Bay K 8644 Research Articles

1,25-Dihydroxyvitamin D3 Attenuates the Effects of Lipopolysaccharide by Causing ADAM10-Dependent Ectodomain Shedding of Toll-Like Receptor 4

Background/Aims: We investigated how 1,25-dihydroxyvitamin D₃ (1,25D₃) inhibits the effects of lipopolysaccharide (LPS) in human aortic endothelial cells. Methods: Cellular signaling was explored by determination of protein abundance with Western blot, measurement of cytosolic Ca²⁺ concentration and immunofluorescence staining for a disintegrin and metalloprotease 10 (ADAM10). Results: LPS stimulated the expression of intercellular adhesion molecule 1 (ICAM-1) through toll-like receptor 4 (TLR4) and subsequent activation of p38 mitogen-activated protein kinase (p38 MAPK). Pretreatment with 1,25D₃ attenuated LPS-induced p38 MAPK activation and ICAM-1 expression by causing ectodomain shedding of TLR4. This effect of 1,25D₃ depended on its ability to induce a rapid extracellular Ca²⁺ influx through L-type calcium channels because the ectodomain shedding was prevented by the absence of extracellular Ca²⁺ or the presence of verapamil. TLR4 ectodomain shedding was also induced by Bay K8644 (L-type calcium channel agonist). Both 1,25D₃ and Bay K8644 caused extracellular Ca²⁺ influx-dependent ADAM10 translocation to the cell surface. Depletion of ADAM10 by siRNA transfection prevented 1,25D₃- and Bay K8644-induced ectodomain shedding of TLR4, and abolished the inhibitory effect of 1,25D₃ on LPS-induced ICAM-1 expression. Conclusion: 1,25D₃ causes ectodomain shedding of TLR4 and thereby decreases the responsiveness of cells to LPS. ADAM10, activated by extracellular Ca²⁺ influx, was implicated in the ectodomain cleavage of TLR4.

Basal and Activated Calcium Sensitization Mediated by RhoA/Rho Kinase Pathway in Rats with Genetic and Salt Hypertension

Calcium sensitization mediated by RhoA/Rho kinase pathway can be evaluated either in the absence (basal calcium sensitization) or in the presence of endogenous vasoconstrictor systems (activated calcium sensitization). Our aim was to compare basal and activated calcium sensitization in three forms of experimental hypertension with increased sympathetic tone and enhanced calcium entry—spontaneously hypertensive rats (SHR), heterozygous Ren-2 transgenic rats (TGR), and salt hypertensive Dahl rats. Activated calcium sensitization was determined as blood pressure reduction induced by acute administration of Rho kinase inhibitor fasudil in conscious rats with intact sympathetic nervous system (SNS) and renin-angiotensin system (RAS). Basal calcium sensitization was studied as fasudil-dependent difference in blood pressure response to calcium channel opener BAY K8644 in rats subjected to RAS and SNS blockade. Calcium sensitization was also estimated from reduced development of isolated artery contraction by Rho kinase inhibitor Y-27632. Activated calcium sensitization was enhanced in all three hypertensive models (due to the hyperactivity of vasoconstrictor systems). In contrast, basal calcium sensitization was reduced in SHR and TGR relative to their controls, whereas it was augmented in salt-sensitive Dahl rats relative to their salt-resistant controls. Similar differences in calcium sensitization were seen in femoral arteries of SHR and Dahl rats.

Sustained Contraction in Vascular Smooth Muscle by Activation of L-type Ca2+ Channels Does Not Involve Ca2+ Sensitization or Caldesmon.

Vascular smooth muscle (VSM) is unique in its ability to maintain an intrinsic level of contractile force, known as tone. Vascular tone is believed to arise from the constitutive activity of membrane-bound L-type Ca2+ channels (LTCC). This study used a pharmacological agonist of LTCC, Bay K8644, to elicit a sustained, sub-maximal contraction in VSM that mimics tone. Downstream signaling was investigated in order to determine what molecules are responsible for tone. Medial strips of swine carotid artery were stimulated with 100 nM Bay K8644 to induce a sustained level of force. Force and phosphorylation levels of myosin light chain (MLC), MAP kinase, MYPT1, CPI-17, and caldesmon were measured during Bay K8644 stimulation in the presence and absence of nifedipine, ML-7, U0126, bisindolylmaleimide (Bis), and H-1152. Nifedipine and ML-7 inhibited force and MLC phosphorylation in response to Bay K8644. Inhibition of Rho kinase (H-1152) but not PKC (Bis) inhibited Bay K8644 induced force. U0126 significantly increased Bay K8644-dependent force with no effect on MLC phosphorylation. Neither CPI-17 nor caldesmon phosphorylation were increased during the maintenance of sustained force. Our results suggest that force due to the influx of calcium through LTCCs is partially MLC phosphorylation-dependent but does not involve PKC or caldesmon. Interestingly, inhibition of MLC kinase (MLCK) and PKC significantly increased MAP kinase phosphorylation suggesting that MLCK and PKC may directly or indirectly inhibit MAP kinase activity during prolonged contractions induced by Bay K8544.

Effects of ZD7288, a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, on term-pregnant rat uterine contractility in vitro

The uterus is a myogenic organ that is able to produce discrete spontaneous action potentials and contractions without any stimuli. Myometrial excitability is governed by ion channels including Ca+2 and K+ channels, but whether or not other channels such as hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which play an important role in regulating cellular excitability, are also involved has not been reported in uterine smooth muscles. The aim of the present study was to examine whether blocking HCN channels with a specific blocker ZD7288 would modulate the uterine contractility in a rat model. Using longitudinal uterine strips from term-pregnant rats, the effects of varying concentrations of ZD7288 (50 μM, 100 μM, and 200 μM) were examined on uterine contractions generated spontaneously or by oxytocin (5 nmol/L) and on uterine strips depolarized by high-KCl (60 mM/L), or activated by L-type Ca2+ channels agonist (Bay K8644; 1 μM). Application of ZD7288 at concentrations of 200 μM and 100 μM, but not 50 μM, significantly decreased the amplitude of spontaneous uterine contractions. In addition, 200 μM of ZD7288 significantly reduced the force of contractions induced by oxytocin with a pronounced reduction while the tissues were depolarized by high-KCl solution, or activated by Bay K8644. The present study provides pharmacological evidence suggesting that pregnant uterine contractility is modulated by HCN channels and that these channels might represent a therapeutic target for controlling premature activation of uterine activity associated with preterm labor.

PP.31.05] ALTERED CALCIUM SENSITIZATION IN HYPERTENSIVE REN-2 TRANSGENIC RATS

Objective: A comparison of BP response to Ca channel opener BAY K8644 prior and after Rho kinase inhibition indicated decreased calcium sensitization in spontaneously hypertensive rats (SHR), although blood pressure (BP) response of intact SHR to Rho kinase inhibitor fasudil was enhanced (Behuliak et al. J Hypertens 31: 2025, 2013). The aim of the present study was to evaluate calcium sensitization in hypertensive transgenic rats with murine Ren-2 gene (TGR) and their normotensive HanSD controls using the same methods. We also tried to elucidate the interference of baroreflex-activated sympathetic activity with BP response to fasudil. Design and method: Blood pressure and heart rate responses to increasing doses BAY K8644 (opener of L type voltage-dependent calcium channels) was determined in conscious rats (subjected to acute RAS and SNS blockade). BP response was recorded prior and after Rho kinase inhibition by fasudil and calcium sensitization was estimated from the difference between both BP response curves. Furthermore, BP-lowering effects of acute administration of increasing fasudil doses were measured in conscious intact rats as well as in rats subjected to combined blockade of RAS, SNS and NOS in which baroreflex efficiency was suppressed. Results: Using the above BAY K8644-based approach we demonstrated moderate attenuation of calcium sensitization in hypertensive TGR. We also observed enhanced acute BP response to fasudil in intact TGR compared to HanSD rats. This different BP response was accompanied by more pronounced heart rate increase in HanSD than in TGR, indicating attenuated baroreflex efficiency in hypertensive TGR. If baroreflex operation was minimized by ganglionic blockade in rats subjected to combined RAS, SNS and NO synthase blockade, fasudil-induced BP fall was manifold augmented in HanSD but unchanged in TGR, suggesting the importance of baroreflex-activated sympathetic activity in compensation of fasudil-induced BP changes. Conclusions: Calcium sensitization is attenuated in hypertensive TGR compared to normotensive controls, but this difference can be seen only in the absence of operating sympathetic nervous system which compensates BP changes elicited by acute Rho kinase inhibition much better in normotensive than in hypertensive animals. Partially supported by grant AZV 15–25396A (MH CR)

OS 05-03 DECREASED RHO-KINASE-BASED CALCIUM SENSITIZATION IN HYPERTENSIVE REN-2 TRANSGENIC RATS (TGR)

Objective: Comparing BP response to Ca channel opener BAY K8644 prior and after Rho kinase inhibition we demonstrated lowered calcium sensitization in spontaneously hypertensive rats (SHR), although BP response of intact SHR to Rho kinase inhibitor fasudil was enhanced (Behuliak et al. J Hypertens 31:2025,2013). The aim of the present study was to evaluate calcium sensitization in hypertensive TGR and their normotensive HanSD controls using the same methods. We also tried to elucidate the interference of baroreflex-activated sympathetic activity with BP response to fasudil. Design and Method: Blood pressure and heart rate responses to increasing doses of BAY K8644 (opener of L type voltage-dependent calcium channels) was determined in conscious heterozygous TGR rats (subjected to acute RAS and SNS blockade). BP response was recorded prior and after Rho kinase inhibition by fasudil and calcium sensitization was estimated from the difference between both BP response curves. Furthermore, BP-lowering effects of acute administration of increasing fasudil doses were measured in conscious intact rats and in rats subjected to combined blockade of RAS, SNS and NOS in which baroreflex efficiency was suppressed. Results: Using the BAY K8644-based approach we demonstrated moderately attenuated calcium sensitization in hypertensive TGR but enhanced acute BP response to fasudil in intact TGR compared to HanSD rats. This was accompanied by greater heart rate increase in HanSD than in TGR, indicating attenuated baroreflex efficiency in hypertensive TGR. If baroreflex operation was minimized by ganglionic blockade, fasudil-induced BP fall was substantially augmented in HanSD but unchanged in TGR. Conclusions: Calcium sensitization is attenuated in hypertensive TGR, this change being evident only in the absence of sympathetic nervous system which compensates BP changes elicited by acute Rho kinase inhibition better in normotensive than in hypertensive animals. Supported by grant AZV 15–25396A (MH CR)

Anticonvulsant mechanism of saponins fraction from adventitious roots of Ficus religiosa: possible modulation of GABAergic, calcium and sodium channel functions

In our previous studies, quantified saponins-rich fraction from adventitious root extract of Ficus religiosa L., Moraceae, showed anticonvulsant effect in acute, as well as chronic mice models of epilepsy. The present study was designed to reveal putative anticonvulsant mechanism of quantified saponins-rich fraction using target specific animal models. The anticonvulsant effect of quantified saponins-rich fraction was initially studied in maximal electroshock and pentylenetetrazol test at 1, 2 and 4mg/kg; i.p. doses. Based on the results of initial anticonvulsant testing, different groups of mice were injected with vehicle or quantified saponins-rich fraction (4mg/kg; i.p.), 30min prior to an injection of N-methyl-d-aspartic acid (100mg/kg; s.c.), bicuculline (5mg/kg; i.p.), strychnine hydrochloride (2mg/kg; i.p.), BAY k-8644 (37.5μg; i.c.v.), veratridine (500μg/kg; i.p.) and the convulsive episodes were studied. Treatment with the extract (1, 2 and 4mg/kg) showed significant protection in maximal electroshock and pentylenetetrazol-induced convulsion tests, in a dose-dependent manner. Moreover, quantified saponins-rich fraction at 4mg/kg dose showed significant increase in latency to clonic convulsions, decrease in seizure severity and increase in average wave amplitude in bicuculline, BAY k-8644 and veratridine tests, respectively, as compared to vehicle control. However, SRF treatment failed to abolish N-methyl-d-aspartic acid and strychnine-induced convulsions, indicated by insignificant change in the appearance of turning behavior and onset of tonic extension, respectively, as compared to vehicle control. From the results of present study, it is concluded that quantified saponins-rich fraction suppress maximal electroshock, pentylenetetrazol, bicuculline, BAY k-8644 and veratridine-induced convulsions, indicating its GABAergic, Na+ and Ca2+ channel modulatory effects. Further it can be correlated that quantified saponins-rich fraction causes deactivation of voltage-gated Na+ and Ca2+ channels, without effecting ligand-gated Na+ and Ca2+ channels. More studies are required at molecular levels using in vitro techniques to understand the exact molecular interactions of quantified saponins-rich fraction with these pathways.

Changes in intracellular Ca2+ in the ureter smooth muscle cells of rats with neuropathic urinary tract dysfunction and its significance

Objective To investigate the changes in intracellular Ca2+ in the ureter smooth muscle cells (USMC) of rats with neuropathic urinary tract dysfunction (NUTD) and their significance. Methods Forty-five rats were randomly and averagely divided into NUTD group, experimental control (EC) group and blank control (BC) group.The NUTD group was operated with a spinal cord transection at the first lumbar level and the sacral cord was destroyed; in EC group the spinal process was partly bitten at the same position, but the spinal cord was not transected; BC group was given no operations.One week later, the video-urodynamic was performed to observe the acontractile detrusor (ACD), vesicoureteral reflux (VUR) and urinary tract dysfunction in rats among the NUTD group, EC group and the BC group.Video-urodynamic assessment was performed at the sixth week after operation.Ureter smooth muscle cells (USMC) were obtained by collagenase digestion.Intracellular Ca2+ in the USMC were observed by laser scanning confocal microscope.Then the effects of Bay K8644(10-8 mol/L, 10-7 mol/L, 10-6 mol/L) on cytosolic Ca2+ concentrations( [Ca2+ ]i) in NUTD group were studied by calculating the fluorescence intensity. Results ACD and no detrusor overactivity were found in all rats in NUTD group and without vesicoureteral reflux.Immunofluorescence method confirmed that the cells were USMC.Compared with BC group(31.44±2.82)and EC group(32.06±3.67), the fluorescence intensity(FI) of intracellular Ca2+ in USMC was much lower in the NUTD group(9.80±1.11), and there was significant difference(P<0.05). Bay K8644 (10-8mol/L, 10-7mol/L, 10-6mol/L) increased the FI of [Ca2+ ]I in a concentration-dependent manner, which were 3.80±1.30, 10.04±2.15, 19.89±2.06, respectively, and there was significant difference (P<0.05). Conclusions The decrease in Ca2+ concentration in the ureter smooth muscle cells may be one of the important factors for the primary ureteral dysfunction of NUTD.And calcium channel agonist can be meaningful for adjusting abnormal Ca2+ concentration in USMC of NUTD. Key words: Neurogenic urinary tract dysfunction; Ureter smooth muscle cell; L-type Ca2+ channel; Bay K8644; Laser scanning confocal microscope

Antispasmodic Effects and Action Mechanism of Essential Oil of Chrysactinia mexicana A. Gray on Rabbit Ileum.

The Chrysactinia mexicana A. Gray (C. mexicana) plant is used in folk medicine to treat fever and rheumatism; it is used as a diuretic, antispasmodic; and it is used for its aphrodisiac properties. This study investigates the effects of the essential oil of C. mexicana (EOCM) on the contractility of rabbit ileum and the mechanisms of action involved. Muscle contractility studies in vitro in an organ bath to evaluate the response to EOCM were performed in the rabbit ileum. EOCM (1–100 µg·mL−1) reduced the amplitude and area under the curve of spontaneous contractions of the ileum. The contractions induced by carbachol 1 µM, potassium chloride (KCl) 60 mM or Bay K8644 1 µM were reduced by EOCM (30 µg·mL−1). Apamin 1 µM and charybdotoxin 0.01 µM decreased the inhibition induced by EOCM. The d-cAMP 1 µM decreased the inhibition induced by EOCM. l-NNA 10 µM, Rp-8-Br-PET-cGMPS 1 µM, d,l-propargylglycine 2 mM, or aminooxyacetic acid hemihydrochloride 2 mM did not modify the EOCM effect. In conclusion, EOCM induces an antispasmodic effect and could be used in the treatment of intestinal spasms or diarrhea processes. This effect would be mediated by Ca2+, Ca2+-activated K+ channels and cAMP.

Perfusion of isolated carotid sinus with hydrogen sulfide attenuated the renal sympathetic nerve activity in anesthetized male rats.

The purpose of the present study was to define the indirect central effect of hydrogen sulfide (H(2)S) on baroreflex control of sympathetic outflow. Perfusing the isolated carotid sinus with sodium hydrosulfide (NaHS), a H(2)S donor, the effect of H(2)S was measured by recording changes of renal sympathetic nerve activity (RSNA) in anesthetized male rats. Perfusion of isolated carotid sinus with NaHS (25, 50, 100 micromol/l) dose and time-dependently inhibited sympathetic outflow. Preconditioning of glibenclamide (20 micromol/l), a ATP-sensitive K(+) channels (K(ATP)) blocker, the above effect of NaHS was removed. With 1, 4-dihydro-2, 6-dimethyl-5-nitro-4-(2-[trifluoromethyl] phenyl) pyridine-3-carboxylic acid methyl ester (Bay K8644, 500 nmol/l) pretreatment, which is an agonist of L-calcium channels, the effect of NaHS was eliminated. Perfusion of cystathionine gamma-lyase (CSE) inhibitor, DL-propargylglycine (PPG, 200 micromol/l), increased sympathetic outflow. The results show that exogenous H(2)S in the carotid sinus inhibits sympathetic outflow. The effect of H(2)S is attributed to opening K(ATP) channels and closing the L-calcium channels.

Sarcolemmal Ca2+-entry through L-type Ca2+ channels controls the profile of Ca2+-activated Cl− current in canine ventricular myocytes

Ca2+-activated Cl− current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood.We aimed to study the profile of ICl(Ca) systematically under physiological conditions (normal Ca2+ cycling and AP voltage-clamp) as well as in conditions designed to change [Ca2+]i. The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied.The profile of ICl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltage-clamp conditions, contained an early fast outward and a late inward component, overlapping early and terminal repolarizations, respectively. Both components were moderately reduced by ryanodine, while fully abolished by BAPTA, but not EGTA. [Ca2+]i was monitored using Fura-2-AM. Setting [Ca2+]i to the systolic level measured in the bulk cytoplasm (1.1μM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Ca2+-entry through L-type Ca2+ channels was essential for activation of ICl(Ca). TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium.Activation of ICl(Ca) in canine ventricular cells requires Ca2+-entry through neighboring L-type Ca2+ channels and is only augmented by SR Ca2+-release. Substantial activation of ICl(Ca) requires high Ca2+ concentration in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.

High-throughput drug profiling with voltage- and calcium-sensitive fluorescent probes in human iPSC-derived cardiomyocytes.

Cardiomyocytes derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) are increasingly used for in vitro assays and represent an interesting opportunity to increase the data throughput for drug development. In this work, we describe a 96-well recording of synchronous electrical activities from spontaneously beating hiPSC-derived cardiomyocyte monolayers. The signal was obtained with a fast-imaging plate reader using a submillisecond-responding membrane potential recording assay, FluoVolt, based on a newly derived voltage-sensitive fluorescent dye. In our conditions, the toxicity of the dye was moderate and compatible with episodic recordings for >3 h. We show that the waveforms recorded from a whole well or from a single cell-sized zone are equivalent and make available critical functional parameters that are usually accessible only with gold standard techniques like intracellular microelectrode recording. This approach allows accurate identification of the electrophysiological effects of reference drugs on the different phases of the cardiac action potential as follows: fast depolarization (lidocaine), early repolarization (nifedipine, Bay K8644, and veratridine), late repolarization (dofetilide), and diastolic slow depolarization (ivabradine). Furthermore, the data generated with the FluoVolt dye can be pertinently complemented with a calcium-sensitive dye for deeper characterization of the pharmacological responses. In a semiautomated plate reader, the two probes used simultaneously in 96-well plates provide an easy and powerful multiparametric assay to rapidly and precisely evaluate the cardiotropic profile of compounds for drug discovery or cardiac safety.

Na+ permeates through L-type Ca2+ channel in bovine airway smooth muscle

Membrane depolarization of airway smooth muscle (ASM) opens L-type voltage dependent Ca2+ channels (L-VDCC) allowing Ca2+ entrance to produce contraction. In Ca2+ free conditions Na+ permeates through L-VDCC in excitable and non-excitable cells and this phenomenon is annulled at µM Ca2+ concentrations. Membrane depolarization also induces activation of Gq proteins and sarcoplasmic reticulum Ca2+ release. In bovine ASM, KCl induced a transient contraction sensitive to nifedipine in Ca2+free medium, indicating an additional mechanism to the SR-Ca2+ release. It is possible that Na+ could permeate through L-VDCC in bovine ASM. KCl induced a transient contraction in Ca2+ free medium with a fast intracellular Ca2+ increment, reduced by TMB-8. This contraction was abolished by caffeine and CPA, diminished with nifedipine and augmented by Bay K8644. Increasing extracellular Na+ concentration in tracheal myocytes, proportionally augmented the SBFI fluorescence ratio, suggesting an increment in the intracellular Na+ concentration ([Na+]i). 50mM Na+ with and without Ca2+ induced a [Na+]i increment, enhanced by Bay K8644 and inhibited with D-600. In Ca2+ free medium, KCl increased [Na+]i. Ba2+ currents corresponding to L-VDCC were observed in myocytes and Na+ permeated in the presence and absence of Ca2+. SBFI-loaded myocytes in Na+ and Ca2+ containing Krebs stimulated with carbachol showed a Na+ increment with a plateau. D-600 and 2-APB almost abolished the carbachol-induced Na+ increment. RT-PCR demonstrated that CaV1.2 is the only L-VDCC subunit present in ASM. Conclusion: under physiological conditions, Na+ permeates through L-VDCC in bovine ASM, probably contributing to sustain membrane depolarization during agonist stimulation.

Glial Cell Line Derived Neurotrophic Factor Protein Secretion by Skeletal Muscles is Altered by Activity of L‐Type Calcium Channels

Glial cell line‐derived neurotrophic factor (GDNF) is an important signaling molecule throughout the peripheral nervous system. GDNF protein is produced and secreted by skeletal muscle cells and helps maintain motor neuron innervation at the neuromuscular junction. Treatment with exogenous GDNF prevents denervation which occurs with aging and neurodegenerative disease. The therapeutic potential of GDNF cannot be fully explored without understanding the mechanisms by which GDNF protein production is regulated. Although the specific cellular events necessary for regulation of GDNF secretion remains to be elucidated, previous studies from our laboratory have shown that increased skeletal muscle activity significantly alters GDNF protein levels in vivo and in vitro. The primary objective of this study was to determine the role of calcium in regulating GDNF protein secretion by skeletal muscle cells. Skeletal muscle cells (C2C12) were grown in culture and allowed to differentiate into myotubes which display a contractile phenotype. The following treatments were given, Nifedipine to block the voltage‐gated L‐type calcium channels, and Bay K8644 to enhance calcium influx through L‐type calcium channels. Both Western Blot and ELISA were employed for protein identification and GDNF protein quantification respectively. Preliminary results indicate that treatment of myotubes with Bay K8644 (100μM) caused an increase in GDNF secretion to 170% of that in untreated controls. Treatment of cells with Nifedipine blocked the stimulatory effect of Bay K8644, and treatment with Nifedipine alone (10μM and 100μM) inhibited GDNF secretion to 77% of that in control cells. The results suggest that secretion of GDNF protein by C2C12 myotubes is regulated by intracellular calcium levels. Acquiring a greater understanding of the role that calcium plays in regulating GDNF production may help to identify potential sites for therapeutic intervention to increase or decrease GDNF production.Support or Funding InformationNSF Grant DBI‐1062883 and NIH Grant 1R15AG022908‐01A2

Spasmolytic effect of Jasonia glutinosa on rodent intestine.

Jasonia glutinosa is an endemic plant species of the Iberian Peninsula and Southern France traditionally used in infusions as a spasmolytic; this plant is also known as "té de roca" (rock tea) but there is no scientific evidence about the effects of this plant. To evaluate the spasmolytic effect of rock tea. We have studied the in vitro effect of a rock tea extract on rat duodenum spontaneous contractions and the in vivo effect on mice gastrointestinal transit. Rock tea extract reduced the spontaneous contractions of rat duodenal smooth muscle, inhibited KCl-induced contractions and blocked the contractions invoked by both extracellular Ca2+ and the agonist of L-type calcium channels Bay K8644. This inhibitory effect was similar to the one observed after the addition of the antagonist of L-type calcium channels verapamil. Rock tea did not modify gastrointestinal transit in healthy mice. However, after the treatment with dextran sulfate sodium, an inducer of colitis, rock tea extract reverted the increase in the gastrointestinal transit associated with this treatment. Rock tea extract relaxed duodenal smooth muscle via L-type calcium channels and normalized gastrointestinal transit in a model of colitis. These results may validate the traditional use of Jasonia glutinosa in patients with gastrointestinal alterations. Thus, rock tea may be used as a spasmolytic agent to treat gastrointestinal disorders.

The effect of cinnamon extract on isolated rat uterine strips

Cinnamon is a spice used by some populations as a traditional remedy to control blood pressure and thus hypertension. Cinnamon extract decreases contractility in some smooth muscles, but its effect on uterine smooth muscle is unknown. The aim of this study was to determine the physiological and pharmacological effects of cinnamon extract (CE) on the contractions of isolated rat uterine strips and to investigate its possible mechanism of action. Isolated longitudinal uterine strips were dissected from non-pregnant rats, mounted vertically in an organ bath chamber, and exposed to different concentrations of CE (10–20mg/mL). The effect of CE was investigated in the presence of each of the following solutions: 60mM KCl, 5nM oxytocin, and 1μM Bay K8644. CE significantly decreased the force of uterine contraction in a concentration-dependent manner and significantly attenuated the uterine contractions elicited by KCl and oxytocin. In addition, CE significantly decreased the contractile force elicited when L-type Ca2+ channels were activated by Bay K8644. CE's major mechanism may be inhibition of L-type Ca2+ channels, which limits calcium influx. These data demonstrate that CE can be a potent tocolytic that can decrease uterine activity regardless of how the force was produced, even when the uterus was stimulated by agonists. As a result, cinnamon may be used to alleviate menstrual pain associated with dysmenorrhoea or prevent unwanted uterine activity in early pregnancy.

Effect of 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzene-sulfonamide on calcium influx in three contraction models.

2,4,6-Trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide (m-3M3FBS) activates phospholipase C and stimulates apoptosis; however, in smooth muscle cells it may increase the perfusion pressure. The main aim of the present study was to evaluate the physiological effect of direct stimulation of phospholipase C on vascular smooth muscle reactivity using three contraction models. Experiments were performed on the isolated and perfused tail artery of Wistar rats. The contraction force in the present model was measured by an increased level of perfusion pressure with a constant flow. Concentration-response curves (CRCs) obtained for phenylephrine, arg-vasopressin, mastoparan-7 and Bay K8644 presented a sigmoidal association. In comparison to the control curves, CRCs in the presence of m-3M3FBS were significantly shifted to the left except for Bay K8644. Analyses of calcium influx suggest that in the presence of m-3M3FBS the calcium influx from intra- and extracellular calcium stores was significantly higher. The results of the present experiments suggest that m-3M3FBS significantly increases the reactivity of vascular smooth muscle stimulated with metabotropic receptors or G-protein by an increase in calcium influx from intra- and extracellular calcium stores. The current knowledge regarding the apoptotic pathway shows the significance of calcium ions involved in this process, thus, m-3M3FBS may induce apoptosis by an increase of cytoplasmic calcium concentration; however, simultaneously, the use of this mechanism in therapy must be preceded by a molecular modification that eliminates a possible vasoconstriction effect.

Abstract 17632: STIM1 Increases Calcium Stores in the Sarcoplasmic Reticulum of Adult Feline Ventricular Myocytes

Background: STIM1 is a Sarcoplasmic Reticulum (SR) membrane resident protein implicated in sensing and maintaining SR Ca 2+ levels. The role of STIM1 in the regulation of SR Ca 2+ stores in the normal and diseased heart is not well described. Previous reports confirm that STIM1 is present at low levels in the healthy adult heart, but expression levels increase after cardiac injury. Objective: To determine if increased STIM1 expression after cardiac injury may be involved in the disturbed Ca 2+ cycling present within diseased cardiomyocytes. Results: Adenovirus was used to express either STIM1 or red fluorescent protein (RFP) in freshly isolated adult feline ventricular myocytes (AFMs). We found that in normally quiescent myocytes, STIM1 induced high resting SR Ca 2+ load and induced Ca 2+ sparks, which lead to spontaneous contractions by triggering action potentials. Concurrently, pacing of AFMs revealed STIM1 also increased fractional shortening and Ca 2+ transient amplitude. However, STIM1 also induced cell death (60% versus only 5% in RFP controls). Blockade of the L-type Ca 2+ channel (LTCC) with verapamil rescued the cell death caused by STIM1, but was unable to reduce SR load or Ca 2+ sparks. In contrast, inhibition of ORAI channels using BTP2 rescued cell death and abolished Ca 2+ sparks, while canonical transient receptor potential channel inhibition had no effect. STIM1 expression reduced baseline LTCC current amplitude by 20%, but there was no difference in current amplitude after Bay K8644 addition, suggesting channel number was not altered. Conclusions: STIM1 induced Ca 2+ influx can load the SR of large mammalian myocytes, causing Ca 2+ sparks and waves that can trigger action potentials. The induced APs lead to cell death and this effect could be blocked LTCC blockade. STIM1-mediated Ca 2+ influx was dependent on ORAI channel function. Similar to previous reports, we found that STIM1 reduced LTCC current but did not alter LTCC availability. These data show that after cardiac injury, STIM1 and ORAI may provide Ca 2+ influx that positively influences contractility but negatively impacts cell survival and arrhythmias.

H2S induces vasoconstriction of rat cerebral arteries via cAMP/adenylyl cyclase pathway

Hydrogen sulfide (H2S), traditionally known for its toxic effects, is now involved in regulating vascular tone. Here we investigated the vasoconstrictive effect of H2S on cerebral artery and the underlying mechanism. Sodium hydrosulfide (NaHS), a donor of H2S, concentration-dependently induced vasoconstriction on basilar artery, which was enhanced in the presence of isoprenaline, a β-adrenoceptor agonist or forskolin, an adenylyl cyclase activator. Administration of NaHS attenuated the vasorelaxant effects of isoprenaline or forskolin. Meanwhile, the NaHS-induced vasoconstriction was diminished in the presence of 8B-cAMP, an analog of cAMP, but was not affected by Bay K-8644, a selective L-type Ca2+ channel agonist. These results could be explained by the revised effects of NaHS on isoprenaline-induced cAMP elevation and forskolin-stimulated adenylyl cyclase activity. Additionally, NaHS-induced vasoconstriction was enhanced by removing the endothelium or in the presence of L-NAME, an inhibitor of nitric oxide synthase. L-NAME only partially attenuated the effect of NaHS which was given together with forskolin on the pre-contracted artery. In conclusion, H2S induces vasoconstriction of cerebral artery via, at least in part, cAMP/adenylyl cyclase pathway.

Flavonoid galetin 3,6-dimethyl ether attenuates guinea pig ileum contraction through K+ channel activation and decrease in cytosolic calcium concentration

Flavonoid galetin 3,6-dimethyl ether (FGAL) has been isolated from the aerial parts of Piptadenia stipulaceae and has shown a spasmolytic effect in guinea pig ileum. Thus, we aimed to characterize its relaxant mechanism of action. FGAL exhibited a higher relaxant effect on ileum pre-contracted by histamine (EC50=1.9±0.4×10−7M) than by KCl (EC50=2.6±0.5×10−6M) or carbachol (EC50=1.8±0.4×10−6M). The flavonoid inhibited the cumulative contractions to histamine, as well as to CaCl2 in depolarizing medium nominally Ca2+-free. The flavonoid relaxed the ileum pre-contracted by S-(–)-Bay K8644 (EC50=9.5±1.9×10−6M) but less potently pre-contracted by KCl or histamine. CsCl attenuated the relaxant effect of FGAL (EC50=1.1±0.3×10−6M), but apamin or tetraethylammonium (1mM) had no effect (EC50=2.6±0.2×10−7 and 1.6±0.3×10−7M, respectively), ruling out the involvement of small and big conductance Ca2+-activated K+ channels (SKCa and BKCa, respectively). Either 4-aminopyridine or glibenclamide attenuated the relaxant effect of FGAL (EC50=1.8±0.2×10−6 and 1.5±0.5×10−6M, respectively), indicating the involvement of voltage- and ATP-sensitive K+ channels (KV and KATP, respectively). FGAL did not alter the viability of intestinal myocytes in the MTT assay and decreased (88%) Fluo-4 fluorescence, indicating a decrease in cytosolic Ca2+ concentration. Therefore, the relaxant mechanism of FGAL involves pseudo-irreversible noncompetitive antagonism of histaminergic receptors, KV and KATP activation and blockade of CaV1, thus leading to a reduction in cytosolic Ca2+ levels.