Absence Of Nifedipine Research Articles

Retrospective study for classifying hypertensive disorders of pregnancy by time of onset

Preeclampsia is characterised by systemic endothelial cell dysfunction thought to be triggered by toxic/dangerous factors from the placenta, including placental extracellular vesicles (EVs). Why placental EVs become toxic is unknown. We previously reported that preeclamptic sera produced toxic/dangerous placental macrovesicles but whether small EVs are also toxic/dangerous in preeclampsia is unknown.First trimester placental explants were treated with 10% preeclamptic or control sera (n = 10) for 24 h. Micro- and nano-vesicles were harvested by sequential centrifugation. Micro- or nano-vesicles were also exposed to monolayers of endothelial cells in the presence or absence of nifedipine (50 μg/ml) or labetalol (0.5 μg/ml) which are well-known anti-hypertensives in clinical practices.The number and size of micro- and nano-vesicles were counted. Endothelial cell-surface intercellular adhesion molecule 1 (ICAM-1) and high mobility group box 1 (HMGB1) levels in micro- or nano-vesicles were measured by immunoassays.Neither the amount nor size of both micro- and nano-vesicles was different after treating placental explants with preeclamptic or control sera. The levels of HMGB1 were significantly increased in both micro- and nano-vesicles from preeclamptic sera treated placental explants (p < 0.03). Exposing endothelial cells to micro- or nano-vesicles from preeclamptic sera-treated placental explants induced endothelial activation, but it was reversed by co-incubation with nifedipine (p = 0.004) or labetalol (p = 0.002).Our data demonstrate that preeclamptic sera produce toxic/dangerous micro- and nano-placental EVs which activated endothelial cells. This effect was reversed by antihypertensives. The increased levels of HMGB1 in EVs may contribute to endothelial cell activation.

Dual role played by Ca 2+ in the cytotoxicity of human metastatic breast cancer cells

Introduction Cancer cells exhibit enhanced potential to metastasize via increased acto-myosin cross bridges formation which requires increased Ca2+ influx via cell surface voltage-gated Ca2+ channels (VGCC) leading to activation of myosin light chain kinase (MLCK). The MLCK then phosphorylates myosin and forms acto-myosin cross-bridges. The role of MLCK, and calcium channel in cancer cell growth has not been extensively tested. Objective In this study, we tested the individual effects of blocking VGCC and MLCK on MCF-7 cell (human metastatic breast cancer cell) survival. We also treated the cells with high KCl to induce increased Ca2+ influx and test if the effects of KCl will be opposite to that of VGCC blocker. Methods The experimental design of this study is primarily focused on the administration of specific inhibitors to MCF-7 cells with an interest in the dose-dependent response of the cells to varying concentrations of the treatments as well as a thorough comparison of the cell morphology, viability, and proliferation to the effects of the 1% BME control. The three drugs that have been investigated so far are Nifedipine, a voltage-gated calcium blocker, Nifedipine in the presence of KCl [in order to artificially depolarize the cell membrane to open the VGCC], and ML-7, a myosin light chain kinase inhibitor. The cell viability was then assessed through an MTT colorimetric assay at 562 nm in order to visually and quantitatively assess the results. Additionally, transfection studies are planned to be carried out to substantiate pharmacological findings of inhibitors. Results & Conclusion Nifedipine dose-dependently inhibited MCF-7 cell viability. Following were the % absorbance values with increasing doses of nifedipine. All values are relative to control without nifediine considered as 100%; [0.625 um Nif – 43.05%, 1.25 um Nif – 42.19%, 2.5 um Nif – 40.15%, 5 um Nif -39.53%, 10 um Nif – 38.19%, 20 um Nif – 25.1%, 40 um Nif – 22.96%, 1% BME –39.26%]. The 1% BME (bitter melon extract) is the positive control in our studies as our lab have found BME to be toxic to MCF-7 cells. ML-7 also dose-dependently inhibited MCF-7 cell viability. Following were the % absorbance values with increasing doses of ML-7. All values are relative to control without ML-7 considered as 100%; (0.5 um ML7- 70.01%, 1 um ML7 – 65.74%, 2 um ML7 – 57.71%, 4 um ML7 – 49.36%, 8 um ML7 – 48.28%, 16 um ML7 – 34.13%, 1% BME – 36.17%]. When we stimulated cells with increasing doses of KCl, increasing toxicity was seen as reflected by the decreasing absorbance values. We tested the effects of KCl in the presence or absence of nifedipine. Following were the % absorbance values with increasing doses of KCl and/or nifedipine. All values are relative to control (neither KCl nor nifedipine) considered as 100%; [40 mm KCl – 29.55%, 20 mm KCl – 44.024%, 10 mm KCl –56.51%, 40 um Nif – 22.81%, 40 mm KCl + 40 um Nif – 13.69%, 20 mm KCl + 40 um Nif –15.87%, 10 mm KCl + 40 um Nif – 30.91%, EtOH – 86.85%, 1% BME – 35.27%.]. Thus, either inhibiting Ca2+ influx with nifedipine or opening VGCC with high KCl both exerted cytotoxic effects on MCF-7 cells. This suggests that Ca2+ is critical for the viability of these cells but at the same time too much Ca2+ is toxic to the cells.

Eucalyptol reduces airway hyperresponsiveness in rats following cigarette smoke-exposed

BackgroundCigarette smoke is the major cause of airway inflammatory disease, including airway hyperresponsiveness. Eucalyptol (EUC), also named 1.8-cineole, is a monoterpenoid found in essential oil of medicinal plants, showing several biological effects. Hypothesis/purposeBased in the eucalyptol protective activity in respiratory diseases as asthma, our hypothesis is that eucalyptol is able to reduce the airway hyperresponsiveness and the respiratory mechanic parameters in rats exposed to cigarette smoke. Study designWistar rats were divided into control and cigarettes smoke (CS) groups. CS group was daily subjected to cigarette smoke and treated by inhalation for 15 min/day with EUC (1 mg/mL) or vehicle during 30 days. After treatment, bronchoalveolar lavage (BAL) was collected to analyze the inflammatory profile, and tracheal rings were isolated for evaluation of the airway smooth muscle hyperresponsiveness. Lung function was analyzed in vivo. MethodsThe inflammatory profile was evaluated by optical microscopy performing total (Neubauer chamber) and differential leukocyte count (smear slides stained in H&E). The hyperresponsiveness was evaluated in tracheal rings contracted with potassium chloride (KCl) carbamoylcholine (CCh), or Barium chloride (BaCl2) in presence or absence of nifedipine. The lung function (Newtonian resistance-RN) was evaluated by bronco stimulation with methacholine (MCh). ResultsBAL from CS group increased the influx of leukocyte, mainly neutrophils and macrophages compared to control group. EUC reduced by 71% this influx. The tracheal contractions induced by KCl, CCh or BaCl2 were reduced by EUC in 59%, 42% and 26%, respectively. The last one was not different of nifedipine activity. Newtonian resistance (RN) was also reduced in 37% by EUC compared to CS group. ConclusionEUC reduces the hyperresponsiveness and the airway inflammatory profile, recovering the lung function.

Use of a microelectrode array to record extracellular pacemaker potentials from the gastrointestinal tracts of the ICR mouse and house musk shrew (Suncus murinus)

BackgroundThe rhythmic contraction and relaxation of smooth muscles in the gastrointestinal (GI) tract is governed by pacemaker electrical potentials, also termed slow waves, which are calcium currents generated by interstitial cells of Cajal (ICCs). Malfunction of pacemaker rhythms contributes to a number of clinically challenging gastrointestinal motility disorders. MethodA microelectrode array (MEA) was used to record slow waves in vitro from intact GI tissues freshly isolated from the ICR mouse and Suncus murinus. The effects of temperature, extracellular calcium and potassium concentrations on pacemaker potentials were quantified using spatiotemporal metrics. ResultsPacemaker frequency decreased from the duodenum to the ileum in the mouse, but this phenomenon was less significant in Suncus murinus. In both the mouse and Suncus murinus, the stomach had a much lower pacemaker frequency than the intestine. Propagation velocity and amplitude were highest in the proximal intestine. Temperature significantly increased pacemaker frequency in the intestinal tissues of both species. Removal of Ca2+ from the medium inhibited pacemaker potential and increasing the Ca2+ concentration increased pacemaker frequency in the mouse ileum. Increasing K+ concentration decreased pacemaker frequency in the absence of nifedipine. ConclusionsThe MEA allows efficient investigation of gut pacemaker frequency and propagation.

Clarification of serotonin-induced effects in peripheral artery disease observed through the femoral artery response in models of diabetes and vascular occlusion: The role of calcium ions.

Recent findings have demonstrated that serotonin is an important participant in the development and progression of peripheral artery diseases. Taking this into consideration, the goals of this study were to investigate the effects of serotonin on isolated Wistar rat femoral arteries in both healthy and diabetic animals, with and without artery occlusion, with a particular focus on determining the role of calcium in this process. Contraction experiments with serotonin on intact and denuded femoral artery rings, in the presence or absence of nifedipine and ouabain (both separately, or in combination), as well as Ca2+ -free Krebs-Ringer bicarbonate solution were performed. The serotonin-induced results were concentration dependent, but only in healthy animals. The endothelium-dependent contraction of the femoral artery was assessed. In healthy animals, the endothelium-reliant part of contraction was dependent on the extracellular calcium, while the smooth muscle-related part was instead dependent on the intracellular calcium. In diabetic animals, both nifedipine and ouabain influenced serotonin-induced vascular effects by blocking intracellular calcium pathways. However, this was diminished after the simultaneous administration of both blockers.

Treating normal early gestation placentae with preeclamptic sera produces extracellular micro and nano vesicles that activate endothelial cells

ObjectivesPreeclampsia is characterised by systemic endothelial cell dysfunction thought to be triggered by toxic/dangerous factors from the placenta, including placental extracellular vesicles (EVs). Why placental EVs become toxic is unknown. We previously reported that preeclamptic sera produced toxic/dangerous placental macrovesicles but whether small EVs are also toxic/dangerous in preeclampsia is unknown. Study designFirst trimester placental explants were treated with 10% preeclamptic or control sera (n=10) for 24h. Micro- and nano-vesicles were harvested by sequential centrifugation. Micro- or nano-vesicles were also exposed to monolayers of endothelial cells in the presence or absence of nifedipine (50μg/ml) or labetalol (0.5μg/ml) which are well-known anti-hypertensives in clinical practices. Main outcomes measuresThe number and size of micro- and nano-vesicles were counted. Endothelial cell-surface intercellular adhesion molecule 1 (ICAM-1) and high mobility group box 1 (HMGB1) levels in micro- or nano-vesicles were measured by immunoassays. ResultsNeither the amount nor size of both micro- and nano-vesicles was different after treating placental explants with preeclamptic or control sera. The levels of HMGB1 were significantly increased in both micro- and nano-vesicles from preeclamptic sera treated placental explants (p<0.03). Exposing endothelial cells to micro- or nano-vesicles from preeclamptic sera-treated placental explants induced endothelial activation, but it was reversed by co-incubation with nifedipine (p=0.004) or labetalol (p=0.002). ConclusionOur data demonstrate that preeclamptic sera produce toxic/dangerous micro- and nano-placental EVs which activated endothelial cells. This effect was reversed by antihypertensives. The increased levels of HMGB1 in EVs may contribute to endothelial cell activation.

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.

7 The antihypertensives, nifedipine and labetalol prevent endothelial cell activation in response to placental extracellular vesicles released from 1st trimester placental explants that had been treated with preeclamptic sera

Objective Preeclampsia is a human pregnancy-specific disorder that presents as maternal hypertension and proteinuria after 20 weeks of gestation. Although the complete pathogenesis of preeclampsia is still unclear, previous studies have shown that placental extracellular vesicles shed from the syncytiotrophoblast into maternal circulation are associated with this disease. These vesicles can be categorised into macro- micro- and nano-vesicles based on their size. Previous studies have also shown that sera from women with preeclampsia changed the nature of placental macro-vesicles to become more dangerous/toxic such that they activated endothelial cells, but whether preeclamptic sera have the same effect on micro and nanovesicles is untested. In this study, we investigated whether preeclamptic sera affect the amount or nature of micro- and nano-vesicles extruded from first trimester placentae. We also investigated whether two drugs commonly used for symptom relief, nifiedipine or labetalol could reverse the activation of endothelial cells induced by these vesicles. Methods 1st trimester placental explants ( n = 8) were treated with 10% preeclamptic or control sera for 24 h. Micro- and nano-vesicles were harvested by sequential centrifugation at 20,000 g or 100,000 g for 1 h, respectively. The number of micro- or nano-vesicles was counted using a Nanosight (NS300) device. Micro- or nano-vesicles were exposed to monolayers of endothelial cells in the presence or absence of nifedipine (50 μg/ml) or labetalol (0.5 μg/ml). Cell-surface intercellular adhesion molecule 1 (ICAM-1) expression was measured by cell-based ELISA as a marker of endothelial cell activation. Results The amount of both micro- and nano-vesicles was significantly ( p p p = 0.004) or labetalol ( p = 0.002). Conclusion Our data demonstrate that an unknown factor(s) in preeclamptic sera causes an increase in the number of both micro- and nano-vesicles released from placentae which is consistent with the known increase in these vesicles in preeclampsia. The vesicles produced after treating placentae with preeclamptic sera were also more dangerous activating endothelial cells but this effect of the vesicles on endothelial cells is reversible by nifedipine or labetalol.

Effects of nifedipine on the pharmacokinetics of repaglinide in rats: Possible role of CYP3A4 and P-glycoprotein inhibition by nifedipine

BackgroundThe aim of this study was to investigate the effects of nifedipine on the bioavailability and pharmacokinetics of repaglinide in rats. MethodsThe effect of nifedipine on P-glycoprotein (P-gp) and cytochrome P450 (CYP) 3A4 activity was evaluated. The pharmacokinetic parameters of repaglinide and blood glucose concentrations were also determined in rats after oral (0.5mg/kg) and intravenous (0.2mg/kg) administration of repaglinide to rats in the presence and absence of nifedipine (1 and 3mg/kg). ResultsAdministration of nifedipine resulted in inhibition CYP3A4 activity with an IC50 value of 7.8μM, and nifedipine significantly inhibited P-gp activity in a concentration-dependent manner. Compared to the oral control group, nifedipine significantly increased the area under the plasma concentration-time curve (AUC0–∞) and the peak plasma concentration (Cmax) of repaglinide by 49.3 and 25.5%, respectively. Nifedipine significantly decreased the total body clearance (CL/F) of repaglinide by 22.0% compared to the oral control group. Nifedipine also increased the absolute bioavailability (AB) of repaglinide by 50.0% compared to the oral control group (33.6%). In addition, the relative bioavailability (RB) of repaglinide was 1.16- to 1.49-fold greater than that of the control group. Compared to the intravenous control, nifedipine significantly increased AUC0–∞ of repaglinide. Blood glucose concentrations had significant differences compared to the oral control groups. ConclusionNifedipine enhanced the oral bioavailability of repaglinide, which may be mainly attributable to inhibition of CYP3A4-mediated metabolism of repaglinide in the small intestine and/or in the liver and to inhibition of the P-gp efflux transporter in the small intestine and/or reduction of total body clearance by nifedipine. The current study has raised awareness of potential drug interactions by concomitant use of repaglinide with nifedipine.

Lipid Raft-dependent Endocytosis of Close Homolog of Adhesion Molecule L1 (CHL1) Promotes Neuritogenesis

CHL1 plays a dual role by either promoting or inhibiting neuritogenesis. We report here that neuritogenesis-promoting ligand-dependent cell surface clustering of CHL1 induces palmitoylation and lipid raft-dependent endocytosis of CHL1. We identify βII spectrin as a binding partner of CHL1, and we show that partial disruption of the complex between CHL1 and βII spectrin accompanies CHL1 endocytosis. Inhibition of the association of CHL1 with lipid rafts by pharmacological disruption of lipid rafts or by mutation of cysteine 1102 within the intracellular domain of CHL1 reduces endocytosis of CHL1. Endocytosis of CHL1 is also reduced by nifedipine, an inhibitor of the L-type voltage-dependent Ca(2+) channels. CHL1-dependent neurite outgrowth is reduced by inhibitors of lipid raft assembly, inhibitors of voltage-dependent Ca(2+) channels, and overexpression of CHL1 with mutated cysteine Cys-1102. Our results suggest that ligand-induced and lipid raft-dependent regulation of CHL1 adhesion via Ca(2+)-dependent remodeling of the CHL1-βII spectrin complex and CHL1 endocytosis are required for CHL1-dependent neurite outgrowth.

Two mechanistically distinct effects of dihydropyridine nifedipine on CaV1.2 L-type Ca2+ channels revealed by Timothy syndrome mutation

Dihydropyridine Ca2+ channel antagonists (DHPs) block CaV1.2 L-type Ca2+ channels (LTCCs) by stabilizing their voltage-dependent inactivation (VDI); however, it is still not clear how DHPs allosterically interact with the kinetically distinct (fast and slow) VDI. Thus, we analyzed the effect of a prototypical DHP, nifedipine on LTCCs with or without the Timothy syndrome mutation that resides in the I–II linker (LI–II) of CaV1.2 subunits and impairs VDI. Whole-cell Ba2+ currents mediated by rabbit CaV1.2 with or without the Timothy mutation (G436R) (analogous to the human G406R mutation) were analyzed in the presence and absence of nifedipine. In the absence of nifedipine, the mutation significantly impaired fast closed- and open-state VDI (CSI and OSI) at −40 and 0mV, respectively, but did not affect channels' kinetics at −100mV. Nifedipine equipotently blocked these channels at −80mV. In wild-type LTCCs, nifedipine promoted fast CSI and OSI at −40 and 0mV and promoted or stabilized slow CSI at −40 and −100mV, respectively. In LTCCs with the mutation, nifedipine resumed the impaired fast CSI and OSI at −40 and 0mV, respectively, and had the same effect on slow CSI as in wild-type LTCCs. Therefore, nifedipine has two mechanistically distinct effects on LTCCs: the promotion of fast CSI/OSI caused by LI–II at potentials positive to the sub-threshold potential and the promotion or stabilization of slow CSI caused by different mechanisms at potentials negative to the sub-threshold potential.

Nifedipine alters the light-rise of the electro-oculogram in man

To determine if the L-type calcium channel participates in the generation of the light-rise of the electro-oculogram (EOG) in man. The aim was to use nifedipine, a specific, L-type calcium channel inhibitor and determine the effects on the light-rise of the EOG in healthy participants. The EOG was recorded in 14 participants before and after a 10 mg oral dose of fast-acting nifedipine. The Arden index, time to peak of the EOGs and pulse were recorded before and after ingestion of nifedipine. The test-retest variability of the EOG's light-rise in the absence of nifedipine was performed on 11 of the participants. The mean ± SEM oral dose was 147 ± 9 μg/kg. Scotopic electroretinograms (ERGs) were recorded at four intensities (0.0067, 0.0849, 0.364 and 1.140 cd.s.m⁻²) in six participants before and after a 10 mg oral dose of nifedipine. The light-rise of the EOG was significantly reduced in five participants by -22.80 ± 5.6% (p = 0.021), whilst in four of the participants the light-rise increased by +15.7 ± 1.9% (p = 0.033). The results of the test-retest EOGs showed a range of -9.1 to +9.8% Arden index in the absence of nifedipine. Thus, the responses of five participants were not included in the analysis, as the change in the EOG with nifedipine was within this range. The differences in the time to peak of the light-rise were not significantly different in those that showed an increase (p = 0.33) or a decrease (p = 0.87) in the EOG after nifedipine. Pulse rate was not significantly different after nifedipine in the group that showed an increased light-rise (p = 0.77) or in those whose light-rise fell after nifedipine (p = 0.33). No significant effect was observed on the a- and b-wave amplitudes and implicit times for the scotopic components of the scotopic ERG. The L-type calcium channel contributes to the light-rise of the EOG in man.

Nifedipine enhances the relaxant effects of cyclo-oxygenase inhibitors on the bovine ciliary muscle.

The inhibition of cyclo-oxygenase (COX) enzymes and the blockade of Ca (2+) channels play an important role in the regulation of smooth muscle relaxation. This study was designed to investigate the relaxant effects of celecoxib, DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone), and indomethacin, cyclo-oxygenase (COX-1 and -2) inhibitors, in the absence or presence of a nifedipine, L-type Ca(2+) channel blocker, on bovine ciliary muscle. Ciliary muscle strips (n = 12) were mounted in organ baths and tested for changes in isometric tension in response to celecoxib, DFU, and indomethacin. The relaxant effects of celecoxib, DFU, and indomethacin on carbachol-induced contractions in the presence or absence of nifedipine were investigated. Celecoxib (10(-7)-10(-4) M), DFU (10(-7)-10(-4) M), indomethacin (10(-7)-10(-4) M), and nifedipine (10(-7)-10(-4) M) inhibited the carbachol-induced contractions in a concentration-dependent manner. The E(max) value of indomethacin was significantly higher than the E(max) values of celecoxib and DFU in ciliary muscle (P < 0.05), with no significant change in pD(2) values (P > 0.05). The relaxation responses by celecoxib, DFU, and indomethacin were significantly increased in the presence of nifedipine (10(-6) M). There were no significant differences between pEC50 and values of celecoxib, DFU, and indomethacin in the absence of nifedipine (10(-6) M) (P > 0.05), but E(max)values were significantly increased (P < 0.05). These results suggest that the celecoxib, DFU, and indomethacin cause relaxation in ciliary muscle precontracted with carbachol. Blockade of calcium channels with nifedipine in ciliary muscle may increase the relaxant effect of celecoxib, DFU, and indomethacin. The topical or systemic use of celecoxib, DFU, and indomethacin with nifedipine can cause blurred near vision due to ciliary muscle relaxation, and in ocular pain conditions caused by ciliary spasm, the pain can be decreased more easily by combined use of these drugs.

Nitric Oxide does not suppress myogenic tone by activating ryanodine sensitive stores in rat middle cerebral artery

Inhibition of NOS in cerebral arteries results in vasoconstriction and the development of vasomotion, indicating that basal NO synthesis suppresses myogenic tone. Recent studies in rat posterior cerebral artery suggest that NO acts via ryanodine-sensitive calcium stores, evoking Ca2+ sparks, which activate smooth muscle BKCa. We investigated whether a similar action of NO was responsible for the suppression of myogenic tone in the rat middle cerebral artery. Segments of the middle cerebral artery from male Wistar rats were mounted in a wire myograph. SMC membrane potential (Em) was recorded with sharp glass microelectrodes and changes in [Ca2+]i measured using a confocal microscope after loading the SMCs with Oregon Green. Asynchronous oscillations in smooth muscle [Ca2+]i were slightly reduced by nifedipine, but not affected further by the addition of L-NAME. In the additional presence of ryanodine (10 μM) the oscillations were abolished. In the absence of nifedipine, L-NAME caused a significant vasoconstriction (~ 75% max) and vasomotion. Both the BKCa blocker iberiotoxin and ryanodine (alone) mimicked the effect of L-NAME. Application of nifedipine decreased tension and markedly reduced L-NAME induced constriction. These results indicate that in rat middle cerebral arteries, ryanodine-sensitive Ca2+ stores are essential for maintaining the asynchronous oscillations in smooth muscle [Ca2+]i observed under resting conditions, NO does not appear to be essential for initiation of these oscillations. Therefore, the effect of basal release of NO to suppress myogenic tone by stimulating BKCa is not due to an action on ryanodine-sensitive stores but may reflect a direct action of NO on these KCa channels.

Endothelium-dependent vasodilatory effect of vitisin C, a novel plant oligostilbene from Vitis plants (Vitaceae), in rabbit aorta.

We investigated the pharmacological properties of vitisin C, a novel plant oligostilbene from Vitis plants. Vitisin C (1-10 microM) dose-dependently inhibited the contractile responses of endothelium-intact rabbit thoracic aorta induced by phenylephrine (1 microM). These inhibitory effects were abolished in the presence of N (G)-nitro-L-arginine methyl ester (L-NAME; 300 microM), a potent inhibitor of nitric oxide synthase, but not atropine (1 microM), a non-selective muscarinic cholinoceptor antagonist. In endothelium-denuded rabbit aorta, vitisin C was ineffective in attenuating phenylephrine-induced contraction. Moreover, vitisin C (10 microM) increased cGMP production in endothelium-intact, but not endothelium-denuded, aorta, and this increase was abolished in the presence of L-NAME (300 microM). To assess Ca(2+) movement across the endothelial cell membrane induced by vitisin C, we further investigated (45)Ca(2+) influx into cultured rabbit aortic endothelial cells in the presence of vitisin C (3 microM), carbachol (1 microM) or A23187 (10 nM). Vitisin C and carbachol significantly enhanced (45)Ca(2+) influx, which was inhibited by nifedipine (10 microM), a blocker of L-type Ca(2+) channels. In the presence of SK&F96365, a blocker of receptor-operated Ca(2+) channels, (45)Ca(2+) influx induced by carbachol was significantly inhibited, whereas that induced by vitisin C was not affected. On the other hand, A23187 enhanced (45)Ca(2+) influx in the presence and absence of nifedipine and SK&F96365. These results suggest that vitisin C evokes endothelium-dependent vasorelaxation through enhancing nitric oxide release, which is facilitated by Ca(2+) influx into endothelial cells via nifedipine-sensitive Ca(2+) channels.

Stroke development in stroke-prone spontaneously hypertensive rats alters the ability of cerebrovascular muscle to utilize internal Ca2+ to elicit constriction.

The ability of middle cerebral arteries (MCAs) to utilize intracellular smooth muscle (SM) Ca2+ to produce constriction in response to pressure and agonists was assessed in relation to hemorrhagic stroke development in Wistar-Kyoto stroke-prone (SHRSP) and stroke-resistant (srSHR) spontaneously hypertensive rats. MCAs were studied with the use of a pressure myograph at 100 mm Hg. MCAs from srSHR and prestroke SHRSP exhibited pressure-dependent constriction and constricted in response to vasopressin or serotonin in the presence of nifedipine or the absence of [Ca2+]o. MCAs from poststroke SHRSP lost the latter functions and could only constrict in response to vasopressin/serotonin in Krebs' solution containing Ca2+ in the absence of nifedipine. This indicated that the SM could not utilize internal Ca2+ for constriction and maintained constriction by Ca2+ entry through L-type channels. The MCAs of poststroke SHRSP could not constrict to [K+]o-induced depolarization, suggesting that the agonist-induced opening of the L-type channels occurred by mechanisms other than SM depolarization. Depletion of the sarcoplasmic SM Ca2+ stores of MCAs from srSHR with cyclopiazonic acid did not prevent pressure-dependent constriction. Stroke in SHRSP produced a defect in the ability of MCAs to constrict in response to vasopressin or serotonin via the use of an intracellular source of Ca2+. This could be promoted by an inability of the SM to release intracellular Ca2+, by the depletion of internal Ca2+ stores, or by a decrease in the contractile sensitivity to Ca2+ released from the internal stores.

Inhibitory effects of intravenous anaesthetic agents on K+-evoked norepinephrine and dopamine release from rat striatal slices: possible involvement of P/Q-type voltage-sensitive Ca2+ channels

The role of the voltage-sensitive Ca2+ channel (VSCC) as a target for anaesthetic action remains controversial. In this study we characterized the VSCC subtypes involved in K+-evoked norepinephrine and dopamine release from rat striatal slices and used this model system to examine the effects of a range of i.v. anaesthetics on release. Nifedipine (L-channel-selective), omega-conotoxin GVI(A) (N-channel-selective), omega-agatoxin IV(A) (P-channel-selective), omega-conotoxin MVIIc (P/Q-channel-selective) and Cd2+ (non-selective), along with alphaxalone, propofol and ketamine, were used in various combinations. Omega-Agatoxin IV(A), omega-conotoxin MVIIc and Cd2+ fully (100%) inhibited norepinephrine and dopamine release. Clinically achievable concentrations of alphaxalone inhibited norepinephrine and dopamine release, with concentrations producing 25 and 50% inhibition (IC25 and IC50) of the maximum of 2.1 and 7.8 microM respectively for norepinephrine and 2.9 and 7.2 microM for dopamine. The effects of propofol were observed at the top of the clinical range and those of ketamine exceeded this range. In addition, IC50 values for alphaxalone in the presence and absence of nifedipine and omega-conotoxin GVI(A) did not differ from the control. Our data suggest that clinically achievable concentrations of alphaxalone and propofol inhibit norepinephrine and dopamine release, which is mediated predominantly through P/Q-type VSCCs, suggesting a role for these channels in anaesthetic action.

The involvement of intracellular Ca(2+) in 5-HT(1B/1D) receptor-mediated contraction of the rabbit isolated renal artery.

5-Hydroxytryptamine(1B/1D) (5-HT(1B/1D)) receptor coupling to contraction was investigated in endothelium-denuded rabbit isolated renal arteries, by simultaneously measuring tension and intracellular [Ca(2+)], and tension in permeabilized smooth muscle cells. In intact arterial segments, 1 nM - 10 microM 5-HT failed to induce contraction or increase the fura-2 fluorescence ratio (in the presence of 1 microM ketanserin and prazosin to block 5-HT(2) and alpha(1)-adrenergic receptors, respectively). However, in vessels pre-exposed to either 20 mM K(+) or 30 nM U46619, 5-HT stimulated concentration-dependent increases in both tension and intracellular [Ca(2+)]. 1 nM - 10 microM U46619 induced concentration-dependent contractions. In the presence of nifedipine (0.3 and 1 microM) the maximal contraction to U46619 (10 microM) was reduced by around 70%. The residual contraction was abolished by the putative receptor operated channel inhibitor, SKF 96365 (2 microM). With 0.3 microM nifedipine present, 100 nM U46619 evoked similar contraction to 30 nM U46619 in the absence of nifedipine, but contraction to 5-HT (1 nM - 10 microM) was abolished. In permeabilized arterial segments, 10 mM caffeine, 1 microM IP(3) or 100 microM phenylephrine, each evoked transient contractions by releasing Ca(2+) from intracellular stores, whereas 5-HT had no effect. In intact arterial segments pre-stimulated with 20 mM K(+), 5-HT-evoked contractions were unaffected by 1 microM thapsigargin, which inhibits sarco- and endoplasmic reticulum calcium-ATPases. In vessels permeabilized with alpha-toxin and then pre-contracted with Ca(2+) and GTP, 5-HT evoked further contraction, reflecting increased myofilament Ca(2+)-sensitivity. Contraction linked to 5-HT(1B/1D) receptor stimulation in the rabbit renal artery can be explained by an influx of external Ca(2+) through voltage-dependent Ca(2+) channels and sensitization of the contractile myofilaments to existing levels of Ca(2+), with no release of Ca(2+) from intracellular stores.

Relationship between L-type Ca2+ current and unitary sarcoplasmic reticulum Ca2+ release events in rat ventricular myocytes.

1. The time courses of Ca2+ current and Ca2+ spark occurrence were determined in single rat ventricular myocytes voltage clamped with patch pipettes containing 0.1 microM fluo-3. Acquisition of line-scan images on a laser scanning confocal microscope was synchronized with measurement of Cd2+-sensitive Ca2+ currents. In most cells, individual Ca2+ sparks were observed by reducing Ca2+ current density with nifedipine (0.1-8 microM). 2. Ca2+ sparks elicited by depolarizing voltage-clamp pulses had a peak [Ca2+] amplitude of 289 +/- 3 nM with a decay half-time of 20.8 +/- 0.2 ms and a full width at half-maximum of 1.40 +/- 0.03 microm (mean +/- s. e.m., n = 345), independent of the membrane potential. 3. The time between the beginning of a depolarization and the initiation of each Ca2+ spark was calculated and data were pooled to construct waiting time histograms. Exponential functions were fitted to these histograms and to the decaying phase of the Ca2+ current. This analysis showed that the time constants describing Ca2+ current and Ca2+ spark occurrence at membrane potentials between -30 mV and +30 mV were not significantly different. At +50 mV, in the absence of nifedipine, the time constant describing Ca2+ spark occurrence was significantly larger than the time constant of the Ca2+ current. 4. A simple model is developed using Poisson statistics to relate macroscopic Ca2+ current to the opening of single L-type Ca2+ channels at the dyad junction and to the time course of Ca2+ spark occurrence. The model suggests that the time courses of macroscopic Ca2+ current and Ca2+ spark occurrence should be closely related when opening of a single L-type Ca2+ channel initiates a Ca2+ spark. By comparison with the data, the model suggests that Ca2+ sparks are initiated by the opening of a single L-type Ca2+ channel at all membrane potentials encountered during an action potential.

Effects of sympathetic nerve stimulation on membrane potential in the circular muscle layer of mouse distal colon.

Little is known about the effects of sympathetic nerve stimulation on the membrane potential of colonic smooth muscle. In the distal colon of the mouse, intracellular microelectrodes were used to record the effects of lumbar colonic (LCN) and intermesenteric nerve (IMN) stimulation on circular muscle membrane potential in vitro. A two-compartment organ bath was used to selectively perfuse the colon and inferior mesenteric ganglion (IMG). In the presence of nifedipine (1-2 microM) (to the colonic compartment only), spontaneous depolarizations (myoelectric complexes, MCs) were recorded about every 4 min. MCs consisted of a prolonged burst of rapid oscillations in membrane potential (approximately 2 Hz) that were superimposed on a slow depolarization (mean amplitude 12 mV). Single electrical stimuli (600 microseconds duration) delivered to the LCN or IMN did not elicit a detectable change in the membrane potential. However, trains of stimuli (e.g., 60 pulses at 10-20 Hz) to the LCN or IMN during the intervals between MCs evoked a depolarization (with superimposed action potentials in the absence of nifedipine). Trains of stimuli delivered during the plateau phase of the MC reduced or abolished the rapid oscillations, without a further membrane depolarization. The MC period was unaffected by stimulation of the IMN or LCN. Responses were abolished by the selective perfusion of guanethidine (1 microM) to the colon, or by severing the LCN. Hexamethonium (500 microM) (to the colon) abolished MCs, induced sustained depolarization and attenuated the amplitude of the sympathetic depolarizations by 74%. In hexamethonium, sympathetic responses remained attenuated when the membrane of the circular muscle was repolarised by sodium nitroprusside (1 microM). Immunohistochemical studies of the colon revealed intense immunoreactivity for tyrosine hydroxylase in the myenteric plexus but not in the circular muscle layer. It is suggested that responses to sympathetic nerve stimulation in the circular muscle layer of the mouse colon are secondary to actions on the enteric nervous system.