Calcium-induced Contractions Research Articles

Hypothermia Inhibits Dexmedetomidine-Induced Contractions in Isolated Rat Aortae.

Dexmedetomidine is widely used to induce sedation in the perioperative period. This study examined the effect of hypothermia (33 and 25 °C) on dexmedetomidine-induced contraction in an endothelium-intact aorta with or without the nitric oxide synthase inhibitor NW-nitro-L-arginine methyl ester (L-NAME). In addition, the effect of hypothermia on the contraction induced by dexmedetomidine in an endothelium-denuded aorta with or without a calcium-free Krebs solution was examined. The effects of hypothermia on the protein kinase C (PKC), myosin light chain (MLC20) phosphorylation, and Rho-kinase membrane translocation induced by dexmedetomidine were examined. Hypothermia inhibited dexmedetomidine-induced contraction in the endothelium-intact aorta with L-NAME or endothelium-denuded aorta. Hypothermia had almost no effect on the dexmedetomidine-induced contraction in the endothelium-denuded aorta with the calcium-free Krebs solution; however, the subsequent contraction induced by the addition of calcium was inhibited by hypothermia. Conversely, the transition from profound hypothermia back to normothermia reversed the hypothermia-induced inhibition of subsequent calcium-induced contractions. Hypothermia inhibited any contraction induced by KCl, PDBu, and NaF, as well as PKC and MLC20 phosphorylation and Rho-kinase membrane translocation induced by dexmedetomidine. These results suggest that hypothermia inhibits dexmedetomidine-induced contraction, which is mediated mainly by the impediment of calcium influx and partially by the attenuation of pathways involving PKC and Rho-kinase activation.

A subfraction obtained from the venom of the tarantula Poecilotheria regalis contains inhibitor cystine knot peptides and induces relaxation of rat aorta by inhibiting L-type voltage-gated calcium channels.

Venoms from tarantulas contain low molecular weight vasodilatory compounds whose biological action is conceived as part of the envenomation strategy due to its propagative effects. However, some properties of venom-induced vasodilation do not match those described by such compounds, suggesting that other toxins may cooperate with these ones to produce the observed biological effect. Owing to the distribution and function of voltage-gated ion channels in blood vessels, disulfide-rich peptides isolated from venoms of tarantulas could be conceived into potential vasodilatory compounds. However, only two peptides isolated from spider venoms have been investigated so far. This study describes for the first time a subfraction containing inhibitor cystine knot peptides, PrFr-I, obtained from the venom of the tarantula Poecilotheria regalis. This subfraction induced sustained vasodilation in rat aortic rings independent of vascular endothelium and endothelial ion channels. Furthermore, PrFr-I decreased calcium-induced contraction of rat aortic segments and reduced extracellular calcium influx to chromaffin cells by the blockade of L-type voltage-gated calcium channels. This mechanism was unrelated to the activation of potassium channels from vascular smooth muscle, since vasodilation was not affected in the presence of TEA, and PrFr-I did not modify the conductance of the voltage-gated potassium channel Kv10.1. This work proposes a new envenomating function of peptides from venoms of tarantulas, and establishes a new mechanism for venom-induced vasodilation.

Tas2R activation relaxes airway smooth muscle by release of Gαt targeting on AChR signaling

Both chronic obstructive pulmonary disease (COPD) and asthma are severe respiratory diseases. Bitter receptor-mediated bronchodilation is a potential therapy for asthma, but the mechanism underlying the agonistic relaxation of airway smooth muscle (ASM) is not well defined. By exploring the ASM relaxation mechanism of bitter substances, we observed that pretreatment with the bitter substances nearly abolished the methacholine (MCh)-induced increase in the ASM cell (ASMC) calcium concentration, thereby suppressing the calcium-induced contraction release. The ASM relaxation was significantly inhibited by simultaneous deletion of three Gαt proteins, suggesting an interaction between Tas2R and AChR signaling cascades in the relaxation process. Biochemically, the Gαt released by Tas2R activation complexes with AChR and blocks the Gαq cycling of AChR signal transduction. More importantly, a bitter substance, kudinoside A, not only attenuates airway constriction but also significantly inhibits pulmonary inflammation and tissue remodeling in COPD rats, indicating its modulation of additional Gαq-associated pathological processes. Thus, our results suggest that Tas2R activation may be an ideal strategy for halting multiple pathological processes of COPD.

An analytical review on the quantitative techniques for estimation of cilostazol in pharmaceutical preparations and biological samples

AbstractCilostazol (CLZ) is a quinolinone derivative possessing anti platelet and vasodilating properties and it is used in the treatment of intermittent claudication. It operates by inhibiting the cyclic guanosine monophosphate (cGMP) dependent phosphodiesterase III which leads to availability of cyclic adenosine monophosphate (cAMP) in blood vessels and platelets owing to vasodilation and reduced calcium-induced contractions. This article illustrates the various reported quantitative methods which can be used to determine cilostazol and its metabolites either alone or in combination forms in pharmaceutical preparation or biological matrices like plasma, serum, and urine. The review covers analytical methods like ultraviolet spectroscopy, fluorescence spectroscopy, electrometric methods, nuclear magnetic resonance spectroscopy, high performance liquid chromatography, high performance thin layer chromatography, ultra-high performance liquid chromatography, and tandem mass spectroscopy from the year 1985 to 2019 with a brief explanation on every analytical method. Among the methods, it was found that most researchers opted for UV and HPLC analytical methods for the estimation of cilostazol.

Endothelium-independent vasorelaxant effect of Asphodelus tenuifolius Cav. via inhibition of myosin light chain kinase activity in the porcine coronary artery

Ethnopharmacological relevanceAsphodelus tenuifolius Cav. (Asphodelaceae), a wild, terrestrial, annual stemless herb, is widely used in traditional medicine for the treatment of hypertension, diabetes, atherosclerosis and circulatory problems. A previous research study from our laboratory revealed that A. tenuifolius has beneficial effects in reducing blood pressure and improves aortic endothelial dysfunction in chronically glucose fed rats. Despite the fact that A. tenuifolius reduces blood pressure and improves endothelial function in vivo, there are no detailed studies about its possible mechanism of action. Aim of the studyThis study was designed to provide pharmacological basis and mechanism of action for the traditional use of A. tenuifolius in hypertension and circulatory problems. We explored the vasorelaxant effect of A. tenuifolius and its underlying vasorelaxation mechanism in porcine coronary artery rings. Materials and methodsAqueous methanolic crude extract of A. tenuifolius was prepared by maceration process and then activity guided fractionation was carried out by using different polarity based solvents. Phytochemical studies were carried out using LC-DAD-MS. Segments of porcine distal coronary artery were set up in a wire myograph for isometric force measurements. Extract/fractions of A. tenuifolius seeds were tested for vasodilator activity by measurement of changes in tone after pre-contraction with the thromboxane mimetic U46619 in the presence or absence of inhibitors of intracellular signaling cascades. ResultsCrude extract/fractions of A. tenuifolius produced dose dependent endothelium independent vasorelaxant response in coronary rings, whereas, the butanol fraction of A. tenuifolius (BS-AT) produced the largest relaxation response with 100% relaxation at 1 mg/ml, therefore the mechanism of relaxation of this fraction was determined. The relaxation to BS-AT was unaffected by removal of the endothelium, pre-contraction with KCl, or the presence of the non-selective potassium channel blocker tetraethylammonium, indicating that the relaxation was endothelium-independent, and does not involve activation of potassium channels. BS-AT (1 mg/ml) inhibited the contractile response to calcium,the L-type calcium channel activator BAY K8664,and ionomycin, indicating that it inhibits calcium-induced contractions. The relaxation response to BS-AT was attenuated in the absence of extracellular calcium. However, relaxations to BS-AT were also reduced after deletion of calcium from intracellular stores with cyclopiazonic acid. Incubation with 1 mg/ml BS-AT also inhibited phosphorylation of myosin light chains in homogenates of coronary artery. ConclusionThe butanol extract of Asphodelus tenuifolius produces a large endothelium-independent relaxation of the porcine coronary artery through inhibition of calcium-induced contractions. The effect appears to be downstream of calcium influx, possibly through inhibition of myosin light chain kinase. This study supports previous studies demonstrating that A. tenuifolius reduces blood pressure. Future studies will aim to determine the active compounds underlying this response.

Mechanism underlying the vasodilation induced by diosmetin in porcine coronary artery

Diosmetin is a flavonoid present naturally in citrus fruit. Plants containing diosmetin have been reported to have anti-hypertensive and vasorelaxant effects. Therefore, experiments were carried out to study the effects of diosmetin in segments of the porcine coronary artery (PCA).PCA rings were mounted for isometric tension recording in isolated tissue baths and pre-contracted with the thromboxane A2 mimetic U46619 or KCl. Cumulative concentration response curves to diosmetin were then carried out in the presence or absence of inhibitors or activators of different signaling pathways. The effect on calcium channels was determined by investigating the effect of a single concentration of diosmetin (30 μM) on calcium-induced contractions or contractions to BAY K8644.Diosmetin caused a concentration-dependent relaxation after pre-contraction with U46619 or KCl, which was unaffected by removal of the endothelium. Tetraethylammonium (TEA), and 4-aminopyridine (4-AP), but not barium chloride, caused significant inhibition of the diosmetin-mediated vasorelaxation, indicating a role for potassium channels. Diosmetin inhibited calcium-induced contractions and contractions to the L-type calcium channel opener BAY K8644. Furthermore, diosmetin inhibited the contractions in response to caffeine, cyclopiazonic acid and ionomycin, indicating a general effect on calcium-induced contractions. Contractions in response to the protein kinase C (PKC) activator Phorbol 12-myristate 13-acetate (PMA) were also inhibited by diosmetin, suggesting that it may inhibit a calcium-activated PKC isoform.In summary, diosmetin produced significant vasodilatory effects. The data indicate a role for potassium channels as well as an effect on calcium-induced contractile pathways, possible through inhibition of PKC.

Mechanisms mediating the vasodilatory effects of juglone in porcine isolated coronary artery

Juglone (5-hydroxy-1, 4-naphthoquinone), is a natural phenolic compound that has been shown to relax smooth muscle. Therefore the aim of this study was to determine the effect of juglone on vascular tone using porcine coronary artery (PCA). Segments of PCA, with or without endothelium, were mounted for isometric tension recording in isolated tissue baths and precontracted with the thromboxane A2 analog U46619 or KCl. After pre-contraction, cumulative concentrations of juglone were added to the tissues, in the presence or absence of a variety of inhibitors on intracellular signaling pathways. Juglone (10-9 to 10-5 M) produced a concentration-dependent relaxation of the PCA which was reduced in endothelium-denuded vessels, as well as in vessels pre-treated with the nitric oxide synthase inhibitor L-NAME, indicating that at least part of the effect of juglone is mediated through an endothelium, NO-dependent mechanism. Juglone also inhibited contractions in response to influx of extracellular calcium and release of intracellular calcium, indicating that juglone may inhibit a common signaling pathway downstream of calcium. Contractions to the protein kinase C activator Phorbol 12-myristate 13-acetate were also reduced by juglone, suggesting that juglone might be acting through inhibition of protein kinase C. In summary, juglone produces a relaxation of the porcine coronary artery through activation of the nitric oxide pathway and inhibition of calcium-induced contractions.

Furanoeremophilanes from Euryops arabicus alleviate metabolic syndrome-associated exaggerated vasoconstriction via direct vasodilatation

• Euryops arabicus alleviated MetS associated exaggerated vasoconstriction. • The chloroform fraction yielded eight furanoeremophilane derivatives. • All isolated compounds showed significant vasodilation activity. • Vasodilation activity may involve calcium channel blocking mechanism. Abstract Exaggerated vasoconstriction plays a critical role in the development of hypertension in metabolic syndrome (MetS). A crude methanolic extract of Euryops arabicus (EA) was found to alleviate MetS-associated exaggerated vasoconstriction. The methanolic extract of EA had a direct vasodilatory effect which might account for the alleviation of vasoconstriction. Bio-guided fractionation revealed that the chloroform fraction is responsible for this effect. The observed vasodilation was not affected by Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), a nitric oxide (NO) synthase inhibitor or methylene blue (MB), a guanylate cyclase inhibitor, while it was increased by tetraethylammonium (TEA), a potassium channel blocker. In addition, the chloroform extract showed concentration-dependent inhibition of calcium-induced contraction. Chemical investigations of the chloroform fraction of EA revealed three new furanoeremophilanes, namely, 6-β-(2`-methyl-2`,3`-epoxy-propionyloxy)-10-α-H-9-oxofuranoeremophilane ( 4 ), 6- α -(2`-methyl, 2`,3`-epoxy-butyryloxy)-10-α-H-9-oxofuranoeremophilane ( 7 ), and 6-β-(2`-methyl, 2`,3`-epoxy-butyryloxy)-euryopsin ( 8 ), in addition to five other known metabolites, namely, 6-isovaleryloxy-euryopsin ( 1) , 6-isopropionyl-euryopsin (adenostylone) ( 2 ), 6-senecioyl-euryopsin-9-one ( 3 ) 6-β -(2`-methyl-2`,3`-epoxy-butyryloxy)-10-α-H-9-oxofuranoeremophilane ( 5 ), and euryopsonol-senecioate ( 6 ). All the isolated compounds showed significant vasodilatory effects that might involve calcium channel blocking, and compounds 7 and 8 were found to be the most bioactive ones.

A simple mechanochemical model for calcium signalling in embryonic epithelial cells

Calcium signalling is one of the most important mechanisms of information propagation in the body. In embryogenesis the interplay between calcium signalling and mechanical forces is critical to the healthy development of an embryo but poorly understood. Several types of embryonic cells exhibit calcium-induced contractions and many experiments indicate that calcium signals and contractions are coupled via a two-way mechanochemical feedback mechanism. We present a new analysis of experimental data that supports the existence of this coupling during apical constriction. We then propose a simple mechanochemical model, building on early models that couple calcium dynamics to the cell mechanics and we replace the hypothetical bistable calcium release with modern, experimentally validated calcium dynamics. We assume that the cell is a linear, viscoelastic material and we model the calcium-induced contraction stress with a Hill function, i.e. saturating at high calcium levels. We also express, for the first time, the “stretch-activation” calcium flux in the early mechanochemical models as a bottom-up contribution from stretch-sensitive calcium channels on the cell membrane. We reduce the model to three ordinary differential equations and analyse its bifurcation structure semi-analytically as two bifurcation parameters vary—the textit{IP}_3 concentration, and the “strength” of stretch activation, lambda . The calcium system (lambda =0, no mechanics) exhibits relaxation oscillations for a certain range of textit{IP}_3 values. As lambda is increased the range of textit{IP}_3 values decreases and oscillations eventually vanish at a sufficiently high value of lambda . This result agrees with experimental evidence in embryonic cells which also links the loss of calcium oscillations to embryo abnormalities. Furthermore, as lambda is increased the oscillation amplitude decreases but the frequency increases. Finally, we also identify the parameter range for oscillations as the mechanical responsiveness factor of the cytosol increases. This work addresses a very important and not well studied question regarding the coupling between chemical and mechanical signalling in embryogenesis.

Effect of simvastatin on vascular tone in porcine coronary artery: Potential role of the mitochondria

Statins induce acute vasorelaxation which may contribute to the overall benefits of statins in the treatment of cardiovascular disease. The mechanism underlying this relaxation is unknown. As statins have been shown to alter mitochondrial function, in this study we investigated the role of mitochondria in the relaxation to simvastatin.Relaxation of porcine coronary artery segments by statins was measured using isolated tissue baths. Mitochondrial activity was determined by measuring changes in rhodamine 123 fluorescence. Changes in intracellular calcium levels were determined in freshly isolated smooth muscle cells with Fluo-4 using standard epifluorescent imaging techniques.Simvastatin, but not pravastatin, produced a slow relaxation of the coronary artery, which was independent of the endothelium. The relaxation was attenuated by the mitochondrial complex I inhibitor rotenone (10μM) and the complex III inhibitor myxothiazol (10μM), or a combination of the two. The complex III inhibitor antimycin A (10μM) produced a similar time-dependent relaxation of the porcine coronary artery, which was attenuated by rotenone. Changes in rhodamine 123 fluorescence showed that simvastatin (10μM) depolarized the membrane potential of mitochondria in both isolated mitochondria and intact blood vessels. Simvastatin and antimycin A both inhibited calcium-induced contractions in isolated blood vessels and calcium influx in smooth muscle cells and this inhibition was prevented by rotenone.In conclusion, simvastatin produces an endothelium-independent relaxation of the porcine coronary artery which is dependent, in part, upon effects on the mitochondria. The effects on the mitochondria may lead to a reduction in calcium influx and hence relaxation of the blood vessel.

Mifepristone is a Vasodilator Due to the Inhibition of Smooth Muscle Cells L-Type Ca2+ Channels.

Derived from the estrane progestins, mifepristone was the first synthetic steroid of this class employed as abortifacient in the first months of pregnancy. Mifepristone reduces high potassium-induced contraction and prevents calcium-induced contraction. At the vascular level, mifepristone induces direct relaxation in rat and human arteries, and this effect seems to be endothelium- and NO independent, suggesting that the vascular smooth muscle is its target. Moreover, mifepristone's effect could involve the modulation of different calcium channels. The aim of the present study is to analyze the involvement of calcium channels in the relaxation induced by mifepristone on vascular smooth muscle cells (VSMCs). Planar cell surface area (PCSA) technique was used to analyze the effect of mifepristone on the VSMC contractility, and the whole cell configuration of patch-clamp technique to measure the activity of L-type Ca(2+) channels (LTCC) in A7r5 cells. Regarding the PCSA technique, mifepristone induced relaxation of the VSMC previously contracted by different agents. Also, a rapid inhibitory effect on basal and BAY K8644-stimulated calcium current was observed, which indicates that this drug has the ability to block LTCC. These results suggest that mifepristone induces relaxation on the VSMCs due to the inhibition of the calcium channels.

Endothelium nitric oxide-independent vasorelaxant effects of the aqueous extract from Stephania abyssinica on the isolated rat thoracic aorta.

Stephania abyssinica (Dillon & A. Rich) Walp (Menispermaceae) is a medicinal plant used in the west region of Cameroon to treat arterial hypertension. The present study evaluated the vasorelaxant effects of the aqueous (AESA) and methanol (MESA) extracts from the fresh leaves of S. abyssinica on aorta rings isolated from Wistar rats. Aorta rings with intact endothelium were contracted with KCl (60 mM) or phenylephrine (10-5 M) and exposed to cumulative concentrations of each extract, ranging from 10 to 1,000 µg/mL. The vasorelaxant effects of AESA were further evaluated in presence of Nω-nitro-L-arginine methyl ester (L-NAME, 10-4 M), tetraethylammonium (TEA, 5 µM), glibenclamide (5 µM), propranolol (5 µM), and the association glibenclamide-propranolol (AGP). In another set of experiments, the effect of AESA was evaluated on calcium-induced contraction in a hyperpotassic milieu. AESA and MESA exhibited a concentration-dependent vasorelaxation on KCl-contracted aortic rings with respective EC50 of 160.10 and 346.50 µg/mL. AESA similarly relaxed aortic rings contracted with phenylephrine (EC50, 176.80 µg/mL). The vasorelaxant activity of AESA was not significantly affected by L-NAME but was markedly reduced by TEA, glibenclamide, propranolol, and AGP. AESA strongly inhibited the Ca2+-induced contraction by 95%. These results support the use of S. abyssinica against arterial hypertension and suggest that the vasorelaxant effect of AESA is not mediated via the endothelium/nitric oxide pathway. AESA relaxant properties might be due to an inhibition of Ca2+ influx and/or the activation of ATP-sensitive K+ channels probably via the stimulation of β-adrenergic receptors.

Effect of Vitis vinifera Leaf Hydroalcoholic Extract on Rat Urinary Bladder Contractility

Introduction: Several reports have shown the various medicinal effects of grape (Vitis vinifera) seed and skin extracts, such as antioxidant, hypotensive and vasodilatory effects. It has been recently shown the relaxatory effect of grape leaf hydroalcoholic extract on rat ileum, uterus, aorta, trachea and vas deferens as well as on frog isolated heart. The aim of the present study was to investigate the effect of Vitis vinifera leaf hydroalcoholic extract (VLHE) on contractility of urinary bladder smooth muscle in male rats and to study the mechanism(s) of its action. Materials and Methods: The extract of dried grape leaf was prepared by macerated method using 70% ethanol for 72h in room temperature. The solvent was then evaporated. Urinary bladder was removed from male adult wistar rats after induction of deep anaesthetize by di-ethyle-ether. Tissues were then suspended in the organ bath containing physiologic solution (37oC, pH 7.4) bubbled with oxygen. Contractions were recorded isometrically under 1g resting tension. Results: VLHE at 1, 2, 4 and 8 mg/ml significantly reduced the urinary bladder contractions evoked by KCl (60 mM P<0.05) and (0.5, 1, 2 and 4 mg/ml) also reduced the contractions evoked by Acetylcholine (5 µM) dose dependently (P<0.05). In calcium free physiologic solution, KCl-induced (120 mM P<0.05) contractions were occurred only after adding calcium (0.312, 0.625, 1.25, 2.5 and 5 mM) to the organ bath solution. VLHE (2 mg/ml) also reduced different concentration of calcium-induced contraction in the presence of KCl (P<0.05). The VLHE- induced urinary bladder relaxation was unaffected by propranolol (1 µM for 20 mins) while the presence of tetraethylamonium (10 mM for 20 mins) could strongly reduce the relaxatory effect of VLHE. Conclusion: The data suggest that VLHE induces spasmolytic effect in rat urinary bladder, possibly via blocking voltage dependent calcium channels and activation of calcium-activated potassium channels.

Prolonged Relaxation Kinetics in Distal Arthrogryposis Skeletal Muscle Myofibrils with a MYH3 R672C Mutation

The mechanisms underlying most forms of Distal Arthrogryposis (DA), a group of congenital contracture syndromes, are unknown. Our previous functional studies from adult individuals with DA caused by the heterozygous myosin heavy chain mutation, MYH3 R672C, showed that the time required by DA skinned skeletal myofibers to relax completely after calcium-induced contraction was several-fold longer than controls (Racca et al. (2010) Biophys J 98:542-3a). Here we measured force and kinetics of activation & relaxation, and compared chemomechanical analysis using myofibrils and myofibers sampled from gastrocnemius muscle from two affected individuals vs. three control (non-DA) individuals. The prolonged relaxation was reflected in isolated myofibrils from DA patients, as 50% relaxation time from maximal activation was 36% longer, and 90% relaxation time was prolonged by 58%. The kinetics of the slow phase of relaxation were significantly slower, in both the rate and duration (DA: kREL,SLOW=0.36±0.07s−1;tREL,SLOW=285±21ms vs. Control: 0.79±0.18s−1;199±23ms), implying slower cross-bridge release. Use of ADP prolonged relaxation of control samples to a greater extent than DA preparations, suggesting that slower ADP release from myosin in DA myofibrils may be the mechanism of slower relaxation and cross-bridge release. We also found that both mRNA and protein for this embryonic myosin (gene MYH3) were present in adult skeletal muscle, such that a small amount of this slower myosin may prolong relaxation. Although the mechanism that leads to the congenital contractures must begin prenatally, these results suggest that MYH3 R672H also affects ongoing function of adult skeletal muscle. Understanding the mechanism by which myosin mutations affect muscle cell contractility could provide a model for exploring the pathogenesis of more common contractures such as idiopathic clubfoot and facilitate the development of novel therapeutic approaches. Supported by F31AR06300(A.R.), 5K23HD057331(A.B.), HD048895(M.B.,M.R.).

Design, Synthesis and Calcium Channel Blocking Activity of Diltiazem- Verapamil Hybrid Molecules

The current manuscript describes the design, synthesis, and in vitro testing of four thioacetanilides with diltiazemverapamil hybrid structural features as potential calcium channel blockers. The current hybrid strategy of drug design aimed to generate compounds that could span, with a single compound, the trans-membrane locales where the two drugs bind, with the ultimate goal of increasing the blocking activity. The latter, was assessed by measuring the inhibitory effects, expressed as IC50, on calcium-induced contractions of potassium depolarized isolated rat aorta strips. The assessment of the binding locales was determined by incubating the test compound with aortic strips for two different periods, 10-minutes and 2-hours, before adding the contractile calcium ions to the assay medium. Diltiazem IC50 values were 0.26 and 0.14 μM, after 10-minutes and 2-hours, respectively, reflecting less than two fold increase in activity and confirming previous reports that its locale of binding in mostly on the exterior side of the membrane. On the other hand, verapamil IC50 values were 0.47 and 0.14 μM after 10-minutes and 2-hour incubation respectively, reflecting approximately a 3-4 fold increase in activity and confirming previous reports that it binds mainly to the interior domains of the membrane. The four designed hybrid compounds showed, after 10-minute incubation, an IC50 value range of 3.7-12.0 μM, and after 2-hour incubation an IC50 range of 0.78-2.12 μM, reflecting approximately a 5-fold increase in activity suggesting more similarity to the verapamil binding profile. The data indicate that the designed compounds are with moderate activities, but generally less active as calcium channel blockers than either of the two parent drugs.

Abstract 373: Mineralocorticoid Receptor and Rhoa/rho Kinase Contribute to Vascular Dysfunction in Obese Db/db Mice

Increasing evidence indicates that adipose tissue modulates vascular function. Our previous studies demonstrated that adipocytes secrete aldosterone. We hypothesized that adipocyte-releasing factors induce vascular dysfunction through mineralocorticoid receptor (MR)-dependent mechanisms. This study explored the small G protein RhoA/Rho kinase as a pathway linking aldosterone/MR and the paracrine effects of adipose tissue in the vasculature, since the activation of this pathway regulates vascular contraction. Diabetic obese mice (db/db) and their control counterparts (db/+) were treated with MR antagonist (MRA) (K canrenoate, 30mg/Kg/day, S.C., 4 weeks) or RhoA/Rho kinase inhibitor, fasudil (30 mg/Kg/day, S.C., 3 weeks). Blood pressure levels were similar between groups. Fasudil, but not MRA, reduced plasma glucose levels in db/db mice (db/+:12.3±6; db/db: 30.1±3; MRA-treated db/db: 32.5±2; fasudil treated db/db: 17.1±2; mmoL/mL). Arteries from db/db mice displayed reduced relaxation to 10 - 5 M acetycholine (Ach; db/+:79.4±3.9% vs db/db: 14.3±3.1%). Arteries from MRA-treated db/db mice exposed to fat conditioned medium (FCM) had improved responses to Ach (MRA-treated db/db: 28.3±1.9% vs MRA-treated db/db +FCM: 42.8±4.3%). Increased norepinephrine (NE)-induced contraction was observed in db/db mice in endothelium-denuded arteries (10 -5 M NE; db/+: 1.5 ± 0.1 vs db/db: 2.2 ± 0.1; mN/mm). NE responses were reduced in MRA-treated db/db mice (1.7±0.1 mN/mm) or fasudil (1.6±0.2 mN/mm). Vascular calcium sensitivity was similar between depolarized arteries from db/+ and db/db. Fasudil treatment reduced calcium-induced contraction in both groups with greater effect in db/db mice (5 mmol/L CaCl 2 ; db/+: 73 % vs db/db: 57 %). MR blockade did not affect vascular RhoA/Rho kinase activity (RhoA membrane to cytosol translocation) in db/db mice. Our data suggest that in db/db mice: MR and RhoA/Rho kinase signaling contribute to adipose modulation of vascular/contraction. While MR plays a role in vascular relaxation, RhoA/Rho kinase signalling is important in contractile dysfunction, adipocyte secretion, and glucose metabolism - processes that may contribute to cardiovascular injury in obesity-associated diabetes.

Fullerenol C60(OH)24 nanoparticles decrease relaxing effects of dimethyl sulfoxide on rat uterus spontaneous contraction

Dimethyl sulfoxide (DMSO) is a widely used solvent and cryoprotectant that can cause impaired blood flow, reduction in intracranial pressure, tissue edema, inflammatory reactions, inhibition of vascular smooth muscle cell migration and proliferation, processes which can lead to atherosclerosis of the coronary, peripheral and cerebral circulation. Although the adverse effects are rare when DMSO is administered in clinically established concentrations, there is no safe antagonist for an overdose. In this work, we treated isolated spontaneous and calcium-induced contractile active rat uteri (Wistar, virgo intacta), with DMSO and fullerenol C60(OH)24 nanoparticle (FNP) in DMSO. FNP is a water-soluble derivative of fullerene C60. Its size is a 1.1 nm in diameter and is a very promising candidate for a drug carrier in nanomedicine. FNP also displays free radical scavenging activity. DMSO decreased both spontaneous and calcium-induced contractions. In contrast, FNP only decreased spontaneous contraction. FNP decreased copper–zinc superoxide dismutase activity and prevented the DMSO-induced increase in glutathione reductase activity. Atomic force microscopy detected that FNP aggregated with calcium ions. Our results indicate that FNP has properties that make it a good candidate to be a modulator of DMSO activity which could minimize side effects of the latter.

The importance of potassium channels in the mechanism of the relaxing effect of pentoxifylline on isolated rat uteri

Background. Pentoxifylline is a methylxanthine derivative that is used to treat peripheral vascular disease. One of the mechanisms of action of pentoxifylline is the vasodilatation of blood vessels. This study examined the effect of increasing pentoxifylline concentrations on the contractility of isolated rat uteri in the presence of a potassium channel antagonist. Methods. The uteri were isolated from virgin Wistar rats (180-220 g) and suspended in an isolated organ bath chamber containing De Jalon's solution and aerated with 95% O2 and 5% CO2. The temperature was maintained at 37єC. Isometric contractions were recorded using an isometric force transducer (Ugo Basile). The preload of the preparation was approximately 1 g. The uteri were allowed to contract spontaneously or in the presence of Ca2+ (0.018 and 0.36 mM) and acetylcholine (ACh) and were treated with pentoxifylline. Results. Pentoxifylline caused concentration-dependent inhibition of spontaneous rhythmic uterine activity and uterine activity caused by calcium Ca2+ (0.018 mM and 0,36 mM). We showed that the inhibitory effects of pentoxifylline depend on the type of muscle contractions activated and that the inhibitory effect is significantly stronger for spontaneous rhythmic activity and forin Ca2 -induced contractions of isolated rat uteri+. The relaxing effect of pentoxifylline depends on the calcium concentration in the medium. Pentoxifylline exerted the weakest relaxant effects on contractions induced by acetylcholine. In contrast to methylene blue, tetraethylammonium, or 4-aminopyridine, glibenclamide did not antagonise the relaxing effect of pentoxifylline on isolated rat uteri. Conclusion. The results obtained suggest that the mechanism of action of pentoxifylline does not lead to the opening of KATP channels. However, the opening of BKCa and voltage-dependent Ca2+ channels had some role, but to varying degrees, in the mechanism of the relaxing effect of pentoxifylline on the spontaneous rhythmic activity and calcium-induced contractions of isolated rat uteri. Our results provide additional confirmation of the dominance of the NO/cGMP signaling pathway in the mechanism of the relaxing effect of pentoxifylline (because the presence of methylene blue significantly antagonised this effect) in relation to the opening of potassium channels, especially KATP channels. These results indicate that pentoxifylline could be a potential tocolytic drug.

The Pharmacological Differences in Antianginal Effects of Long-lasting Calcium Channel Blockers

We examined antianginal effects of azelnidipine and amlodipine in an arginine vasopressin-induced rat anginal model. Oral administration of azelnidipine or amlodipine produced long lasting inhibition of arginine vasopressin-induced ST-segment depression in electrocardiogram. The degrees of inhibition with azelnidipine at doses of 1 and 3 mg/kg were comparable to those with amlodipine at 3 and 10 mg/kg. Both drugs lowered mean blood pressure in a dose-related manner, whereas only azelnidipine decreased heart rate. Azelnidipine at 3 mg/kg and amlodipine at 10 mg/kg produced a similar decrease in the rate pressure product, an index for cardiac oxygen consumption. Their inhibitory effects on calcium-induced vascular contraction were compared in isolated porcine coronary arteries. Both drugs produced a slow-developing inhibition of calcium-induced contraction. Although their inhibitory effects were similar, the way the both drugs inhibited calcium-induced contraction differed with each other. After removing the drug from bathing solution, the inhibitory effects of azelnidipine were not blunted but were sustained for a long time, which indicates that azelnidipine has high vascular affinity. On the other hand, those of amlodipine were rapidly blunted. These results suggest that the mechanisms underlying antianginal effects of azelnidipine differ from those of amlodipine. The antianginal effect with azelnidipine may be accounted for by its high affinity to the coronary blood vessels and the heart rate slowing effect, both of which are not shared with amlodipine.

The pharmacological differences in anti-anginal effects of long-lasting calcium channel blockers: azelnidipine and amlodipine.

ABSTRACT: We examined anti-anginal effects of azelnidipine and amlodipine in an arginine vasopressin (AVP)-induced rat anginal model. Oral administration of azelnidipine or amlodipine produced long-lasting inhibition of AVP-induced ST-segment depression in ECG. The degrees of inhibition with azelnidipine at doses of 1 and 3 mg/kg were comparable to those with amlodipine at 3 and 10 mg/kg. Both drugs lowered mean blood pressure in a dose related manner, while only azelnidipine decreased heart rate. Azelnidipine at 3 mg/kg and amlodipine at 10 mg/kg produced a similar decrease in the rate pressure product, an index for cardiac oxygen consumption. Their inhibitory effects on calcium-induced vascular contraction were compared in isolated porcine coronary arteries. Both drugs produced a slow-developing inhibition of calcium-induced contraction. Although their inhibitory effects were similar, the way the both drugs inhibited calcium-induced contraction differed with each other. After removing the drug from bathing solution, the inhibitory effects of azelnidipine were not blunted but were sustained for a long time which indicates that azelnidipine has high vascular affinity. On the other hand, those of amlodipine were rapidly blunted. These results suggest that the mechanisms underlying anti-anginal effects of azelnidipine differ from those of amlodipine. The anti-anginal effect with azelnidipine may be accounted for by its high affinity to the coronary blood vessels and the heart rate slowing effect, both of which are not shared with amlodipine.