Aerobic metabolism on muscle contraction in porcine gastric smooth muscle.

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.

Phosphodiesterase 9 (PDE9) regulates bovine tracheal smooth muscle relaxation.

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.

Imidazole-induced contractions in bovine tracheal smooth muscle are not dependent on the cAMP pathway.

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.

Effects of chlorogenic acid on carbachol-induced contraction of mouse urinary bladder.

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.

Phloridzin inhibits high K+-induced contraction via the inhibition of sodium: glucose cotransporter 1 in rat ileum.

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.

Aerobic metabolism on muscle contraction in porcine iris sphincter.

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.

Endothelium-Dependent and -Independent Vasodilator Effects of Dimethyl Sulfoxide in Rat Aorta.

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 hypoxia and glucose-removal condition on muscle contraction of the smooth muscles of porcine urinary bladder.

To elucidate the dependence of aerobic energy metabolism and utilization of glucose in contraction of urinary bladder smooth muscle, we investigated the changes in the reduced pyridine nucleotide (PNred) fluorescence, representing glycolysis activity, and determined the phosphocreatine (PCr) and ATP contents of the porcine urinary bladder during contractions induced by high K(+) or carbachol (CCh) and with and without hypoxia (achieved by bubbling N2 instead of O2) or in a glucose-free condition. Hyperosmotic addition of 65 mM KCl (H-65K(+)) and 1 µM CCh induced a phasic contraction followed by a tonic contraction. A glucose-free physiological salt solution (PSS) did not change the subsequent contractile responses to H-65K(+) and CCh. However, hypoxia significantly attenuated H-65K(+)- and CCh-induced contraction. H-65K(+) and CCh induced a sustained increase in PNred fluorescence, representing glycolysis activity. Hypoxia enhanced H-65K(+)- and CCh-induced increases in PNred fluorescence, whereas glucose-free PSS decreased these increases, significantly. In the presence of H-65K(+), hypoxia decreased the PCr and ATP contents; however, the glucose-free PSS did not change the PCr contents. In conclusion, we demonstrated that high K(+)- and CCh-induced contractions depend on aerobic metabolism and that an endogenous substrate may be utilized to maintain muscle contraction in a glucose-free PSS in the porcine urinary bladder.

Effects of papaverine on carbachol- and high K+ -induced contraction 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 Ca(2+) from 1.5 mM to 7.5 mM reduced verapamil-induced relaxation in high K(+)-depolarized muscles, but papaverine-induced relaxation did not change. Furthermore, 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.

PDE4 and PDE5 regulate cyclic nucleotide contents and relaxing effects on carbachol-induced contraction in the bovine abomasum.

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.

Key role of glycogen storage in high K+-induced contraction of the smooth muscles of the bovine trachea.

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.

Effects of papaverine on twitches in mouse diaphragm.

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.

Effects of rolipram on U46619-induced contraction and cyclic nucleotide content in the porcine coronary artery.

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.

Effects of various selective phosphodiesterase inhibitors on relaxation and cyclic nucleotide contents in porcine iris sphincter.

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 IC(50) was zaprinast > Ro20-1724 > EHNA >/= milrinone > vinpocetine. In the presence of CCh (0.3 muM), 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.

Effects of high-K(+), Na(+)-deficient solution on contractility of the smooth muscles of the porcine trachea.

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.

Ibudilast-induced decreases in cytosolic Ca(2+) level and contraction in rat aorta.

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 [Ca(2+)](i) level in a concentration-dependent manner, to the same degree. 3-Isobutyl-1-methylxanthine (IBMX) inhibited PE-induced contraction and [Ca(2+)](i) level in a concentration-dependent manner, but it inhibited high K(+)-induced contraction without decrease of [Ca(2+)](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 microM ibudilast inhibited the extracellularly added Ca(2+)-induced muscle tension and increases in [Ca(2+)](i) level during high K(+) depolarization. These results suggested that ibudilast inhibited PE- and high K(+)-induced muscle contractions mainly by the inhibition of [Ca(2+)](i) level in endothelium-denuded rat aorta.

Effects of high-K+, Na+-deficient solution on contractility of the smooth muscles of the bovine trachea.

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.

Inhibitory mechanism of monensin on high K+-induced contraction in guniea-pig urinary bladder.

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 microM) was more potent than those of NaCN (100 microM - 1 mM) and forskolin (3 - 10 microM). Monensin (0.1 microM), NaCN (300 microM), or forskolin (10 microM) 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 microM but not at 0.1 microM that induced maximum relaxation. In the alpha-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.

Effects of various selective phosphodiesterase inhibitors on carbachol-induced contraction and cyclic nucleotide contents in the guinea pig gall bladder.

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 Ro20-1724 > vinpocetine > EHNA > milrinone > zaprinast, 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.

Inhibitory mechanism of papaverine on carbachol-induced contraction in bovine trachea.

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 [Ca(2+)](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 microM) remarkably inhibited muscle tension, but slightly decreased creatine phosphate and ATP contents. Iberiotoxin restored the inhibitions of muscle contraction and [Ca(2+)](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.