Contraction Of Aortic Smooth Muscle Research Articles

Sevoflurane and isoflurane inhibit KCl-induced, Rho kinase-mediated, and PI3K-participated vasoconstriction in aged diabetic rat aortas

BackgroundThe mechanism of volatile anesthetics on vascular smooth muscle (VSM) contraction in the setting of diabetes mellitus (DM) remains unclear. The current study was designed to determine the effects of sevoflurane (SEVO) and isoflurane (ISO) on phosphoinositide 3-kinase (PI3K) and Rho kinase (ROCK) mediated KCl-induced vasoconstriction in aged type 2 diabetic rats.MethodsKCl-induced (60 mM) contractions were examined in endothelium-denuded aortic rings from aged T2DM Otsuka Long-Evans Tokushima Fatty (OLETF) rats (65–70 weeks old), control age-matched nondiabetic Long-Evans Tokushima Otsuka (LETO) rats and young Wistar rats (6–8 weeks old). The effects of SEVO or ISO (1–3 minimum alveolar concentration, MAC) on KCl-induced vasoconstriction, as well as those of LY294002 (PI3K inhibitor) and Y27632 (ROCK inhibitor) were measured in aortic rings from the three groups using an isometric force transducer.ResultsKCl induced rapid and continuous contraction of aortic smooth muscle in the three groups, and the contraction was more obvious in OLETF rats.SEVO and ISO inhibited KCl-induced vasoconstriction in a concentration-dependent manner and were suppressed by LY294002 (10 µM) and Y27632 (1 µM). SEVO had a stronger inhibitory effect on the aortas of young Wistar rats than ISO, especially at 2 MAC and 3 MAC (P < 0.05). In aged rats, the inhibitory effect of ISO was stronger than that of SEVO, especially OLETF rats. There was no significant difference in the effects of different concentrations of ISO on arterial contraction among the three groups (P > 0.05). The effects of 1 MAC SEVO on Wistar rats and 3 MAC SEVO on OLETF rats, however, were noticeably and significantly different (P < 0.05).Compared with the control condition, LY294002 and Y27632 had the most noticeable effect on the KCl-induced contraction of aortic rings in OLETF rats (P < 0.01).ConclusionSEVO (3 MAC), ISO (1, 2, 3 MAC), LY294002 and Y27632 have more significant inhibitory effect on the contraction of vascular smooth muscle in aged T2MD rats. The mechanism of SEVO and ISO in vascular tension in T2DM is partly due to changes in PI3K and/or Rho kinase activity.

Green Synthesis of Bioactive CuO@Fe3O4@Walnut Shell Nanocomposite Using Crataegus azarolus Var. aronia L. Extract and its Antivasoconstrictive Action on Rat’s Aortic Smooth Muscle

The antioxidant potential of Crataegus azarolus Var. aronia aqueous extract was used as reducing and stabilizing media to the green synthesis of bioactive CuO@Fe3O4@Walnut shell nanocomposite (NCs). The ability of the plant extract as bioreducing medium was proved using Fourier-transform infrared spectroscopy and ultraviolet–visible spectroscopy. While, the structural identification of synthesized CuO@Fe3O4@Walnut shell NCs was proved using the scanning electron microscopy, electron dispersive spectroscopy, elemental mapping, and point analysis. Then, the antivasoconstriction effects of CuO@Fe3O4@Walnut shell NCs (5*10−2 mg/ml) and (1*10−1 mg/ml) on rats isolated thoracic aortic smooth muscle cells were studied. Green synthesized CuO@Fe3O4@Walnut shell NCs showed effective antivasoconstriction activity against norepinephrine (NE) (1*10−9–10−4 M) induced contraction in endothelium-intact and endothelium-denuded rats aortic smooth muscle. In conclusion, the aqueous extract of C. aronia acts as potential reducing and stabilizing media to the green synthesis of bioactive CuO@Fe3O4@Walnut shell NCs and the NCs play an important role against contractile effects of NE on rats’ aortic smooth muscle.

Nimodipine inhibits intestinal and aortic smooth muscle contraction by regulating Ca2+-activated Cl− channels

Nimodipine is a clinically used dihydropyridine L-type calcium channel antagonist that effectively inhibits transmembrane Ca2+ influx following the depolarization of smooth muscle cells, but the detailed effect on smooth muscle contraction is not fully understood. Ca2+-activated Cl− channels (CaCCs) in vascular smooth muscle cells (VSMCs) may regulate vascular contractility. We found that nimodipine can inhibit transmembrane protein 16A (TMEM16A) activity in a concentration-dependent manner by cell-based fluorescence-quenching assay and short-circuit current analysis, with an IC50 value of ~5 μM. Short-circuit current analysis also showed that nimodipine prevented Ca2+-activated Cl− current in both HT-29 cells and mouse colonic epithelia accompanied by significantly decreased cytoplasmic Ca2+ concentrations. In the absence of extracellular Ca2+, nimodipine still exhibited an inhibitory effect on TMEM16A/CaCCs. Additionally, the application of nimodipine to CFTR-expressing FRT cells and mouse colonic mucosa resulted in mild activation of CFTR-mediated Cl− currents. Nimodipine inhibited basolateral CCh-activated K+ channel activity with no effect on Na+/K+-ATPase activity. Evaluation of intestinal smooth muscle contraction showed that nimodipine inhibits intestinal smooth muscle contractility and frequency, with an activity pattern that was similar to that of non-specific inhibitors of CaCCs. In aortic smooth muscle, the expression of TMEM16A in thoracic aorta is higher than that in abdominal aorta, corresponding to stronger maximum contractility in thoracic aorta smooth muscle stimulated by phenylephrine (PE) and Eact. Nimodipine completely inhibited the contraction of aortic smooth muscle stimulated by Eact, and partially inhibited the contraction stimulated by PE. In summary, the results indicate that nimodipine effectively inhibits TMEM16A/CaCCs by reduction transmembrane Ca2+ influx and directly interacting with TMEM16A, explaining the mechanisms of nimodipine relaxation of intestinal and aortic smooth muscle contraction and providing new targets for pharmacological applications.

Trimethylamine-N-oxide acutely increases cardiac muscle contractility.

Cardiovascular disease is a major cause of morbidity and mortality among patients with chronic kidney disease (CKD). Trimethylamine-N-oxide (TMAO), a uremic metabolite that is elevated in the setting of CKD, has been implicated as a nontraditional risk factor for cardiovascular disease. While association studies have linked elevated plasma levels of TMAO to adverse cardiovascular outcomes, its direct effect on cardiac and smooth muscle function remains to be fully elucidated. We hypothesized that pathological concentrations of TMAO would acutely increase cardiac and smooth muscle contractility. These effects may ultimately contribute to cardiac dysfunction during CKD. High levels of TMAO significantly increased paced, ex vivo human cardiac muscle biopsy contractility (P < 0.05). Similarly, TMAO augmented contractility in isolated mouse hearts (P < 0.05). Reverse perfusion of TMAO through the coronary arteries via a Langendorff apparatus also enhanced cardiac contractility (P < 0.05). In contrast, the precursor molecule, trimethylamine (TMA), did not alter contractility (P > 0.05). Multiphoton microscopy, used to capture changes in intracellular calcium in paced, adult mouse hearts ex vivo, showed that TMAO significantly increased intracellular calcium fluorescence (P < 0.05). Interestingly, acute administration of TMAO did not have a statistically significant influence on isolated aortic ring contractility (P > 0.05). We conclude that TMAO directly increases the force of cardiac contractility, which corresponds with TMAO-induced increases in intracellular calcium but does not acutely affect vascular smooth muscle or endothelial function of the aorta. It remains to be determined if this acute inotropic action on cardiac muscle is ultimately beneficial or harmful in the setting of CKD.NEW & NOTEWORTHY We demonstrate for the first time that elevated concentrations of TMAO acutely augment myocardial contractile force ex vivo in both murine and human cardiac tissue. To gain mechanistic insight into the processes that led to this potentiation in cardiac contraction, we used two-photon microscopy to evaluate intracellular calcium in ex vivo whole hearts loaded with the calcium indicator dye Fluo-4. Acute treatment with TMAO resulted in increased Fluo-4 fluorescence, indicating that augmented cytosolic calcium plays a role in the effects of TMAO on force production. Lastly, TMAO did not show an effect on aortic smooth muscle contraction or relaxation properties. Our results demonstrate novel, acute, and direct actions of TMAO on cardiac function and help lay the groundwork for future translational studies investigating the complex multiorgan interplay involved in cardiovascular pathogenesis during CKD.

Sibutramine facilitates apoptosis and contraction of aortic smooth muscle cells through elevating production of reactive oxygen species

Sibutramine had been prescribed as an oral anorexiant that reduces dietary intake, but was withdrawn from the market due to frequent occurrence of severe cardiovascular events including hypertension. To elucidate the pathogenic mechanism of hypertension, we here investigated whether sibutramine facilitates damage and contraction of human aortic smooth muscle (HASM) cells or not. Treatment with sibutramine provoked HASM cell apoptosis, which was attributed to production of reactive oxygen species and mitochondria dysfunction. In addition, the drug treatment of the cell promoted calcium influx, phosphorylation of myosin light chain and contraction, which were abrogated by pretreating the cells with antioxidant and nitric oxide (NO) donor. Thus, the drug-evoked contraction is likely due to a preceding disturbance of balance between the drug-elicited reactive oxygen species production and exogenous NO supply. Compared to sibutramine, its N-desmethyl and N-didesmethyl metabolites exhibited much less toxicity and contraction against HASM cells, in which sibutramine was hardly demethylated. The low metabolic capacity of the cells may also be pertinent to the damage and contraction elicited by sibutramine. Taken together, our data suggest that sibutramine facilitates apoptosis and contraction of aortic smooth muscle cells through elevating production of reactive oxygen species and decreasing exogenous NO supply, leading to pathogenesis of hypertension.

Inhibition of ICa,L by DHEA in Aortic Smooth Muscle Cells: Voltage‐dependency, Modulation by GPCR Signaling and Inhibition of Glucose‐6‐phosphate Dehydrogenase

Dehydroepiandrosterone (DHEA) is a hormone abundantly released from adrenal cortex as DHEA‐sulfate (DHEA‐S). DHEA‐S is released also in brain. DHEA and epiandrosterone (EPI), a metabolite of DHEA, produces relaxation of high‐K+ induced contraction of aortic smooth muscle (ASM). EPI increases voltage‐dependent inactivation (voltage‐dependent inhibition, VDI) of ICa,L in ventricular myocytes. Whole‐cell ICa,L was recorded as IBa from ASM cells (ASMCs) of A7r5 cell line and enzymatically isolated bovine coronary ASMCs to study mechanism and modulation of DHEA‐induced inhibition of ICa,L. DHEA produced VDI; acceleration of time course of inactivation, increase of inhibition by depolarization of holding potential (HP) and leftward shift of steady state inactivation curve; f∞, 2s‐V, obtained by 2 s conditioning pulse and f∞, HP‐V as a function HP. In addition, DHEA produced voltage‐independent inhibition (VIDI) represented by inhibition of peak amplitude (ICa,L,peak) of ICa,L elicited by pulses from hyperpolarized HPs. Window ICa,L (IWD) supplies Ca2+ in the E‐C coupling of ASM. IWD obtained from I–V of ICa,L,peak and f∞, HP‐V fitted well with recorded IWD and they were strongly inhibited by DHEA. Efficacy of DHEA‐induced VDI was reduced by rightward shift of f∞, 2s‐V curve produced by depletion of cholesterol by MβCD. Blocking of GPCR signaling by intracellular dialysis of GDP‐β‐S also shifted f∞, 2s‐V to the right and it produced decrease of DHEA‐induced inhibition of ICa,L at depolarized HP (VDI). DHEA and EPI are inhibitors of glucose‐phosphate‐dehydrogenase (G6PD), a redox‐related pentose‐phosphate pathway enzyme. 6‐Aminonicotinamide (6‐AN), another inhibitor of G6PD produced VIDI without inducing VDI. DHEA‐S that does not inhibit G6PD induced small but significant VDI at an order higher concentration of DHEA without generating VIDI. GPCR signaling and membrane cholesterol are indirectly involved in the modulation of DHEA‐induced VDI by affecting basal voltage‐dependent inactivation. The occurrence of VIDI by 6‐AN indicates that G6PD inhibition underlies DHEA‐induced VIDI. DHEA‐induced inhibition of IWD by VDI and VIDI contributes for DHEA‐induced relaxation of ASM.Support or Funding InformationNational Heart, Lung, and Blood Institute Grant RO1 HL‐085352 to S. A. Gupte.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genetic approaches to identify pathological limitations in aortic smooth muscle contraction.

Aortic smooth muscle contains limiting amounts of myosin light chain kinase (MLCK) for myosin regulatory light chain (RLC) phosphorylation and contraction that predisposes to thoracic aortic disease in humans containing heterozygous loss-of-function mutations in MYLK. We tested the hypothesis that thoracic aortic smooth muscle contraction may also be susceptible to variations in the smooth muscle-specific isoform of the motor protein myosin where inactivation of one Myh11 allele or the presence of one Myh11 missense variant associated with an increased risk of human aortic disease may result in a reduced force development response. Additionally, other kinds of smooth muscles may be less sensitive to the effects of mutations in one smooth muscle myosin allele, similar to results obtained with Mylk. Force development responses were reduced in aortic tissue from a conditional knockout of smooth muscle myosin heavy chain in adult mice (Myh11+/- or Myh11-/-) with a greater reduction with homozygous vs heterozygous tissues. Similar reductions in force responses were obtained with tissues containing either a heterozygous or homozygous knockin mutation in smooth muscle myosin heavy chain (Myh11+/R247C or Myh11R247C/R247C mutations that cause human aortic disease) with no significant changes in RLC phosphorylation. Agonist-dependent force responses were not reduced significantly in urinary bladder, ileal, or tracheal tissues from Myh11+/- mice while only ileal tissue showed a reduced force response in Myh11R247C/R247C mice. Thus, heterozygous mutations in Myh11 associated with reduced myosin function result in compromised contractile function primarily in aortic smooth muscle.

The Role of Tyrosine Kinase in Ca^(2+)-Independent Contraction of the Ropivacaine on Rat Aortic Smooth Muscle

The tyrosine kinase signaling pathway plays an important role in the mediation of Ca²⁺ independent mechanisms of smooth muscle contraction. Several components of this pathway, including protein kinase C (PKC), p44/42 mitogen-activated protein kinase (p44/42 MAPK) and Rho-kinase are involved in Ca²⁺ independent mechanisms. Whether the tyrosine kinase pathway mediates vasoconstriction induced by the anesthetic ropivacaine remains unclear. The present study was designed to examine the role of tyrosine kinase in ropivacaine-induced, Ca²⁺-independent contraction of rat aortic smooth muscle. The effects of tyrosine kinase inhibitor on ropivacaine-induced contractile response were observed by isometric force measurement. The protein tyrosine phosphorylation, PKC, p44/42 MAPK, and membrane translocation of Rho-kinase were examined by Western blotting. Ropivacaine induced a concentration-dependent contractile response, and showed a number of effects on protein tyrosine phosphorylation. In this study, phosphorylation levels were shown to increase at lower concentrations of ropivacaine, but the levels decline at higher concentrations in rat aortic rings attenuated by the tyrosine kinase inhibitor genistein in a concentration-dependent fashion. Ropivacaine-induced phosphorylation of PKC and p44/42 MAPK and Rho-kinase membrane translocation were also significantly attenuated by genistein in similar decreasing manner as the PKC inhibitor bisindolylmaleimide I (Bis I) and the Rho-kinase inhibitor, Y27632, but to a lesser degree than that by the p44/42 MAPK inhibitor, PD 098059. Our results showed that the ropivacaine-induced, Ca²⁺ independent-mediated contraction of rat aortic smooth muscle is, in part, regulated by tyrosine kinase-catalyzed protein tyrosine phosphorylation.

Signaling through Myosin Light Chain Kinase in Smooth Muscles

Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates smooth muscle myosin regulatory light chain (RLC) to initiate contraction. We used a tamoxifen-activated, smooth muscle-specific inactivation of MLCK expression in adult mice to determine whether MLCK was differentially limiting in distinct smooth muscles. A 50% decrease in MLCK in urinary bladder smooth muscle had no effect on RLC phosphorylation or on contractile responses, whereas an 80% decrease resulted in only a 20% decrease in RLC phosphorylation and contractile responses to the muscarinic agonist carbachol. Phosphorylation of the myosin light chain phosphatase regulatory subunit MYPT1 at Thr-696 and Thr-853 and the inhibitor protein CPI-17 were also stimulated with carbachol. These results are consistent with the previous findings that activation of a small fraction of MLCK by limiting amounts of free Ca(2+)/calmodulin combined with myosin light chain phosphatase inhibition is sufficient for robust RLC phosphorylation and contractile responses in bladder smooth muscle. In contrast, a 50% decrease in MLCK in aortic smooth muscle resulted in 40% inhibition of RLC phosphorylation and aorta contractile responses, whereas a 90% decrease profoundly inhibited both responses. Thus, MLCK content is limiting for contraction in aortic smooth muscle. Phosphorylation of CPI-17 and MYPT1 at Thr-696 and Thr-853 were also stimulated with phenylephrine but significantly less than in bladder tissue. These results indicate differential contributions of MLCK to signaling. Limiting MLCK activity combined with modest Ca(2+) sensitization responses provide insights into how haploinsufficiency of MLCK may result in contractile dysfunction in vivo, leading to dissections of human thoracic aorta.

Pravastatin normalizes ET-1-induced contraction in the aorta of type 2 diabetic OLETF rats by suppressing the KSR1/ERK complex

Endothelin (ET)-1 is a likely candidate for a key role in diabetic vascular complications. In the present study, we hypothesized that treatment with pravastatin (an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase) would normalize the ET-1-induced contraction in aortas isolated from type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats. Contractile responses were examined by measuring isometric force in endothelium-denuded aortic helical strips from four groups: Long-Evans Tokushima Otsuka (LETO; genetic control), OLETF (type 2 diabetic), pravastatin-treated LETO, and pravastatin-treated OLETF rats. Both immunoblot analysis and immunoprecipitation assays were used to examine Src, protein phosphatase (PP)2A, kinase suppressor of Ras (KSR)1, and ERK signaling pathway protein levels and activities. In endothelium-denuded aortas isolated from OLETF rats at the chronic stage of diabetes (56-60 wk) (vs. those from age-matched LETO rats), we found the following: 1) ET-1-induced contraction was enhanced, 2) ERK1/2 phosphorylation was increased, 3) phosphorylations of KSR1 and PP2A were reduced (i.e., enhancement of the kinase active state), 4) ERK1/2-KSR1 complexes were increased, and 5) Src tyrosine kinase activity was diminished. Endothelium-denuded aortas isolated from OLETF rats treated with pravastatin (10 mg/kg po, daily for 4 wk) exhibited normalized ET-1-induced contractions and suppressed ET-1-stimulated ERK phosphorylation, with the associated phosphorylated KSR1 and phosphorylated PP2A levels being increased toward normal levels. These results suggest that in type 2 diabetic rats, pravastatin normalizes ET-1-induced contraction in aortic smooth muscle via a suppression of PP2A/KSR1/ERK activities after an enhancement of Src kinase activity.

Propofol attenuates angiotensin II-induced vasoconstriction by inhibiting Ca2+-dependent and PKC-mediated Ca2+ sensitization mechanisms

Angiotensin II (Ang II)-induced vascular contraction is mediated by Ca(2+)-dependent mechanisms and Ca(2+) sensitization mechanisms. The phosphorylation of protein kinase C (PKC) regulates myofilament Ca(2+) sensitivity. We have previously demonstrated that sevoflurane inhibits Ang II-induced vasoconstriction by inhibiting PKC phosphorylation, whereas isoflurane inhibits Ang II-induced vasoconstriction by decreasing intracellular Ca(2+) concentration ([Ca(2+)](i)) in vascular smooth muscle. Propofol also induces vasodilation; however, the effect of propofol on PKC-mediated myofilament Ca(2+) sensitivity is poorly understood. The aim of this study is to determine the mechanisms by which propofol inhibits Ang II-induced vascular contraction in rat aortic smooth muscle. An isometric force transducer was used to investigate the effect of propofol on vasoconstriction, a fluorometer was used to investigate the change in [Ca(2+)](i), and Western blot testing was used to analyze Ang II-induced PKC phosphorylation. Ang II (10(-7) M) elicited a transient contraction of rat aortic smooth muscle, which was associated with an elevation of [Ca(2+)](i). Propofol (10(-6 )M) inhibited Ang II-induced vascular contraction (P < 0.01) and increase in [Ca(2+)](i) (P < 0.05) in rat aortic smooth muscle. Ang II also induced a rapid increase in [Ca(2+)](i) in cultured vascular smooth muscle cells, which was suppressed by propofol (P < 0.05). Propofol (10(-6) M) attenuated Ang II-stimulated PKC phosphorylation (P < 0.05). These results suggest that the inhibitory effect of propofol on Ang II-induced vascular contraction is mediated by the attenuation of a Ca(2+)-dependent pathway and Ca(2+) sensitivity through the PKC signaling pathway.

Levobupivacaine-induced contraction of isolated rat aorta is calcium dependent

Levobupivacaine is a long-acting local anesthetic that intrinsically produces vasoconstriction in isolated vessels. The goals of this study were to investigate the calcium-dependent mechanism underlying levobupivacaine-induced contraction of isolated rat aorta in vitro and to elucidate the pathway responsible for the endothelium-dependent attenuation of levobupivacaine-induced contraction. Isolated rat aortic rings were suspended to record isometric tension. Cumulative levobupivacaine concentration-response curves were generated in either the presence or absence of the antagonists verapamil, nifedipine, SKF-96365, 2-aminoethoxydiphenylborate, Gd(3+), N(W)-nitro-l-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and methylene blue, either alone or in combination. Verapamil, nifedipine, SKF-96365, 2-aminoethoxydiphenylborate, low calcium concentrations, and calcium-free Krebs solution attenuated levobupivacaine-induced contraction. Gd(3+) had no effect on levobupivacaine-induced contraction. Levobupivacaine increased intracellular calcium levels in vascular smooth muscle cells. L-NAME, ODQ, and methylene blue increased levobupivacaine-induced contraction in endothelium-intact aorta. SKF-96365 attenuated calcium-induced contraction in a previously calcium-free isotonic depolarizing solution containing 100mmol/L KCl. Levobupivacaine-induced contraction of rat aortic smooth muscle is mediated primarily by calcium influx from the extracellular space mainly via voltage-operated calcium channels and, in part, by inositol 1,4,5-trisphosphate receptor-mediated release of calcium from the sarcoplasmic reticulum. The nitric oxide - cyclic guanosine monophosphate pathway is involved in the endothelium-dependent attenuation of levobupivacaine-induced contraction.

E0708 IGFBP-7 contributes to vascular remolding during hypertension development in ouabain-induced hypertensive rats

PurposesThe aim of this study was to elucidate the potential new targets for vascular remolding with hypertension.Methods and ResultsIn this study, we established the ouabain-induced hypertensive SD rats (OHR), the...

The Direct Effect of Levobupivacaine in Isolated Rat Aorta Involves Lipoxygenase Pathway Activation and Endothelial Nitric Oxide Release

Levobupivacaine is a long-acting local anesthetic with a clinical profile similar to that of racemic bupivacaine but with a greater margin of safety. Levobupivacaine produces dose-dependent vasoconstriction in vivo. Our goal in this in vitro study was to investigate the role of pathways involved in arachidonic acid metabolism in the levobupivacaine-induced contraction of isolated rat aorta and to determine which endothelium-derived vasodilators are involved in the modulation of levobupivacaine-induced contraction. Rat thoracic aortic rings were isolated and suspended for isometric tension recording. Cumulative levobupivacaine dose-response curves over a range of 10(-6) to 3 x 10(-4) M were constructed in 1) aortic rings with no drug pretreatment; 2) endothelium-denuded rings pretreated with quinacrine dihydrochloride (nonspecific phospholipase A(2) inhibitor: 2 x 10(-5), 4 x 10(-5) M), nordihydroguaiaretic acid (NDGA) (lipoxygenase inhibitor: 10(-5), 3 x 10(-5) M), indomethacin (nonspecific cyclooxygenase inhibitor: 10(-5) M), AA-861 (5-lipoxygenase inhibitor: 10(-5), 5 x 10(-5) M), fluconazole (cytochrome P450 epoxygenase inhibitor: 10(-5) M), verapamil (10(-5) M), or calcium-free solution; and 3) endothelium-intact rings pretreated with N(omega)-nitro-L-arginine methyl ester (L-NAME) (nitric oxide synthase inhibitor: 5 x 10(-5) M), indomethacin, or fluconazole. Levobupivacaine-induced contractile response at each concentration (10(-4), 3 x 10(-4) M) was assessed in endothelium-denuded rings. Dose-response curves for potassium chloride in endothelium-denuded rings were generated in the presence or absence of NDGA and AA-861. Intracellular Ca(2+) levels were monitored by Ca(2+) image analysis using Fluo-4 fluorescence in vascular smooth muscle cells treated with levobupivacaine alone or AA-861 plus levobupivacaine. Levobupivacaine produced a tonic contraction in isolated rat aorta rings; this response was maximal at 10(-4) M levobupivacaine and gradually attenuated at 3 x 10(-4) M levobupivacaine. Levobupivacaine-induced contractions of endothelium-denuded rings were larger than those of endothelium-intact rings. Levobupivacaine-induced contraction of endothelium-denuded rings was attenuated by quinacrine dihydrochloride, NDGA, AA-861, verapamil, and calcium-free solution and, to a lesser extent, by indomethacin. L-NAME enhanced levobupivacaine-induced contraction of endothelium-intact rings and indomethacin slightly attenuated this contraction. NDGA and AA-861 attenuated the potassium chloride-induced contraction. AA-861 attenuated the levobupivacaine-induced intracellular calcium increase in vascular smooth muscle cells. Our data indicate that levobupivacaine-induced contraction of rat aortic smooth muscle is mediated mainly by activation of the lipoxygenase pathway and in part by activation of the cyclooxygenase pathway. In addition, activation of the lipoxygenase pathway seems to facilitate calcium influx via L-type calcium channels. Endothelial nitric oxide attenuates levobupivacaine-induced contraction.

Diminished expression of dihydropteridine reductase is a potent biomarker for hypertensive vessels

To identify the new targets for hypertension, we analyzed the protein expression profiles of aortic smooth muscle in spontaneously hypertensive rats (SHR) of various ages during the development of hypertension, as well as in age-matched normotensive Wistar-Kyoto (WKY) rats, using a proteomic analysis. The expressions of seven proteins were altered in SHR compared with WKY rats. Of these proteins, NADH dehydrogenase 1alpha, GSTomega1, peroxi-redoxin I and transgelin were upregulated in SHR compared with WKY rats. On the other hand, the expression of HSP27 and Ran protein decreased in SHR. The diminution of dihydrobiopterin reductase, an enzyme located in the regeneration pathways of tetrahydrobiopterin (BH4), was also prominent in SHR. The results from a PCR analysis revealed that the expression of BH4 biosynthesis enzymes - GTP cyclohydrolase-1 and sepiapterin reductase - decreased and increased, respectively, in SHR compared with WKY rats. The level of BH4 was less in aortic strips from SHR than from WKY rats. Moreover, treatment with BH4 inhibited aortic smooth muscle contraction induced by serotonin. These results suggest that the deficiency in BH4 regeneration produced by diminished dihydrobiopterin reductase expression is involved in vascular disorders in hypertensive rats.

Volatile Anesthetics Inhibit Angiotensin II-Induced Vascular Contraction by Modulating Myosin Light Chain Phosphatase Inhibiting Protein, CPI-17 and Regulatory Subunit, MYPT1 Phosphorylation

Vascular contraction is regulated by myosin light chain (MLC) phosphorylation. Inhibition of MLC phosphatase (MLCP) increases MLC phosphorylation for a given Ca(2+) concentration, and results in promoting myofilament Ca(2+) sensitivity. MLCP activity is mainly determined by protein kinase C (PKC) and Rho kinase through the phosphorylation of both PKC-potentiated inhibitory protein (CPI-17) and myosin phosphatase target subunit (MYPT1). We have previously demonstrated that sevoflurane inhibits PKC phosphorylation and membrane translocation of Rho kinase. This study was designed to investigate the effects of sevoflurane and isoflurane on CPI-17, MYPT1, and MLC phosphorylation in response to angiotensin II (Ang II) in rat aortic smooth muscle. The effects of sevoflurane or isoflurane (1-3 minimum alveolar concentration) on the vasoconstriction and phosphorylation of MLC, CPI-17, MYPT1 at Thr853 and MYPT1 at Thr696 in response to Ang II were investigated using isometric force transducer and Western blotting, respectively. Ang II (10(-7) M) elicited a transient contraction of rat aortic smooth muscle that was inhibited by both sevoflurane and isoflurane in a concentration-dependent manner. Ang II also induced an increase in the phosphorylation of MLC, CPI-17, MYPT1/Thr853 and MYPT1/Thr696. Sevoflurane inhibited the phosphorylation of MLC, CPI-17, and MYPT1/Thr853 in response to Ang II in a concentration-dependent manner. Isoflurane also inhibited MLC phosphorylation in response to Ang II, which was associated with decreases in MYPT1/Thr853, but not in CPI-17. Neither sevoflurane nor isoflurane affected the Ang II-induced phosphorylation of MYPT1/Thr696. Although both volatile anesthetics inhibited Ang II-induced vasoconstriction and MLC phosphorylation to similar extent, the mechanisms behind the inhibitory effects of each anesthetic on MLCP activity appear to differ.

FRET analysis of actin–myosin interaction in contracting rat aortic smooth muscle

We examined the interaction of smooth muscle myosin with alpha-actin and beta-actin isoforms during the contraction of A7r5 smooth muscle cells and rat aortic smooth muscle. The techniques of confocal microscopy and fluorescence resonance energy transfer (FRET) analysis were utilized in examining A7r5 cells and rat aortic rings contracted with phorbol 12,13-dibutyrate. Visual evaluation of confocal images of A7r5 smooth muscle cells contracted by phorbol 12,13-dibutyrate indicated significant disassociation of myosin from alpha-actin but not beta-actin. Whole-cell FRET analysis confirmed these observations (alpha-actin-myosin -67%, beta-actin-myosin -2%). Time course studies further showed that alpha-actin-myosin complex increased significantly (40%) within 1.5 min after the addition of phorbol 12,13-dibutyrate and then declined as contraction progressed. FRET analysis of rat aortic rings at different intervals of contraction indicated significant increases in alpha-actin-myosin at the initiation (79%) and plateau (67%) in force development, but not during the intermediate period of slowly developing tension (-4%). By comparison, beta-actin-myosin complex was unchanged except during slow force development, in which the association was significantly decreased (-30%). Similar to that of alpha-actin-myosin, Alexa 488 - phalloidin staining fluorescence indicated increased tissue F-actin content at the initiation (21%) and plateau (62%) in force. FRET images indicated the development of thickened cables and patches of alpha-actin-myosin in tissue throughout the interval of contraction. The results provide direct evidence of dynamic remodeling of the contractile protein during vascular smooth muscle contraction and suggest that FRET analysis may be a powerful tool for assessment of tissue protein-protein associations.

Purification and characterization of a cysteine-rich secretory protein from Philodryas patagoniensis snake venom

Cysteine-rich secretory proteins (CRiSPs) are widespread in reptile venoms, but most have functions that remain unknown. In the present study we describe the purification and characterization of a CRiSP (patagonin) from the venom of the rear-fanged snake Philodryas patagoniensis, and demonstrate its biological activity. Patagonin is a single-chain protein, exhibiting a molecular mass of 24,858.6 Da, whose NH 2-terminal and MS/MS-derived sequences are nearly identical to other snake venom CRiSPs. The purified protein hydrolyzed neither azocasein nor fibrinogen, and it could induce no edema, hemorrhage or inhibition of platelet adhesion and aggregation. In addition, patagonin did not inhibit contractions of rat aortic smooth muscle induced by high K +. However, it caused muscular damage to murine gastrocnemius muscle, an action that has not been previously described for any snake venom CRiSPs. Thus, patagonin will be important for studies of the structure-function and evolutionary relationships of this family of proteins that are widely distributed among snake venoms.

Un effet direct de la ropivacaïne implique l’activation des voies de la lipoxygénase dans le muscle lisse de l’aorte du rat

Ropivacaine is a long-acting amino-amide local anesthetic that induces vasoconstriction in vitro and in vivo. The aim of this study was to investigate the pathways involved in arachidonic acid metabolism associated with S-ropivacaine-induced contraction of rat aortic smooth muscle in vitro. Rat thoracic aortic rings without endothelium were isolated and suspended for isometric tension recording. Cumulative dose-response curves were generated with concentrations of 10(-5) to 10(-3) M ropivacaine enantiomer in the presence or absence of quinacrine dihydrochloride, nordihydroguaiaretic acid, quinacrine dihydrochloride plus nordihydroguaiaretic acid, indomethacin, fluconazole, AA-861, and verapamil. The maximal S-ropivacaine-induced contractile response achieved at 3x10(-4) M was also assessed in aortic rings pretreated with normal or calcium-free Krebs solution. Ropivacaine enantiomers induced dose-dependent biphasic contractions in aortic rings. S-ropivacaine (10(-4), 3x10(-4) M) induced a stronger contraction than R-ropivacaine. Quinacrine dihydrochloride (2x10(-5), 4x10(-5) M) attenuated the S-ropivacaine-induced biphasic contraction in a dose-dependent manner. Indomethacin (3x10(-5), 6x10(-5) M), nordihydroguaiaretic acid (10(-5) M), and AA-861 (10(-5) M) also attenuated the S-ropivacaine-induced dose-dependent biphasic contraction, whereas fluconazole (3x10(-5)) had no effect. Combined pretreatment with quinacrine dihydrochloride and nordihydroguaiaretic acid almost completely abolished the S-ropivacaine-induced contraction. S-ropivacaine-induced contractile responses were attenuated by verapamil (10(-5) M) and calcium-free Krebs solution. S-ropivacaine induces dose-dependent biphasic contractions in rat aortic smooth muscle through a mechanism requiring extracellular calcium that is mediated by activation of the lipoxygenase pathway and, to a lesser extent, the cyclooxygenase pathway.

Inhibition of rat aortic smooth muscle contraction by 2-methoxyestradiol

Recent studies suggest that 2-methoxyestradiol (2-ME), an estrogen metabolite, has a similar inhibitory effect as 17beta-estradiol (E2) on vascular tone. However, it is not known whether 2-ME mediates the effects of E2 or by what mechanism 2-ME regulates smooth muscle contraction. Therefore, we compared the effects of 2-ME and E2 on rat aortic smooth muscle contraction. A preincubation with 2-ME (10 microM) for 1 h inhibited phenylephrine (PE)-induced tension in endothelium-intact, but not -denuded, tissues, whereas E2 inhibited PE-induced contraction in both preparations. The effects of 2-ME and E2 on endothelium-intact preparations were prevented by L-NAME hydrochloride (a nitric oxide synthase inhibitor). The 2-ME treatment reduced PE-induced phosphorylation of the 20-kDa myosin regulatory light chain. The inhibitory effects of 2-ME and E2 were not affected by ICI-182780 (an estrogen receptor antagonist) or actinomycin D (a gene transcription inhibitor); however, the effect of 2-ME, but not E2, was prevented by cycloheximide (a protein synthesis inhibitor). Furthermore, the effect of E2 was not blocked by 1-aminobenzotriazole (a cytochrome P-450 inhibitor) or Ro 41-0960 (a catechol-O-methyltransferase inhibitor). The effect of 2-ME was not mimicked by microtubule-interfering agents (nocodazole or Taxol). We conclude that 2-ME inhibits smooth muscle contractility through an endothelium- and nitric oxide-dependent mechanism, which does not involve estrogen receptors or microtubule disruption. The effect of 2-ME, but not E2, involves de novo protein synthesis. 2-ME does not mediate the inhibitory effect of E2 on smooth muscle contraction. These results support a potentially important role of 2-ME in the regulation of smooth muscle tone in the vasculature.