraction by f-2 or f-4 was recovered by adding 5.5 mM pyruvate. The delta PD increased by 5.5 mM glucose in the inverted intestines of guinea-pig and rat were equally suppressed by 0.1 mg/ml of f-2 or f-4 to 40%. In a rat sucrose tolerance test, f-2 and f-4 suppressed the elevation of blood glucose level. Both f-2 and f-4 suppressed the contraction of guinea-pig ileal longitudinal muscle, interfered with the increase in delta PD induced by glucose in the inverted intestines of guinea-pig and rat, and inhibited the elevation of blood glucose level. In conclusion, it is suggested that some of the extracts containing gymnemic acids from GS leaves suppress the elevation of blood glucose level by inhibiting glucose uptake in the intestine.

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.

Tiamulin with an IC50 of 1.7 x 10(-6) M inhibited both the rapid and sustained contractions induced by hyperosmotically added 60 mM K+ (Hyper 60 K+) without changing the membrane potential in the intestinal muscle. Tiamulin inhibition (2 x 10(-6)-2 x 10(-5) M) of the Ca(2+)-induced contraction in depolarized muscle was competitively antagonized by raising external Ca2+. Tiamulin (2 x 10(-5) M) slightly affected the Hyper 60 K(+)-induced phasic contraction under hypoxia and the carbachol-induced phasic contraction. Moreover, tiamulin (2 x 10(-5) M) inhibited the Hyper 60 K(+)-induced contraction with decreasing [Ca2+]cyt level. Although the inhibitory effect of 10(-7)-10(-5) M monesin, an inhibitor of mitochondrial respiration, on the Hyper 60 K(+)-induced contraction was reduced under hypoxia, the effect of tiamulin (2 x 10(-7)-2 x 10(-4) M) was not modified. Tiamulin changed neither the intracellular Na+ and K+ content of the depolarized muscle nor the Ca(2+)-induced contraction in the chemically skinned preparations. These results suggest that the inhibitory action of tiamulin on the Hyper 60 K(+)-induced tonic contraction is possibly due to the competitive inhibition of Ca2+ entry through the voltage-dependent Ca2+ channel of the intestinal smooth muscle cell.

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 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.

Ouabain was examined for effect on the mechanical response of the ileum isolated from eleven animal species, dog, cat, rabbit, guinea pig, rat, vole, mouse, cock, pigeon, frog and toad. In isotonic recording, ouabain induced a sustained contraction in ileum isolated from some species. It caused a transient contraction on following by a relaxation in others. The ouabain sensitivities of a contractile response in the ilea from eleven species, as demonstrated by the estimation of ED50, was divided into four groups. That is, a high sensitive group; frog (ED50=9.0×10-8M), cat (9.3×10-8M), pigeon (3.5×10-7M) and cock (5.1×10-7M), a sensitive group; rabbit (1.1×10-6M), vole (2.3×10-6M) and guinea pig (2.7×10-6M), a low sensitive group; mouse (1.7×10-5M) and rat (9.7×10-5M) and an insensitive group; toad (>10-3M). Although ED50 in the dog was not able to be calculated, its sensitivity seems to belong to the high sensitive group. On the other hand, ED50 of acetylcholine (Ach) in thc ilea, isolated from these species were estimated in a narrow range of concentration from 5×10-9M to 5×10-8M. There were species differences in ouabain sensitivity for ileal contractile response but not in Ach sensitivity. A distinct correlation was found between the mechanical response to ouabain and the inhibitory potency of ouabain for Na, K-ATPase reported by Repke et al. These results seem to confirm that ouabain may induce a contraction by a mechanism which inhibits an electrogenic Na pump or Na, K-ATPase in the intestinal smooth muscle cell and which induces a depolarization.

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.

The effects of verapamil and sodium nitroprusside on muscle tension and 45Ca uptake activated in different ways were compared in rabbit aorta, rat aorta and guinea-pig taenia coli. In rabbit aorta, K-induced contraction was specifically inhibited by verapamil and noradrenaline-induced contraction by sodium nitroprusside. In rat aorta, both K-induced and noradrenaline-induced contractions were inhibited by verapamil or by sodium nitroprusside also. In taenia, both K- and histamine-induced sustained contractions were inhibited by verapamil but not by sodium nitroprusside. The effect of verapamil was competitively antagonized by external Ca, while that of sodium nitroprusside was not. High K, noradrenaline and histamine increased the rate of 45Ca uptake in aortae and taenia. In rabbit aorta the increment in response to high K was specifically inhibited by verapamil and the increment induced by noradrenaline was specifically inhibited by sodium nitroprusside. In rat aorta, increments induced by both high K and noradrenaline were inhibited by verapamil and by sodium nitroprusside. In taenia, the increments induced by high K and by histamine were inhibited by verapamil but not by sodium nitroprusside. These results suggest different characteristics of Ca entry systems in these smooth muscles. In rabbit aorta, there seem to be two Ca channels, one of which is activated by high K and inhibited by verapamil, while the other is activated by noradrenaline and inhibited by sodium nitroprusside. In rat aorta, both K- and noradrenaline-activated Ca pathways are sensitive to both verapamil and sodium nitroprusside whereas, in taenia, both K- and histamine-activated Ca pathways are sensitive only to verapamil.

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.

Age-related changes in the sensitivity to verapamil and sodium nitroprusside were examined in isolated aortic strips of the rabbit. In the aortae of newborn rabbits within 10 days of birth, the resting tone of the muscle was strongly reduced by sodium nitroprusside but not by either Ca-deficient solution or by verapamil. High K-induced contraction and noradrenaline-induced contraction were both inhibited by verapamil or sodium nitroprusside. In the aortae of 24 day-old rabbits, resting tension was slightly reduced by sodium nitroprusside but not by verapamil. High K-induced contraction was less sensitive to sodium nitroprusside than to verapamil whereas noradrenaline-induced contraction was less sensitive to verapamil than to sodium nitroprusside. In the aortae isolated from 60 day-old or older rabbits, resting tension was not affected by either sodium nitroprusside or verapamil. High K-induced contraction was inhibited by verapamil whereas sodium nitroprusside showed only a weak inhibitory effect. Noradrenaline-induced contraction was inhibited by sodium nitroprusside although verapamil had only a slight inhibitory effect. In the aortae of 1 day-old and also in adult rabbits, noradrenaline induced an additional increase in muscle tension when applied during the sustained contraction induced by high K. It is suggested that, in the newborn rabbit aorta, the voltage-dependent Ca channel is sensitive to both verapamil and sodium nitroprusside and the sensitivity to sodium nitroprusside gradually decreases during maturation whereas the receptor-linked Ca channel is also sensitive to both of the inhibitors at birth but the sensitivity to verapamil gradually decreases with age.

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. 1. PTX (10 −8 M) induced a rapid increase followed by a gradual decrease in muscle tension in normal physiological salt solution (PSS), while it induced a slow increase in muscle tension in low-Na + solution. These contractions were inhibited by Ca 2+ channel blockers, verapamil and nicardipine. 2. 2. PTX rapidly increased tissue Na + and decreased tissue K + contents in normal PSS. In low-Na + solution, PTX decreased tissue K + content with a slower rate than that in normal PSS. 3. 3. PTX increased uptake of 45 Ca 2+ in normal as well as low-Na + solutions with similar time course as the increase in muscle tension. However, 45 Ca 2+ uptake still remained high when the PTX-induced transient contraction ceased. 4. 4. These results suggest that PTX increases Ca 2+ influx through voltage-dependent Ca 2+ channels to cause contraction. After a prolonged exposure to PTX, however, muscle tension is uncoupled from Ca 2+ influx.

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.

Effect of removal of the external Potassium ion (K removal) was examined on the mechanical response of the ileum isolated from thirteen animal species; monkey, dog, cat, rabbit, guinea pig, rat, mouse, volt, deer, cock, pigeon, frog and toad. In isotonic recording, K removal induced a sustained contraction in ileum isolated from cat and deer, however, a transient contraction followed by a relaxation in the ileum isolated from other species. Readmission of potassium to the K-free solution produced a quick relaxation in the muscles of pigeon, frog, guinea pig, rat and mouse. Ouabain (1×10-5 M) inhibited the relaxation induced by the readmission of K. There were species differences in contractile activity to K removal; the relative height of ileal contraction induced by K removal was in the order of pigeon, frog, vole, dog, cat, deer, monkey, cock, guinea pig, rat, rabbit, mouse and toad, respectively. Furthermore, the magnitude of the contractile responses to K removal in the ilea isolated from the various kinds of animals correlated with the ED50 of ouabain for a contractile response in the ilea from these animals, as reported by Shimizu et al. (1979). These results probably confirm the concept that K removal and ouabain induce a contraction which is due to the inhibition on an electrogenic Na pump in muscle cells of the ileum.

The effects of Li+ substitution for external Na+ in the medium on a 60 mM KCl (60K+)-induced contraction were compared in the ileal longitudinal smooth muscles isolated from monkey, rabbit, guinea pig and rat. Li+ substitution inhibited the 60K+-induced contraction in the ileum of all the animal species. Greater inhibitory effect was observed with the increase in external Li+ concentration and the increase in incubation period with Li+. Intracellular Li+ contents of these muscles also increased with the increase in external Li+ concentration and the incubation period. Thus, a good correlation was obtained between Li + -induced inhibition of 60K+-induced contraction and the increase in Li+ content. However, close examination revealed that the contraction in monkey and rat ileum was inhibited by much lower Li+ accumulation than that in guinea pig and rabbit ileum, suggesting the existence of species difference in the sensitivity of the ileal smooth muscle to the inhibitory effect of Li+ substitution, and a mechanism for the species differences in the inhibitory action of Li+-substitution was discussed.

Effects of ouabain, Na+ depletion and K+ depletion on the tension development of rabbit aorta were studied. Contraction in the strips was induced by ouabain and Na+-free solution substituted with Li+, tris or sucrose. The contractions were rarely seen under alpha-adrenergic blockade, in reserpinized aorta and mechanically denervated aorta. Contractions induced by norepinephrine were potentiated by Na+ depletion, K+ depletion and ouabain. Contractions induced by high K+ solution were also potentiated by Na+ depletion and ouabain. The potentiation in Li+ solution, K+-free solution and with ouabain was not seen under alpha-adrenergic blockade, in reserpinized aorta and denervated aorta although the potentiation by tris and sucrose solutions was still observed. These data suggest that Na+ depletion, K+ depletion and ouabain release endogenous catecholamines which affect the contraction of the vascular smooth muscle of rabbit aorta.

1. In guinea-pig aorta, removal of external K produced contraction which reached maximum (about 1 g) after about 3 h. Excess K (65.4 mM) produced contraction (about 1 g) at a faster rate of increase than was observed in K-free solution. 2. Verapamil, nifedipine and diltiazem (10−7–10−5 M) and the raising of [Mg]0 (8–32 mM) had no effect on the contraction induced by K-free solution, although they relaxed the contraction induced by high-K solution. Verapamil (10−6 M) also relaxed the contractions induced by other depolarizing agents, TEA (40 mM) and Ba (15 mM). 3. Following incubation in Ca- and K-free solution for 3 h, the readdition of Ca produced contraction at a faster rate of increase than in a K-free normal Ca containing solution. Verapamil (10−6 M) and Mg (16 mM) had exhibited little effect on these Ca-induced contractions. The Ca-induced contractions in high-K solution, on the other hand, were significantly suppressed in the presence of verapamil (10−6 M) and Mg (16 mM). 4. After incubation of the muscle in K-free solution for 1 h, Na depletion caused a more rapid and greater contraction than was seen in untreated muscle. This tension development was also unaffected by verapamil (10−6 M) and Mg (16 mM). 5. Substitution of Sr for Ca produced contraction in K-free or high-K solution. Sr-induced contraction in K-free solution was scarcely affected by verapamil (10−6 M), while Sr-induced contraction in high-K solution was inhibited. 6. 45Ca entry into smooth muscle during the contractions was measured by the modified “La method”. The 45Ca uptake was markedly increased both in K-free and high-K solutions. Verapamil (10−6 M) and Mg (16 mM) had no significant effect on the 45Ca uptake increase clicited by K-free solution, whereas they significantly inhibited the 45Ca uptake increased by high-K solution. 7. It is suggested that the contraction induced by K-free solution is initiated by a mechanism different from that underlying the depolarization-induced contraction, and that it is produced by an influx of Ca through Na-Ca exchange mechanism. The data also suggest that the Ca binding site on the Na-Ca exchange carrier is occupied by Sr ions but not by Mg ions. It is likely that such a carrier system is not influenced by organic “Ca antagonists”.

1 Several putative calmodulin antagonists have been examined for their inhibitory action on muscle tension and cellular Ca content in the K-depolarized vascular and intestinal smooth muscles. 2 The 65.4 mM K-induced sustained contraction in the media-intimal layer of rabbit aorta and the 45.4 mM K-induced sustained contraction in guinea-pig taenia coli were inhibited by the calmodulin antagonists, prenylamine, chlorpromazine, N2-dansyl-L-arginine-4-t-butylpiperadine amide (No. 233), and N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), and also by the organic Ca antagonists, verapamil and diltiazem. 3 The cellular Ca content in rabbit aorta and guinea-pig taenia coli as measured by a modified lanthanum technique increased in the high-K solutions. The increments were inhibited by these antagonists at concentrations similar to those required to inhibit the K-induced contractions. However, W-7 did not change (in aorta) or only slightly decreased (in taenia coli) the K-induced increase in the cellular Ca content. 4 A high concentration (2 X 10(-4)M) of W-7 increased the resting cellular Ca content without increasing the muscle tension in aorta. The increment was inhibited by verapamil, sodium nitroprusside or hypoxia (N2 aeration). 5 It is suggested that the inhibitory effects of prenylamine, chlorpromazine and No. 233 may be attributed mainly to the Ca antagonistic effect whereas W-7 may inhibit the process beyond the transmembrane Ca influx.

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.

Papaverine (Pap) inhibited a tonic tension development in guinea pig taenia coli induced by an elevation of the potassium concentration of the medium to 40 mM (40-K). The ID50 of Pap was 4.5 × 10-6 M. Inhibition of the 40-K induced tonic tension by Pap was dependent on the calculated concentration of non-ionized form, but not the ionized form. Inhibition of the tonic tension by Pap and various metabolic inhibitors was not antagonized by raising the Ca++ concentration in the medium. Results obtained from simultaneous measurement of the 40-K induced O2 consumption and tension illustrated that the inhibition of O2 consumption rapidly induced the inhibition of the tension. Furthermore, glucose removal, amytal or Pap resulted in a progressive relationship between the inhibition of O2 consumption and the inhibition of tension. The inhibition of Pap as determined by O2 consumption was not reversed by application of 10-4 M 2,4-dinitrophenol (DNP). These results plus graphical analysis of the mechanical response suggest that Pap may penetrate the cell membrane and inhibit mitochondrial respiration, thereby inhibiting this very respiration-dependent contracture.

It was reported that an application of high-K (isotonic, 40 mM) caused an increase in oxygen consumption accompanied by an increase in tension change in guinea pig taenia coli (1) and that these changes were reversibly abolished by a depletion of Ca from the high-K medium and Sr could replace Ca only for oxygen consumption (2). These results suggest that Ca entering smooth muscle treated with high-K probably increases oxygen consumption, independent of an increase in muscle tension. In a previous paper (3), effects of factors inhibiting tension development on oxygen consumption of the muscle in high-K medium were reported. Glucose removal from the high-K medium abolished the already developed tension corresponded with a decrease in the elevated oxygen consumption rate. Addition of DNP (1×10-4M) to high-K medium further increased or maintained the increased oxygen consumption although it abolished the already developed tension, and the change in oxygen consumption was not modified by Ca removal. On the other hand ouabain (2.5×10-6 M) maintained the increased oxygen consumption in high-K medium, but almost abolished the developed tension. The increase in oxygen consumption was dependent on external calcium. In the present paper, effects of factors inhibiting high-K induced tension and/or oxygen consumption on Ca distribution in taenia coli are studied and this throws some light on a participation of Ca distribution in changes in tension and oxygen consumption of the muscle in high-K medium. A study on ouabain and DNP with high-K on Ca exchange in taenia coli has been reported by Pfaffman and Holland (4), which is incompatible with the present data.

In smooth muscle of guinea pig taenia coli, showing spontaneous activity, the curves of rhythmic changes in muscle tension appears parallel to the curves of changes in Po2 level in the medium (1-3). On the other hand, Saito et al. (3) reported that an application of high K (isotonically, 40 mM) caused an increase in oxygen consumption accompanying increase in tension change in taenia coli, and that this was in accordance with the concept (4, 5) that the K-induced tonic response resulted from a transmembrane transport of Ca that depended on the aerobic breakdown of carbohydrates. In the present paper, effects of Ca depletion on the oxygen consumption accompanying muscle tension change both spontaneous and that induced by high K were studied, and a dissociation of the changes in oxygen consumption and muscle tension change was attempted by substituting strontium (Sr) for Ca in the external medium. Some of the results have already been reported (6, 7).

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.