Calcium Channel Activator BAY-K8644 Research Articles

Calcium channel inhibitor and extracellular calcium improve aminoglycoside-induced hair cell loss in zebrafish.

Aminoglycosides are commonly used antibiotics for treatment of gram-negative bacterial infections, however, they might act on inner ear, leading to hair-cell death and hearing loss. Currently, there is no targeted therapy for aminoglycoside ototoxicity, since the underlying mechanisms of aminoglycoside-induced hearing impairments are not fully defined. This study aimed to investigate whether the calcium channel blocker verapamil and changes in intracellular & extracellular calcium could ameliorateaminoglycoside-induced ototoxicity in zebrafish. The present findings showed that a significant decreased number of neuromasts in the lateral lines of zebrafish larvae at 5days' post fertilization after neomycin (20μM) and gentamicin (20mg/mL) exposure, which was prevented by verapamil. Moreover, verapamil (10-100μM) attenuated aminoglycoside-induced toxic response in different external calcium concentrations (33-3300μM). The increasing extracellular calcium reduced hair cell loss from aminoglycoside exposure, while lower calcium facilitated hair cell death. In contrast, calcium channel activator Bay K8644 (20μM) enhanced aminoglycoside-induced ototoxicity and reversed the protective action of higher external calcium on hair cell loss. However, neomycin-elicited hair cell death was not altered by caffeine, ryanodine receptor (RyR) agonist, and RyR antagonists, including thapsigargin, ryanodine, and ruthenium red. The uptake ofneomycininto hair cells was attenuated by verapamil and under high external calcium concentration. Consistently, the production of reactive oxygen species (ROS) in neuromasts exposed to neomycin was also reduced by verapamil and high external calcium. Significantly, zebrafish larvae when exposed to neomycin exhibited decreased swimming distances in reaction to droplet stimulus when compared to the control group. Verapamil and elevated external calcium effectively protected the impaired swimming ability of zebrafish larvae induced by neomycin. These data imply that prevention of hair cell damage correlated with swimming behavior against aminoglycoside ototoxicity by verapamil and higher external calcium might be associated with inhibition of excessive ROS production andaminoglycoside uptake through cation channels. These findings indicate that calcium channel blocker and higher external calcium could be applied to protect aminoglycoside-induced listening impairments.

Peppermint and caraway oils have muscle inhibitory and pro-secretory activity in the human intestine in vitro.

Herbal medicinal products with a broad activity spectrum may be promising alternatives to treat functional gastrointestinal disorders (FGD). Menthacarin® is a drug with a fixed combination of peppermint and caraway oils, which is clinically used to treat FGD-associated symptoms. We studied the effects of peppermint and caraway oils on contractile and secretory activity in 255 human small and large intestinal preparations derived from surgical resections (73 patients). Motility was recorded in circular smooth muscle strips and secretion with the Ussing chamber-voltage clamp technique. Electrical field stimulation evoked nerve induced contractile responses. KEYRESULTS: Peppermint and caraway oil concentrations dependently inhibited muscle contractility as indicated by sustained muscle relaxation and decrease in phasic contractility. These effects occurred in small and large intestinal preparations with IC50 values ranging between 17 and 90µg/mL for peppermint oil and between 7 and 127µg/mL for caraway oil. Neither peppermint nor caraway oil influenced the nerve evoked contractile response. The inhibition of contractile activity, but not the muscle relaxation, was prevented by the L-type calcium channel activator Bay K8644 but not by the neurotoxin tetrodotoxin. Both peppermint oil and caraway oil increased epithelial secretion, which remained in tetrodotoxin. The findings revealed a strong muscle inhibitory and pro-secretory action of peppermint and caraway oils at clinically relevant concentrations. Both actions were nerve-independent. The inhibition of contractility was mediated by inhibition of L-type calcium channels. The effects on muscle and epithelial activity may contribute to the beneficial effects observed in patients with FGD.

Functional Effects of Alagebrium (ALT-711)-Isolated Rat Carotid Artery.

In our study, the effects of glycosylated protein cross-link breaker, alagebrium was investigated on isolated rat carotid artery using myography. Alagebrium showed vasodilator effect on carotid artery rings; particularly, this effect was significantly increased in endothelium-intact rings. To clarify the vasodilator mechanism of alagebrium, different antagonists such as N(G)-Nitro-L-arginine methyl ester (L-NAME), glibenclamide, indomethacin, metoprolol, propranolol, tetraethylammonium, and calcium channel activator BAYK-8644 were used to reverse this effect. Relaxation% responses to alagebrium were more significantly increased in intact endothelium than in denuded arteries. Blocking vasodilation related to channels (K-ATP, PGI2, BKca) and receptors (ß1, ß2) did not reverse the relaxation response to alagebrium. Vasodilator response to alagebrium was only slightly decreased after L-NAME incubation and significantly decreased after BAYK-8644 incubation. Results of present study suggest that the mechanism of alagebrium-induced vasodilator effect may include the blockage of L-type calcium channels and partially of the nitric oxide synthase enzyme.

Phosphoinositide 3-kinase β, phosphoinositide 3-kinase δ, and phosphoinositide 3-kinase γ mediate the anti-inflammatory effects of magnesium sulfate

BackgroundWe previously demonstrated that inhibiting phosphoinositide 3-kinase (PI3K) or activating L-type calcium channels blocked the anti-inflammatory effects of magnesium sulfate (MgSO4). However, the question as which class I PI3K isoform (PI3Kα, PI3Kβ, PI3Kδ, or PI3Kγ) is involved in this regard remains unstudied. The question as whether MgSO4 and L-type calcium channels interact to influence PI3K activation also remains unstudied. We therefore designed this study to test two hypotheses: (1) inhibiting PI3Kα, PI3Kβ, PI3Kδ, or PI3Kγ would block the anti-inflammatory effects of MgSO4 and (2) activating L-type calcium channels would block the effects of MgSO4 on activating PI3K. Materials and methodsPI3K isoform investigation: macrophages (RAW264.7 cells) were treated with endotoxin, endotoxin plus MgSO4, or endotoxin plus MgSO4 plus the selective inhibitor of PI3Kα (PIK-75), PI3Kβ (TGX-221), PI3Kδ (IC-87114), or PI3Kγ (AS-252424). Calcium channel investigation: macrophages were treated with endotoxin, endotoxin plus MgSO4, or endotoxin plus MgSO4 plus the L-type calcium channel activator BAY-K8644. ResultsThe endotoxin plus MgSO4 group presented lower concentrations of inflammatory mediators (macrophage inflammatory protein 2, tumor necrosis factor α, and interleukin 6, lower nuclear concentration of phosphorylated nuclear factor κB, lower cytosolic concentration of phosphorylated inhibitor κBα, and higher concentration of phosphorylated Akt (PI3K activation marker) than the endotoxin group (all P < 0.05). These effects of MgSO4 were significantly reduced by TGX-221, IC-87114, or AS-252424, but not PIK-75. Additionally, BAY-K8644 blocked the effect of MgSO4 on activating PI3K. ConclusionsMgSO4 exerts its anti-inflammatory effects through activating PI3Kβ, PI3Kδ, and PI3Kγ. The underlying mechanism appears to involve inhibition of L-type calcium channels.

Magnesium sulfate provides neuroprotection in lipopolysaccharide-activated primary microglia by inhibiting NF-κB pathway

BackgroundMagnesium sulfate has been used as an anticonvulsant in severe preeclamptic or eclamptic women prior to surgical trauma, but its effects on neuroinflammation is not well defined. In the present study, we investigated the neuroprotective effects of magnesium sulfate in lipopolysaccharide (LPS)-induced microglia and explored the underlying mechanism. Materials and methodsMicroglia was incubated with LPS in the presence or absence of various concentrations of magnesium sulfate, or L-type calcium channel activator BAY-K8644. The levels of inflammatory mediators, such as nitric oxide, prostaglandin E2, interleukin 1β, and tumor necrosis factor α, were measured using enzyme-linked immunosorbent assay. The expression of inducible nitric oxide synthase mRNA was detected by reverse-transcription polymerase chain reaction. Nuclear factor κB (NF-κB) activity in the nuclear extract of microglia was detected by NF-κB p50/p65 transcription factor assay kit. ResultsMagnesium sulfate at 5 and 10 mmol/L significantly inhibited the release of nitric oxide, prostaglandin E2, interleukin 1β, and tumor necrosis factor α, and the expression of inducible nitric oxide synthase mRNA in LPS-activated microglia. Furthermore, magnesium sulfate inhibited the translocation of NF-κB from the cytoplasm to the nucleus in a dose-dependent manner. Notably, these effects were significantly reversed by L-type calcium channel activator BAY-K8644. ConclusionsMagnesium sulfate protects microglia against LPS-induced release of inflammatory mediators, and these effects may be mediated by inhibiting L-type calcium channels and NF-κB signaling.

In-vitro recording of adult zebrafish heart electrocardiogram — A platform for pharmacological testing

Background Recently zebrafish has become a powerful vertebrate model system for cardiac arrhythmia and another advantage is that the heart could be rapidly excised for in-vitro electrophysiological recording. Methods We made direct in-vitro recordings of adult zebrafish heart ECG using the microelectrode and tested the effects of various drugs on zebrafish heart ECG. Results The QT interval change in the ECG was verified by optically mapped action potential duration (APD). The mean in-vitro heart rate (HR) of the adult zebrafish was 118 ± 22 beats/min. The mean QT interval was 312 ± 36 ms and mean HR corrected QT interval (QTc) 439 ± 39 ms. The mean optically mapped APD was 308 ± 30 ms, which was close to the QT interval in ECG (p = 0.815). We found that sympathomimetic drug isoproterenol increased HR, whereas L-type calcium channel blocker verapamil decreased HR. Sodium channel blocker quinidine and L-type calcium channel activator BayK8644 prolonged QTc interval in a dose-dependent manner (515 ± 24 ms and 519 ± 27 ms, respectively, both p < 0.01). The APD was also prolonged accordingly. Both rapidly and slowly activating delayed rectifier potassium channel (IKr and IKs) blockers E4031 and chromanol 293B, respectively, also prolonged QTc interval in a dose-dependent manner (523 ± 25 ms and 529 ± 27 ms, respectively, both p < 0.01). Quinidine, E4031 and chromanol 293B also decreased HR. Conclusions Direct in-vitro recording of adult zebrafish heart is an efficient platform for test of drugs which exert electrophysiological effects on cardiomyocytes, and perturbation of ionic channels that are responsible for human long QT syndrome also modulates QT interval in adult zebrafish heart.

Magnesium Sulfate Mitigates Acute Lung Injury in Endotoxemia Rats

Magnesium sulfate (MgSO4) possesses potent anti-inflammation capacity. We sought to elucidate the effects of MgSO4 on mitigating acute lung injury induced by endotoxemia. MgSO4 is an antagonist of the L-type calcium channels and the N-methyl-D-aspartate (NMDA) receptor. The roles of the L-type calcium channels and NMDA receptor in this regard were also elucidated. Ninety-six adult male rats were randomized to receive normal saline, MgSO4 (100 mg/kg), lipopolysaccharide (LPS), LPS plus MgSO4 (10, 50, or 100 mg/kg), LPS plus MgSO4 (100 mg/kg) plus the L-type calcium channel activator BAY-K8644, or LPS plus MgSO4 (100 mg/kg) plus exogenous NMDA (n=12 in each group). Between-group differences in lung injury were evaluated. Histologic findings, in concert with assays of leukocyte infiltration (polymorphonuclear leukocytes/alveoli ratio and myeloperoxidase activity) and lung water content (wet/dry weight ratio), confirmed that LPS induced acute lung injury. LPS also caused significant inflammatory response (increases in chemokine, cytokine, and prostaglandin E2 concentrations) and imposed significant oxidative stress (increases in nitric oxide and malondialdehyde concentrations) in rat lungs. MgSO4 at the dosages of 50 mg/kg and 100 mg/kg, but not at 10 mg/kg, significantly mitigated the acute lung injury, lung inflammatory response, and oxidative stress caused by endotoxemia. Moreover, the protective effects of MgSO4 were counteracted by BAY-K8644 and exogenous NMDA. MgSO4 mitigates lung inflammatory response, oxidative stress, and acute lung injury in endotoxemia rats in a dose-dependent manner. The mechanisms may involve antagonizing the L-type calcium channels and the NMDA receptor.

In-vitro Recording of Adult Zebrafish Heart Electrocardiogram—A Platform for Pharmacological Testing

Background: Recently zebrafish has become a powerful vertebrate model system for cardiac arrhythmia and another advantage is that the heart could be rapidly excised for in-vitro electrophysiological recording. Methods: We made direct in-vitro recordings of adult zebrafish heart ECG using the microelectrode array and tested the effects of various drugs on zebrafish heart ECG. Results: The QT interval change in the ECG was verified by optically mapped action potential duration (APD). The mean optically mapped APD was 308±30 ms, which was close to the QT interval in ECG (p=0.815). We found that sympathomimetic drug isoproterenol increased HR, whereas L-type calcium channel blocker verapamil decreased HR. Sodium channel blocker quinidine and L-type calcium channel activator BayK8644 prolonged QTc interval in a dose-dependent manner (515±24 ms and 519±27 ms, respectively, both p<0.01). The APD was also prolonged accordingly. Both rapidly and slowly activating delayed rectifier potassium channel (IKr and IKs) blockers E4031 and Chromanol 293B, respectively, also prolonged QTc interval in a dose-dependent manner (523±25 ms and 529±27 ms, respectively, both p<0.01). Quinidine, E4031 and Chromanol 293B also decreased HR. Conclusions: Direct in-vitro recording of adult zebrafish heart is an efficient platform for test of drugs which exert electrophysiological effects on cardiomyocytes, and perturbation of ionic channels that are responsible for human long QT syndrome also modulates QT interval in adult zebrafish heart.

Magnesium Sulfate Inhibits Activator Protein-1 Upregulation in Endotoxin-activated Murine Macrophages

Objective The expression of inflammatory molecules is regulated by the transcription factor activator protein-1 (AP-1), a heterodimeric protein that consists of proteins from various families, including c-Jun and c-Fos. We sought to elucidate whether MgSO 4 regulates the activation of AP-1 in endotoxin-activated RAW264.7 cells, a murine macrophage-like cell line. The possible roles of the L-type calcium channels in this process were also elucidated. Materials and Methods RAW264.7 cells were treated with phosphate buffered saline, MgSO 4, lipopolysaccharide (LPS), LPS plus MgSO 4 (20 mM), or LPS plus MgSO 4 plus the L-type calcium channel activator BAY-K8644 (1 μM). After harvesting, expression of AP-1 was evaluated. Results LPS induced AP-1 activation based on the fact that the nuclear protein concentrations of AP-1 components, including c-Jun and c-Fos, as well as the AP-1 DNA-binding activity, were significantly increased in LPS-treated RAW264.7 cells. MgSO 4, in contrast, significantly inhibited LPS-induced AP-1 activation in activated RAW264.7 cells. Moreover, the effect of MgSO 4 on AP-1 was reversed by BAY-K8644. Conclusion MgSO 4 inhibited AP-1 activation in LPS-treated macrophages and the mechanism may involve the L-type calcium channels.

L-type calcium channels are involved in mediating the anti-inflammatory effects of magnesium sulphate

Magnesium sulphate (MgSO(4)) has potent anti-inflammatory capacity. It is a natural calcium antagonist and a potent L-type calcium channel inhibitor. We sought to elucidate the possible role of calcium, the L-type calcium channels, or both in mediating the anti-inflammatory effects of MgSO(4). RAW264.7 cells, an immortalized murine macrophage-like cell line, were treated with phosphate buffered saline, MgSO(4), lipopolysaccharide (LPS), LPS plus MgSO(4), LPS plus MgSO(4) plus extra-cellular supplement with calcium chloride (CaCl(2)), or LPS plus MgSO(4) plus the L-type calcium channel activator BAY-K8644. After harvesting, the production of inflammatory molecules was evaluated. Because the production of endotoxin-induced inflammatory molecules is regulated by the crucial transcription factor nuclear factor (NF)-kappaB, we also evaluated the expression of NF-kappaB. LPS significantly induced the production of inflammatory molecules, including macrophage inflammatory protein-2, tumour necrosis factor-alpha, interleukin (IL)-1beta, IL-6, nitric oxide/inducible nitric oxide synthase, and prostaglandin E(2)/cyclo-oxygenase-2. LPS also induced NF-kappaB activation, as inhibitor-kappaB degradation, NF-kappaB nuclear translocation, and NF-kappaB-DNA binding activity were significantly increased in LPS-treated RAW264.7 cells. MgSO(4), in contrast, significantly inhibited the LPS-induced inflammatory molecules production and NF-kappaB activation. Moreover, the effects of MgSO(4) on inflammatory molecules and NF-kappaB were reversed by extra-cellular calcium supplement with CaCl(2) and L-type calcium channel activator BAY-K8644. MgSO(4) significantly inhibited endotoxin-induced up-regulation of inflammatory molecules and NF-kappaB activation in activated RAW264.7 cells. The effects of MgSO(4) on inflammatory molecules and NF-kappaB may involve antagonizing calcium, inhibiting the L-type calcium channels, or both.

Dimemorfan prevents seizures induced by the L-type calcium channel activator BAY k-8644 in mice

A dextromethorphan (3-methoxy-17-methylmorphinan) analog, dimemorfan (3-methyl- N-methylmorphinan) that is not metabolized to dextrorphan [3-hydroxy-17-methylmorphinan, which induces phencyclidine (PCP)-like behavioral effects], attenuates maximal electroshock seizures. However, the pharmacological mechanism of action of dimemorfan remains to be determined. In this study, we assessed the locomotor activity mediated by these morphinans. Circling behavior was pronounced in mice treated with PCP or dextrorphan, while animals treated with dextromethorphan exhibited moderate behaviors. Dimemorfan did not show any significant behavioral effects. We used BAY k-8644 (an L-type Ca 2+ channel agonist in the dihydropyridine class) to explore the effects of dextromethorphan and dimemorfan on the convulsant activity regulated by calcium channels. Intracerebroventricular injection of BAY k-8644 (37.5 μg) significantly induced seizures in mice. As with dextromethorphan (6.25 or 12.5 mg/kg), dimemorfan (6.25 or 12.5 mg/kg) pretreatment significantly attenuated BAY k-8644-induced seizures in a dose-dependent manner. BAY k-8644-induced seizure activity paralleled increased expression of c- fos and c- jun, AP-1 DNA binding activity, and fos-related antigen immunoreactivity. Pretreatment with dextromethorphan or dimemorfan significantly attenuated the expression induced by BAY k-8644. Therefore, our results suggest that the anticonvulsant effects of dextromethorphan and dimemorfan are mediated, at least in part, via L-type calcium channel, and that dimemorfan is equipotent to dextromethorphan in preventing BAY k-8644-induced seizures, while it lacks behavioral side effects related to psychotomimetic reactions.

Acidosis-induced coronary constriction in the rat heart: evidence for the activation of L-type calcium channels.

Perfused rat hearts were used to study the effects of acidosis on coronary tone. When pH was decreased, over the range pH 7.4 to pH 6.2, by reducing perfusate bicarbonate levels, under constant flow conditions, there was a transient decrease in coronary perfusion pressure (CPP), followed by a sustained acidosis-dependent increase in CPP, which reversed when pH was returned to pH 7.4. This increase in CPP was seen at perfusion rates of 5, 10, and 20 ml/min(-1). When using constant pressure perfusion acidosis reduced coronary flow. In a HEPES-buffered bicarbonate-free solution, acidosis did not cause a transient fall in CPP but it did produce a sustained increase in CPP. Addition of ammonium chloride (10 mM) reduced CPP, while washout of ammonium chloride increased CPP. The acidosis-induced increase in CPP was not affected by indomethacin, nitro-L-arginine, the nonselective adenosine receptor antagonist, 8-phenyl theophylline, or the thromboxane receptor antagonist, ZD 1542. The acidosis-induced increase in CPP was independent of the myocardial depressant effects of acidosis, but was attenuated by three different L-type calcium channel blockers. These results demonstrate that the coronary circulation of the rat constricts in response to acidosis. Experiments performed with L-type calcium channel blockers, and the calcium channel activator BAY K8644, suggest that constriction occurs via activation of L-type calcium channels. This would not be expected on the basis of electrophysiological studies, which have shown an inhibition of L-type calcium channels by acidosis.

Muscimol-induced death of GABAergic neurons in rat brain aggregating cell cultures

During brain development, spontaneous neuronal activity has been shown to play a crucial role in the maturation of neuronal circuitries. Activity-related signals may cause selective neuronal cell death and/or rearrangement of neuronal connectivity. To study the effects of sustained inhibitory activity on developing inhibitory (GABAergic) neurons, three-dimensional primary cell cultures of fetal rat telencephalon were used. In relatively immature cultures, muscimol (10 μM), a GABA A receptor agonist, induced a transient increase in apoptotic cell death, as evidenced by a cycloheximide-sensitive increase of free nucleosomes and an increased frequency of DNA double strand breaks (TUNEL labeling). Furthermore, muscimol caused an irreversible reduction of glutamic acid decarboxylase activity, indicating a loss of GABAergic neurons. The muscimol-induced death of GABAergic neurons was attenuated by the GABA A receptor blockers bicuculline (100 μM) and picrotoxin (100 μM), by depolarizing potassium concentrations (30 mM KCl) and by the L-type calcium channel activator BAY K8644 (2 μM). As compared to the cholinergic marker (choline acetyltransferase activity), glutamic acid decarboxylase activity was significantly more affected by various agents known to inhibit neuronal activity, including tetrodotoxin (1 μM), flunarizine (5 μM), MK 801 (50 μM) and propofol (40 μM). The present results suggest that the survival of a subpopulation of immature GABAergic neurons is dependent on sustained neuronal activity and that these neurons may undergo apoptotic cell death in response to GABA A autoreceptor activation.

Angiotensin II and potassium regulate human CYP11B2 transcription through common cis-elements.

Aldosterone synthase is a mitochondrial enzyme that catalyzes the conversion of 11-deoxycorticosterone to the potent mineralocorticoid aldosterone. The gene encoding aldosterone synthase, CYP11B2, is expressed in the zona glomerulosa of the adrenal cortex. Although the major physiological regulators of aldosterone production are angiotensin II (ANG II) and potassium (K+), the mechanisms by which these compounds regulate CYP11B2 transcription are unknown. Therefore we analyzed the human CYP11B2 5'-flanking region using a transient transfection expression system in the H295R human adrenocortical cell line. ANG II and K+ increased expression of a luciferase reporter construct containing 2015 bp of human CYP11B2 5'-flanking DNA. This response was mimicked by treatment with the calcium channel activator BAYK8644, whereas activation of the protein kinase C pathway with 12-o-tetradecanoylphorbol-13-acetate had no effect. Reporter gene activity was also increased after activation of cAMP-dependent pathways by (Bu)2cAMP. Deletion, mutation, and deoxyribonuclease I footprinting analyses of the CYP11B2 5'-flanking region identified two distinct elements at positions -71/-64 (TGACGTGA) and -129/-114 (CTCCAGCCTTGACCTT) that were both required for full basal reporter gene activity and for maximal induction by either cAMP or calcium-signaling pathways. The -71/-64 element, which resembles a consensus cAMP response element (CRE), bound CRE-binding proteins from H295R cell nuclear extracts as determined by electrophoretic mobility shift analysis. Analysis of the -129/-114 element using electrophoretic mobility shift analysis demonstrated binding of the orphan nuclear receptors steroidogenic factor 1 and chicken ovalbumin upstream promoter transcription factor. These data demonstrate that ANG II, K+, and cAMP-signaling pathways utilize the same SF-1 and CRE-like cis-elements to regulate human CYP11B2 expression.

Angiotensin II and Potassium Regulate Human CYP11B2 Transcription through Common cis-Elements

Aldosterone synthase is a mitochondrial enzyme that catalyzes the conversion of 11-deoxycorticosterone to the potent mineralocorticoid aldosterone. The gene encoding aldosterone synthase, CYP11B2, is expressed in the zona glomerulosa of the adrenal cortex. Although the major physiological regulators of aldosterone production are angiotensin II (ANG II) and potassium (K+), the mechanisms by which these compounds regulate CYP11B2 transcription are unknown. Therefore we analyzed the human CYP11B2 5′-flanking region using a transient transfection expression system in the H295R human adrenocortical cell line. ANG II and K+ increased expression of a luciferase reporter construct containing 2015 bp of human CYP11B2 5′-flanking DNA. This response was mimicked by treatment with the calcium channel activator BAYK8644, whereas activation of the protein kinase C pathway with 12-o-tetradecanoylphorbol-13-acetate had no effect. Reporter gene activity was also increased after activation of cAMP-dependent pathways by (Bu)2cAMP. Deletion, mutation, and deoxyribonuclease I footprinting analyses of the CYP11B2 5′-flanking region identified two distinct elements at positions −71/−64 (TGACGTGA) and −129/−114 (CTCCAGCCTTGACCTT) that were both required for full basal reporter gene activity and for maximal induction by either cAMP or calcium-signaling pathways. The −71/−64 element, which resembles a consensus cAMP response element (CRE), bound CRE-binding proteins from H295R cell nuclear extracts as determined by electrophoretic mobility shift analysis. Analysis of the −129/−114 element using electrophoretic mobility shift analysis demonstrated binding of the orphan nuclear receptors steroidogenic factor 1 and chicken ovalbumin upstream promoter transcription factor. These data demonstrate that ANG II, K+, and cAMP-signaling pathways utilize the same SF-1 and CRE-like cis-elements to regulate human CYP11B2 expression.

The effect of BAY K-8644 on cytotoxic edema induced by total ischemia of rat brain.

The calcium channel activator BAY K-8644, a dihydropyridine (DHP) derivative, has been shown to possess neurochemical and behavioral activities, but its effect on ischemic brain damage has remained unknown. This report describes the effect of the drug on the progression of cytotoxic edema induced by total ischemia of the brain, evaluated by measuring the time constant, k, of elongation of the 1H-NMR relaxation time (T2) after brain biopsy. Twenty-six male Wistar rats were divided into four groups, (a) control (saline) group (n = 10), (b) BAY K-8644 vehicle group (n = 4), (c) BAY K-8644 0.03 mg/kg group (n = 6) and (d) BAY K-8644 0.3 mg/kg group (n = 6). The k value of group (d), 18.2 +/- 5.8 min (mean +/- SD), was significantly higher compared with those of groups (a) 10.3 +/- 1.6, (b) 11.8 +/- 1.5 and (c) 9.8 +/- 3.3 min (p < 0.01 by ANOVA). These results indicate that BAY K-8644 delayed the progression of cytotoxic edema induced by total ischemia of the rat brain.

Calcium Antagonistic Properties of the Sesquiterpene T‐Cadinol: A Comparison with Nimodipine in the Isolated Rat Aorta

(+)-T-Cadinol is a sesquiterpene with smooth muscle relaxing properties. In the isolated rat aorta, T-cadinol relaxed contractions induced by 60 mM K+ in a concentration-dependent fashion. The dihydropyridine calcium antagonist nimodipine was approximately 4,000 times more potent than T-cadinol. While both drugs nearly abolished the K(+)-induced contractions, they only partially relaxed contractions induced by phenylephrine. The relaxation induced by T-cadinol and nimodipine in K(+)-contracted aortic rings, was completely reversed by the calcium channel activator Bay K8644. In aortic preparations partially depolarized by 20 mM K+, Bay K8644 induced a concentration-dependent contraction. Nimodipine shifted the Bay K8644 concentration-response curve to the right in a parallel manner, consistent with a competitive mode of inhibition. T-cadinol at concentrations less than 10(-3.5) M also produced a right-ward shift of the Bay K8644 concentration-response curve with a maintained maximum response. However, the highest T-cadinol concentration used 10(-3.5 M) significantly reduced the maximum response. In conclusion, although T-cadinol and nimodipine display marked structural differences, their pharmacological profiles of action have several features in common, suggesting that T-cadinol is a calcium antagonist, possibly interacting with the dihydropyridine binding sites on the calcium channels.

Influence of Bay K8644 on vascular responses mediated by α1- and α2-adrenoceptors

1. The calcium channel activator Bay K8644 increased the potency of noradrenaline in cat middle cerebral ( α 2-adrenoceptors) and mesenteric (atypical or mixed α 1- and α 2-adrenoceptor population) arteries, but not in rat middle cerebral and mesenteric arteries ( α 1-adrenoceptors). 2. In cat arteries, exposure to 15mM K + solution shifted the noradrenaline concentration-response curve to the left in an almost identical manner as did Bay K8644. 3. Bay K8644 completely reversed the relaxation produced by nifedipine in K +-contracted cat middle cerebral arteries, whereas the relaxation induced by verapamil, diltiazem or flunarizine was only partially reversed. This suggests a specific interaction between Bay K8644 and the dihydropyridine receptors on the calcium channels. 4. It is concluded that the degree to which noradrenaline promotes calcium influx through membrane channels is at least partly related to the α-adrenoceptor subtype mediating the response.

Accurate freehand injection into the lateral brain ventricle of the conscious mouse.

Accurate freehand injection into the lateral brain ventricle of the conscious mouse.