金田 剛治

Background: We previously reported that myocardial fibrosis may be one of the causes of left ventricular hypertrophy and cardiac dysfunction in dogs with hyperglucocorticism (HGC). The detailed mechanism by which myocardial fibrosis of the left ventricle occurs in dogs with hyperglucocorticism (HGC) remains unclear. Aim: This study investigated the mechanism by which HGC causes fibrosis of the left ventricle.Methods: The impacts of HGC on the heart by comparing samples obtained from high-dose glucocorticoid-treated (P) and untreated (C) dogs. The P group included healthy Beagle dogs (n=6) treated with prednisolone (2 mg/kg, bid, po) for 84 days, and the C group included healthy Beagle dogs (n=6) euthanized for unrelated reasons. In three of the P group dogs, serum was collected before the start of administration (Day 0) and on Day 84 to measure angiotensin II concentrations and oxidative stress markers (8-hydroxy-2’-deoxyguanosine (8OHdG), NADPH oxidase, and superoxide levels). Samples of the left ventricular free wall (LVFW), right ventricular free wall (RVFW), interventricular septum (IVS), and aortic root were harvested from both groups (n = 6 for each group). Using these tissue samples, angiotensin II type 1 receptor (AT1R), 8OHdG, and transforming growth factor β1 (TGFβ1) immunohistochemical stains were performed.Results: The blood NADPH oxidase concentration was significantly higher (P=0.027) in the P group 84 days after initiation of the medication compared to that before prednisolone treatment. By contrast, there was no significant difference in serum angiotensin II (P=0.450), 8OHdG (P=0.068), and superoxide (P=0.057) concentrations. The positive staining rates of AT1R, 8OHdG, and TGFβ1 in the heart (LVFW, RVFW, IVS, and aortic root) were significantly higher in the P group than those in the C group.Conclusion: Angiotensin II and oxidative stress in HGC may cause left ventricular fibrosis in dogs.

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 concentrationdependent 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.

Angiotensin II (100 nM) induced bi-phasic increases in cytosolic Ca2+ level ([Ca2+]i) through the activation of angiotensin II type 1 receptor. Pharmacological examinations using 10 µM verapamil, 30 µM La3+, and 1 µM thapsigargin indicated that the first phase of the [Ca2+]i-increase was mediated by Ca2+ release from sarcoplasmic reticulum (SR) and Ca2+ influx independently of voltage dependent Ca2+ channel (VDC). In contrast, the second phase of [Ca2+]i-increase was mediated by Ca2+ influx through VDC. Although both [Ca2+]i and myosin light chain (MLC)-phosphorylation at the first phase was apparently exceeded the threshold for contraction as estimated by high K+-induced responses, there was no appreciable contraction, indicating the dissociation between MLC phosphorylation and force during this phase. In contrast, the second phase of [Ca2+]i was associated with the increases in both MLC phosphorylation and force. These results suggest that angiotensin II is a unique agonist which dissociates MLC-phosphorylation from muscle force during the Ca2+ releases from SR.

Pectenotoxin-2 (PCTX-2) is one of the polyether macrolide toxins isolated from scallops involved in diarrheic shellfish poisoning via actin depolymerization. In the present study, we examined the bioactive mechanism of PCTX-2 in smooth muscle cells and clarify mode of action of the PCTX-2-induced actin depolymerization using purified skeletal actin. PCTX-2 (300 nM-3 µM) non-selectively inhibited vascular smooth muscle contractions elicited by high K+ or phenylephrine in a dose-dependent manner. However, elevated cytosolic Ca2+ and myosin light chain phosphorylation stimulated by high K+ were only slightly inhibited by PCTX-2. By monitoring the fluorescent intensity of pyrenyl-actin, PCTX-2 was found to inhibit both the velocity and degree of actin polymerization. The critical concentration of G-actin was linearly increased in accordance with the concentration of PCTX-2, indicating sequestration of G-actin with 1 to 1 ratio. The kinetics of F-actin depolymerization by dilution assay indicated that PCTX-2 does not sever F-actin. Transmission electron microscopic and confocal microscopic observations demonstrated that PCTX-2 selectively depolymerized filamentous actin without affecting tublin. In conclusion, PCTX-2 is a potent natural actin depolymerizer which sequesters G-actin without severing F-actin.

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.

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.

Canine prostate cancer (cPCa) is an untreatable malignant neoplasm resulting in local tissue invasion and distant metastasis. MicroRNAs (miRs) are small non-coding RNAs that function as oncogenes or tumor suppressors. The purpose of this study was to characterize the expression of miRs that are altered in cPCa tissue. The expression levels of 277 mature miRs in prostatic tissue (n=5, respectively) were compared between the non-tumor and tumor groups using real-time PCR. Five miRs (miR-18a, 95, 221, 222 and 330) were up-regulated, but 14 miRs (miR-127, 148a, 205, 299, 329b, 335, 376a, 376c, 379, 380, 381, 411, 487b and 495) were down-regulated specifically in cPCa (P<0.05). These miRs have potential use as early diagnosis markers for cPCa and in miR-based therapy.

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 coil, 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; I 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 O-2 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. O-2 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 N-2 instead of O-2 or by addition of sodium cyanide (NaCN). H-65K(+)- and CCh-induced muscle contraction, involved increasing O-2 consumption. Hypoxia and NaCN significantly decreased H-65K(+)- and CCh-induced muscle contraction and/or O-2 consumption and PCr contents. Our results suggest that the contractile behavior in porcine iris sphincter highly depends on mitogen oxidative metabolism.

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 N-2 instead of O-2) or in a glucose-free condition. Hyperosmotic addition of 65 mM KCl (H-65K(+)) and 1 mu M 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.

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 mu mol/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-N-G-nitroarginine methyl ester (L-NAME, 100 mu mol/l) and by the removal of the endothelium. In the aorta with endothelium, DMSO (3%) and CCh (3 mu mol/l) increased cGMP contents, significantly and L-NAME (100 mu mol/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. (C) 2016 S. Karger AG, Basel

The effects of papaverine on carbachol (CCh) -and high K+-induced contraction in the bovine abomasum were investigated. Papaverine inhibited CCh (1 mu M) -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 mu M. Changes in the extracellular Ca2+ from 1.5 mM to 7.5 mM reduced verapamil-induced relaxation in high K+-depolarized muscles, but papaverine-induced relaxation did not change. Futhermore, papaverine (30 mu M) and NaCN (300 mu M) 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.

飢餓状態などのケトン体生成が増加する状況では肝臓での酢酸生成が増加する。特に血糖維持を糖新生に依存するネコではケトン体、酢酸生成が増加しやすいと考えられる。本研究ではネコの栄養状態および病態評価に血中酢酸濃度を利用する目的で酢酸測定法を検討し、健常ネコでの標準値を測定するとともに糖尿病ネコでの変動を調べた。健常ネコの血中酢酸濃度は 0.71 ± 0.23mM であり、雌雄間で有意な差が認められた（p<0.01）。また糖尿病 8 症例の平均は 0.91 ± 0.34mM であり、健常ネコよりも高値を示す傾向が見られた。

モルモット回腸輪走筋と縦走筋の受容体作動薬のカルバコール(CCh)およびヒスタミン(His)に対する収縮蛋白系のCa2+感受性の差異について検討した。 その結果，モルモット回腸輪走筋および縦走筋の受容体作動薬に対する感受性は縦走筋の方が高かった。β-escin処理スキンドファイバー標本の実験において，両筋の収縮要素に対するCa2+感受性に差異はなかったが，受容体作動薬に対するCa2+感受性の増加の差異はG蛋白を介した細胞内情報伝達系によるCa2+感受性の変化に関連し，縦走筋のCキナーゼの関与は輪走筋に比べ少ないことが考えられた。

Papaverine (0.3-100 mu M) more potently inhibited phenylephrine (1 mu M)-induced contraction than 65 mM K+-induced contraction of the aorta, while it equally inhibited contractions induced by 65 mM K+ and carbachol (1 mu M) in ileal smooth muscle. In phenylephrine-treated aorta, papaverine (1-10 mu M) increased the cAMP and cGMP content. However, in carbachol-treated ileum, 30 mu M papaverine partially increased the cAMP content while it maximally relaxed the preparation. In fura2-loaded aorta, papaverine (0.3-10 mu M) 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 mu M) 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. (C) 1998 Elsevier Science B.V. All rights reserved.

The effects of various selective phosphodiesterase (PDE) inhibitors on muscle contractility in guinea pig ileal longitudinal smooth muscle were investigated. 1) 3-Isobutyl-1-methyl xanthine (IBMX) or zaprinast markedly inhibited the high K+- or carbachol (CCh)-induced contraction and increased cGMP content of the muscle strip in a concentration-dependent manner. However, these agents only slightly increased the cAMP content. Milrinone or Ro20-1724 also slightly inhibited the high K+- or CCh-induced contraction and increased the cAMP content, but did not increase cGMP. 2) In a fura2-loaded muscle, IBMX or zaprinast inhibited both contractions and the increase in intracellular Ca2+ ([Ca2+](i)) level induced by high K+ or CCh, although the inhibitory effect on the [Ca2+](i) level was smaller than that on muscle tension. 3) In alpha-toxin-permeabilized muscles, cGMP, IBMX or zaprinast significantly inhibited the Ca2+-induced contraction. These results suggest that IBMX and zaprinast inhibit muscle contraction in the ileal longitudinal smooth muscles mainly through an increase in cGMP and the inhibitory mechanism of IBMX or zaprinast is involved in the decreases in the [Ca2+](i) level and sensitivity of contractile elements to 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 Ca2+-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.

A contractile propertiy of phenylephrine (PE), α1 agonist, on rat anococcygeus muscle was compared with that on rat aorta by simultaneously measuring changes in intracellular Ca2+ ([Ca2+] 1 level and muscle tension.(1) PE (0.1~30, μ M) and high K+induced a sus tained increases in [Ca2+] 1 level and muscle tension of both the muscles. (2) An application of verapamil (10μM) and EGTA (4mM) decreased the PE or high K+-increased tension and [Ca2+] 1 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+] 1 and developed tension. The developed tension by PE was greater than that by high K+at the same level of [Ca2+] 1 only in the aorta. A difference of regression slopes in the relationship between [Ca2+] 1 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 anococ cygeus muscle in Ca2+free medium elicited a small transient contractile tension and increase in [Ca2+] 1 level, but that to aorta showed a large and transient increase in both the parameters. (5) Phorbol ester, DPB (1μM), did not affect muscle tension or [Ca2+] 1 level in anococcygeus muscle, but DPB induced greater increases in aorta. (6) An application of PE (10μM) with GTP produced a left shift in the pCa-tension curve in the β-escin-permeabilized fiber of the anococcygeus muscle. In summary, it is suggested that the sustained contraction induced by PE in anococ cygeus muscle is involved with the increases in [Ca2+] 1 which is due to Ca2+influx mediated by a, receptor, but scarecely 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 Ca2+-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. © 1995, Japan Society of Smooth Muscle Research. All rights reserved.