Calcium Channel Agonist Bay Research Articles

Hydroxychloroquine and azithromycin alter the contractility of living porcine heart slices.

The cardiotoxicity risk of hydroxychloroquine (HCQ) and azithromycin (AZM) has been the subject of intensive research triggered by safety concerns in COVID-19 patients. HCQ and AZM have been associated with QT interval prolongation and drug-induced arrhythmias, however other cardiotoxicity mechanisms remain largely unexplored. Our group has pioneered the living heart slice preparation, an ex-vivo platform that maintains native cardiac tissue architecture and physiological electrical and contractile properties. Here, we evaluated the cardiotoxic effect of HCQ and AZM applied alone or in combination on cardiac contractility by measuring contractile force and contraction kinetics in heart slices prepared from porcine hearts. Our results show that clinically relevant concentrations of HCQ monotherapy (1-10µM) reduced contractile force and contraction kinetics in porcine slices in a dose-dependent manner. However, AZM monotherapy decreased contractile force and contraction kinetics only at higher concentrations (30µM). Combination of HCQ and AZM induced a dose-dependent effect similar to HCQ alone. Furthermore, pre-treating porcine heart slices with the L-type calcium channel agonist Bay K8644 prevented the effect of both drugs, while administration of Bay K8644 after drugs interventions largely reversed the effects, suggesting a mechanism involving inhibition of L-type calcium channels. These findings indicate that HCQ and AZM alter cardiac function beyond QT prolongation with significant contractile dysfunction in intact cardiac tissue. Our porcine heart slices provide a powerful platform to investigate mechanisms of drug cardiotoxicity.

Calcium-Prolactin Secretion Coupling in Rat Pituitary Lactotrophs Is Controlled by PI4-Kinase Alpha

The role of calcium, but not of other intracellular signaling molecules, in the release of pituitary hormones by exocytosis is well established. Here, we analyzed the contribution of phosphatidylinositol kinases (PIKs) to calcium-driven prolactin (PRL) release in pituitary lactotrophs: PI4Ks - which control PI4P production, PIP5Ks - which synthesize PI(4, 5)P2 by phosphorylating the D-5 position of the inositol ring of PI4P, and PI3KCs – which phosphorylate PI(4, 5)P2 to generate PI(3, 4, 5)P3. We used common and PIK-specific inhibitors to evaluate the strength of calcium-secretion coupling in rat lactotrophs. Gene expression was analyzed by single-cell RNA sequencing and qRT-PCR analysis; intracellular and released hormones were assessed by radioimmunoassay and ELISA; and single-cell calcium signaling was recorded by Fura 2 imaging. Single-cell RNA sequencing revealed the expression of Pi4ka, Pi4kb, Pi4k2a, Pi4k2b, Pip5k1a, Pip5k1c, and Pik3ca, as well as Pikfyve and Pip4k2c, in lactotrophs. Wortmannin, a PI3K and PI4K inhibitor, but not LY294002, a PI3K inhibitor, blocked spontaneous action potential driven PRL release with a half-time of ~20 min when applied in 10 µM concentration, leading to accumulation of intracellular PRL content. Wortmannin also inhibited increase in PRL release by high potassium, the calcium channel agonist Bay K8644, and calcium mobilizing thyrotropin-releasing hormone without affecting accompanying calcium signaling. GSK-A1, a specific inhibitor of PI4KA, also inhibited calcium-driven PRL secretion without affecting calcium signaling and Prl expression. In contrast, PIK93, a specific inhibitor of PI4KB, and ISA2011B and UNC3230, specific inhibitors of PIP5K1A and PIP5K1C, respectively, did not affect PRL release. These experiments revealed a key role of PI4KA in calcium-secretion coupling in pituitary lactotrophs downstream of voltage-gated and PI(4, 5)P2-dependent calcium signaling.

The calcium channel agonist Bay K 8644 promotes the growth of human liver cancer HepG2 cells in vitro: suppression with overexpressed regucalcin.

Hepatocellular carcinoma is one of the most prevalent malignant diseases and causes a third of cancer-related death. The consequences of altered calcium homeostasis in cancer cells may contribute to tumor progression. Regucalcin plays an inhibitory role in calcium signaling linked to transcription regulation. Regucalcin gene expression is downregulated in the tumor tissues of liver cancer patients, suggesting an involvement as a suppressor in hepatocarcinogenesis. We investigated whether Bay K 8644, an agonist of the L-type Ca2+ channel, promotes the growth of human liver cancer and if the effect of Bay K 8644 is suppressed by overexpressed regucalcin using the HepG2 cell model. The colony formation and growth of HepG2 cells were promoted by culturing with Bay K 8644 (0.1-10nM). This effect was suppressed by inhibitors of signaling processes linked to cell proliferation, including PD98059 and wortmannin. Death of HepG2 cells was stimulated by Bay K 8644 with higher concentrations (25 and 100nM). The effects of Bay K 8644 on cell growth and death were abolished by verapamil, an antagonist of calcium channel. Mechanistically, culturing with Bay K 8644 increased levels of mitogen-activated protein kinase (MAPK) and phospho-MAPK. Notably, overexpressed regucalcin suppressed Bay K 8644-promoted growth and death of HepG2 cells. Furthermore, overexpressed regucalcin prevented growth and increased death induced by thapsigargin, which induces the release of intracellular stored calcium. Thus, higher regucalcin expression suppresses calcium signaling linked to the growth of liver cancer cells, providing a novel strategy in treatment of hepatocellular carcinoma with delivery of the regucalcin gene.

Taurine–magnesium coordination compound, a potential anti-arrhythmic complex, improves aconitine-induced arrhythmias through regulation of multiple ion channels

Taurine–magnesium coordination compound (TMCC) exhibits antiarrhythmic effects in cesium–chloride-and ouabain-induced arrhythmias; however, the mechanism underlying these effects on arrhythmia remains poorly understood. Here, we investigated the effects of TMCC on aconitine-induced arrhythmia in vivo and the electrophysiological effects of this compound in rat ventricular myocytes in vitro. Aconitine was used to induce arrhythmias in rats, and the dosages required to produce ventricular premature contraction (VPC), ventricular tachycardia (VT), ventricular fibrillation (VF), and cardiac arrest (CA) were recorded. Additionally, the sodium current (INa) and L-type calcium current (ICa,L) were analyzed in normal and aconitine-treated ventricular myocytes using whole-cell patch-clamp recording. In vivo, intravenous administration of TMCC produced marked antiarrhythmic effects, as indicated by the increased dose of aconitine required to induce VPC, VT, VF, and CA. Moreover, this effect was abolished by administration of sodium channel opener veratridine and calcium channel agonist Bay K8644. In vitro, TMCC inhibited aconitine-induced increases in INa and ICa,L. These results revealed that TMCC inhibited aconitine-induced arrhythmias through effects on INa and ICa,L.

Phenytoin inhibits contractions of rat gastrointestinal and portal vein smooth muscle by inhibiting calcium entry.

Phenytoin is widely used as a second-line treatment for status epilepticus. Besides its well-known cardiac pro-arrhythmogenicity, side effects on other organ systems have received less attention. This study investigates the effects of phenytoin on gastrointestinal tissue function using an invitro model of smooth muscle preparations from rats by combining registrations of pharmacological effects on mechanical contractions, electric field potentials, and dynamic intravital fluorescence microscopy. When added to the bathing solution at a concentration of 30μM, phenytoin reduced the frequency of spontaneous activity significantly in antrum and portal vein preparations to 72.2±36.5% (p=0.022) and 80.7±24.4% (p=0.037) of control values, respectively. At a concentration of 100μM, theheight of spontaneous contractions declined to 9.8±19.6% (p=0.005) (antrum), 15.7±28.2% (p=0.004) (portal vein), and 31.8±31.3% (p= 0.005) (colon) in comparison to the control conditions before the application of phenytoin. Depolarization triggered increases in calcium dependent fluorescence signals were reduced by 52.8±39.1% (p=0.012) The inhibition of spontaneous activity caused by phenytoin was reduced in the presence of the L-type calcium channel agonist BAY K8644(-). Phenytoin exerts strong inhibitory effects on the spontaneous and stimulated contractile activity of smooth muscles from both the upper and lower gastrointestinal tract. The mechanism underlying thiseffect is not related to the sodium channel blocking activity of phenytoin, but is rather causedby an inhibition of calcium entry through voltage dependent L-type calcium channels. The results of this study should raise vigilance to gastrointestinal complications in patients treated with phenytoin.

The calcium channel agonist BAY K8644 enhances the responsiveness of human airway muscle to KCl and histamine but not to carbachol.

The role of calcium channel antagonists in asthma remains controversial. A new compound BAY K8644 is an agonist at the calcium voltage-dependent channel (VDC). Therefore, we studied the effect of BAY K8644 on contraction to KCl, histamine, and carbachol in human bronchus in vitro to assess the role of the VDC in this tissue. Cumulative concentration response curves were obtained to the agonists in bronchial spiral strips in the absence and in the presence of BAY K8644. Contractile responses to histamine were significantly (p less than 0.05) enhanced by BAY K8644 (10(-6) M) at concentrations between 10(-7) M and 3 X 10(-6) M and there was a significant decrease in the histamine EC50 (the concentration producing half the maximal response). Responses to low concentrations of KCl (10(-4) M to 3 X 10(-3) M) were also potentiated. BAY K8644 did not affect carbachol-induced contractions. We conclude that calcium influx through VDC occurs in response to KCl and histamine but not to carbachol. Such findings could explain the variability in efficacy of calcium channel antagonists in inhibiting various forms of bronchospasm.

Physiological and Pharmacological Modulation of the Embryonic Skeletal Muscle Calcium Channel Splice Variant CaV1.1e

CaV1.1e is the voltage-gated calcium channel splice variant of embryonic skeletal muscle. It differs from the adult CaV1.1a splice variant by the exclusion of exon 29 coding for 19 amino acids in the extracellular loop connecting transmembrane domains IVS3 and IVS4. Like the adult splice variant CaV1.1a, the embryonic CaV1.1e variant functions as voltage sensor in excitation-contraction coupling, but unlike CaV1.1a it also conducts sizable calcium currents. Consequently, physiological or pharmacological modulation of calcium currents may have a greater impact in CaV1.1e expressing muscle cells. Here, we analyzed the effects of L-type current modulators on whole-cell current properties in dysgenic (CaV1.1-null) myotubes reconstituted with either CaV1.1a or CaV1.1e. Furthermore, we examined the physiological current modulation by interactions with the ryanodine receptor using a chimeric CaV1.1e construct in which the cytoplasmic II-III loop, essential for skeletal muscle excitation-contraction coupling, has been replaced with the corresponding but nonfunctional loop from the Musca channel. Whereas the equivalent substitution in CaV1.1a had abolished the calcium currents, substitution of the II-III loop in CaV1.1e did not significantly reduce current amplitudes. This indicates that CaV1.1e is not subject to retrograde coupling with the ryanodine receptor and that the retrograde coupling mechanism in CaV1.1a operates by counteracting the limiting effects of exon 29 inclusion on the current amplitude. Pharmacologically, CaV1.1e behaves like other L-type calcium channels. Its currents are substantially increased by the calcium channel agonist Bay K 8644 and inhibited by the calcium channel blocker nifedipine in a dose-dependent manner. With an IC50 of 0.37 μM for current inhibition by nifedipine, CaV1.1e is a potential drug target for the treatment of myotonic dystrophy. It might block the excessive calcium influx resulting from the aberrant expression of the embryonic splice variant CaV1.1e in the skeletal muscles of myotonic dystrophy patients.

Antispasmodic effects of myrrh due to calcium antagonistic effects in inflamed rat small intestinal preparations.

Myrrh is the oleo-gum resin of mainly Commiphora molmol and as a powdered substance, one compound in the traditional medicinal product Myrrhinil-Intest®, which has been used for the treatment of unspecific, inflammatory intestinal disorders. The aim of the present study was to evaluate the antispasmodic effect of myrrh under healthy and inflamed conditions, and to evaluate a calcium-antagonistic effect as a possible mode of action. Therefore, an ethanolic myrrh extract was tested for its effects on muscle tone and acetylcholine-induced contractions in untreated and inflamed rat ileum/jejunum preparations. Inflammation was experimentally induced by 2,4,6-trinitrobenzene sulfonic acid (10 mM, 30 min). Additionally, the effect of the calcium channel agonist Bay K8644 in the presence of varying myrrh extract concentrations was examined. Myrrh extract (0.99 mg/mL) suppressed the acetylcholine-induced contraction down to 25.8 % in untreated and 15.2 % in inflamed preparations. Myrrh extract (0.15; 0.25 and 0.35 mg/mL) induced a concentration-dependent rightward shift of the Bay K8644 concentration-response curve in untreated and inflamed preparations with a significant EC50 shift. Schild analysis resulted in a pA2 value of 0.93 for untreated preparations. Increasing myrrh extract concentrations induced a concentration-dependent decrease of the agonistic maximum effect in untreated and inflamed preparations down to 15.8 % and 25.8 %, respectively, for the highest concentration leading to a pD2 value of 0.58. Myrrh extract reduced intestinal muscle tone and acetylcholine-induced contraction of untreated and inflamed ileum/jejunum preparations based on dual calcium antagonism characterized by a right shift of the agonistic dose-response curve and a depression of the maximum effect. The resulting reduction of intestinal motility and spasmolytic effects provide a rationale for the symptom treatment of intestinal disorders such as irritable bowel syndrome.

Cardiotoxic Electrophysiological Effects of the Herbicide Roundup(®) in Rat and Rabbit Ventricular Myocardium In Vitro.

Roundup (R), a glyphosate (G)-based herbicide (GBH), containing unknown adjuvants is widely dispersed around the world. Used principally by farmers, intoxications have increasingly been reported. We have studied R effects (containing 36% of G) on right ventricular tissues (male Sprague-Dawley rats, up to 20,000ppm and female New Zealand rabbits, at 25 and 50ppm), to investigate R cardiac electrophysiological actions in vitro. We tested the reduced Ca(++) intracellular uptake mechanism as one potential cause of the electrical abnormalities after GBH superfusion, using the Na(+)/K(+)-ATPase inhibitor ouabain or the 1,4-dihydropyridine L-type calcium channel agonist BAY K 8644 which increases I Ca. R concentrations were selected based on human blood ranges found after acute intoxication. The study showed dose-dependent V max, APD50 and APD90 variations during 45min of R superfusion. At the highest concentrations tested, there was a high incidence of conduction blocks, and 30-min washout with normal Tyrode solution did not restore excitability. We also observed an increased incidence of arrhythmias at different doses of R. Ouabain and BAY K 8644 prevented V max decrease, APD90 increase and the cardiac inexcitability induced by R 50ppm. Glyphosate alone (18 and 180ppm) had no significant electrophysiological effects. Thus, the action potential prolonging effect of R pointing to I Ca interference might explain both conduction blocks and proarrhythmia in vitro. These mechanisms may well be causative of QT prolongation, atrioventricular conduction blocks and arrhythmias in man after GBH acute intoxications as reported in retrospective hospital records.

A Calcium-Activated Nonselective Cation Conductance Underlies the Plateau Potential in Rat Substantia Nigra GABAergic Neurons

Plateau potentials can be elicited in nigral GABAergic neurons by injection of 500 ms depolarizing current pulses from hyperpolarized holding potentials in whole-cell recordings in vitro. In approximately one-third of these neurons, plateau potentials were observed under control conditions and could be elicited in the remaining neurons after blocking potassium conductances. Application of the L-type calcium channel agonist Bay K 8644 or activation of NMDA receptors enhanced plateau potentials observed under control conditions and caused a plateau to be elicited in neurons not exhibiting it previously. The plateau potential was abolished in calcium-free buffer, as well as by nickel or cadmium. The L-type calcium channel blockers nimodipine and nifedipine abolished the plateau potential observed under control conditions but did not affect plateaus unmasked by tetraethylammonium. Plateau potentials observed under control conditions as well as those observed in the presence of Bay K 8644, NMDA, or tetraethylammonium were abolished in low-sodium buffer and by the calcium-activated nonselective cation conductance blocker flufenamic acid. These data suggest that nigral plateau potentials are mediated by a calcium-activated nonselective cation conductance (I(CAN)) that is activated by calcium entry predominantly through L-type calcium channels. In many nigral neurons, I(CAN) is masked by tetraethylammonium-sensitive potassium conductances, but plateaus can be evoked after increasing calcium conductances. The I(CAN)-mediated plateau potential in nigral GABAergic neurons likely affects the way these neurons integrate input and may represent a mechanism contributing to the rhythmic firing of these neurons seen in pathological conditions such as Parkinson's disease.

Using Dihydropyridine-Release Strategies to Enhance Load Effects in Engineered Human Bone Constructs

We report on the development of a novel biodegradable scaffold capable of enhancing mechanical signals for tissue-engineering applications. It has been shown that mechanotransduction enhances bone formation in vitro and in vivo; in tissue-engineering applications, this phenomenon is exploited through the use of mechanical bioreactors to generate bone tissue. The dihydropyridine agonist Bay K8644 (Bay) acts to increase the opening time of mechanosensitive voltage-operated calcium channels (VOCCs), specifi- cally L-type VOCCs, which are known to play a fundamental role in the early mediation of mechanotransduction. We have produced porous 3-dimensional, Bay-encapsulated biodegradable poly(L-lactide) acid scaffolds using a solvent-casting and salt-leaching technique. The effects of the released Bay on osteoid production and mineralization in human bone cell-seeded constructs following incubation in a perfusion-compression bioreactor in vitro was investigated using Western blotting techniques and a calcium assay protocol developed in our lab. Our newly developed scaffolds act by slowly releasing the calcium channel agonist Bay K8644 as observed using ultraviolet spectroscopy, maintaining the open state of mechanosensitive VOCCs responding to load, which augments the load signal at sites of strain across the scaffold. Our results demonstrate that, in the presence of physiological loading regimes in vitro, release of Bay enhances collagen I protein production and osteoid calcification more than non-Bay control constructs do. Osteopontin and alpha2delta1 VOCC subunit protein levels were also higher as a result of perfusion-compression conditioning. These results indicate that Bay-encapsulated scaffolds can be used in the presence of load to enhance the production of load-bearing engineered tissue.

Calcium-dependent expression of TNF-α in neural cells is mediated by the calcineurin/NFAT pathway

We report induction of TNF-α via the calcium/calcineurin/NFAT pathway in PC12 neural cells. In PC12, expression of TNF-α mRNA, protein and TNF-α gene promoter activity was induced by co-stimulation with phorbol ester and either calcium ionophore A23187 or the L-type Voltage Gated Calcium Channel agonist Bay K 8644. Pre-treatment with calcineurin inhibitors CsA or FK506 inhibited the dominant calcium-dependent component of this induction, limiting it to the level achieved with phorbol ester alone. Promoter activation by Bay was abolished by nifedipine, a specific inhibitor of L-type Voltage Gated Calcium Channels. Exogenous NFAT protein transactivated the TNF-α promoter, and the peptide VIVIT–a specific inhibitor of calcineurin/NFAT binding–blocked calcium-inducible transactivation of the TNF-α promoter. Given proposed functions of TNF-α in spatial learning, memory and the pathogenesis of neurodegenerative diseases, the data presented suggest an important role for calcineurin/NFAT signaling in these key neurological processes.

Different antioxidative potencies of dihydropyridine calcium channel modulators in various models

There is evidence that dihydropyridine calcium antagonists (DHP) play a beneficial role during the development of atherosclerosis. Since antioxidative properties of this substance class may be important, we investigated the antioxidative potency of the DHP prototype calcium channel antagonist nifedipine, the long acting calcium channel antagonist lacidipine, the DHP calcium channel agonist Bay K 8644 and the bulky DHP derivate Bay O 5572 (negligible effects on L-type calcium channels) in three different models. Additionally, we examined the potential correlation between lipophilic and antioxidative properties. In an in vitro model, Bay K 8644 was significantly more effective in scavenging superoxide anions (hypoxanthine/xanthine-oxidase-assay) than lacidipine, Bay O 5572 or nifedipine (micro- to millimolar concentration range). Addition of artificial membrane preparations (dimyristoylphosphatidylcholine) to mimic a physiological environment resulted in an enhanced antioxidative effect, with lacidipine being the most effective DHP to quench radicals (low micromolar concentration range). Thirdly, in a more physiological model of hyperglycemia (30 mmol/l) induced release of reactive oxygen species (ROS) from native endothelial cells of porcine coronary arteries, we showed that nifedipine was a significantly more potent antioxidant (therapeutical nanomolar concentration range) than the other DHP. Calculation of the lipophilicity of the four substances (lacidipine>Bay O 5572>Bay K 8644>nifedipine) showed a positive correlation between the antioxidative potency and the lipophilicity in the model with the artificial membranes but not in the other models. We conclude that it seems necessary to access antioxidative properties of substances in physiological models in which we could demonstrate that nifedipine exhibits ROS-quenching properties in a therapeutic concentration range.

The phototransduction cascade in the isolated chick pineal gland revisited

It is well established that the isolated chick pineal gland is directly light sensitive and that melatonin synthesis of the gland can be inhibited by exposing the gland to light during scotophase. Since not all the steps of the phototransduction cascade have been clarified to the same extent as in the retina, we have treated isolated chick pineal glands with 90 min of light during scotophase and with drugs that affect key-components of vertebrate phototransduction, i.e., cyclic guanosine monophosphate (cGMP) phosphodiesterase 6 (PDE6), cGMP levels and cGMP-gated calcium channels. The endpoint measured was the activity of the rate-limiting enzyme of melatonin synthesis, arylalkylamine N-acetyltransferase (AA-NAT), which is inhibited by light. The effects on AA-NAT activity of light were negated by addition of dipyridamol and zaprinast, either of which inhibits the light-induced activation of PDE6. The effect of light was also counteracted by the nitric oxide donor sodium nitroprusside and C-type natriuretic peptide, both of which increase cGMP levels, and by the calcium channel agonist Bay K 8644, which prevents the cGMP-decrease-induced closure of cGMP-gated calcium channels. Inhibition of nitric oxide synthase (NOS) by N G-nitro- l-arginine did not influence the inhibitory effect of light, suggesting that the NOS pathway does not play a role. Since the light effect on AA-NAT activity involves both cGMP and cyclic adenosine monophosphate (cAMP) hydrolysis, we have also studied whether the cGMP-inhibited cAMP phosphodiesterase 3 (PDE3) is involved. As the specific PDE3 inhibitor cilostamide is without effect, we assume that the light-induced decrease of cAMP levels does not involve PDE3. These results taken together strongly suggest that the investigated steps of the phototransduction cascade in the isolated chick pineal gland are basically similar to those in the retina.

Cortisol rapidly suppresses intracellular calcium and voltage-gated calcium channel activity in prolactin cells of the tilapia (Oreochromis mossambicus).

Cortisol was previously shown to rapidly (10-20 min) reduce the release of prolactin (PRL) from pituitary glands of tilapia (Oreochromis mossambicus). This inhibition of PRL release by cortisol is accompanied by rapid reductions in (45)Ca(2+) and cAMP accumulation. Cortisol's early actions occur through a protein synthesis-independent pathway and are mimicked by a membrane-impermeable analog. The signaling pathway that mediates rapid, nongenomic membrane effects of glucocorticoids is poorly understood. Using the advantageous characteristics of the teleost pituitary gland from which a nearly pure population of PRL cells can be isolated and incubated in defined medium, we examined whether cortisol rapidly reduces intracellular free calcium (Ca(i)(2+)) and suppresses L-type voltage-gated ion channel activity in events that lead to reduced PRL release. Microspectrofluorometry, used in combination with the Ca(2+)-sensitive dye fura 2 revealed that cortisol reversibly reduces basal and hyposmotically induced Ca(i)(2+) within seconds (P < 0.001) in dispersed pituitary cells. Somatostatin, a peptide known to inhibit PRL release through a membrane receptor-coupled mechanism, similarly reduces Ca(i)(2+). Under depolarizing [K(+)], the L-type calcium channel agonist BAY K 8644, a factor known to delay the closing of L-type Ca(2+) channels, stimulates PRL release in a concentration-dependent fashion (P < 0.01). Cortisol (and somatostatin) blocks BAY K 8644-induced PRL release (P < 0.01; 30 min), well within the time course over which its actions occur, independent of protein synthesis and at the level of the plasma membrane. Results indicate that cortisol inhibits tilapia PRL release through rapid reductions in Ca(i)(2+) that likely involve an attenuation of Ca(2+) entry through L-type voltage-gated Ca(2+) channels. These results provide further evidence that glucocorticoids rapidly modulate hormone secretion via a membrane-associated mechanism similar to that observed with the fast effects of peptides and neurotransmitters.

Ligand-activated signal transduction in the 2-cell embryo.

Platelet-activating factor (PAF) is an autocrine trophic/survival factor for the preimplantation embryo. PAF induced an increase in intracellular calcium concentration ([Ca2+]i) in the 2-cell embryo that had an absolute requirement for external calcium. L-type calcium channel blockers (diltiazem, verapamil, and nimodipine) significantly inhibited PAF-induced Ca2+ transients, but inhibitors of P/Q type (omega-agatoxin; omega-conotoxin MVIIC), N-type (omega-conotoxin GVIA), T-type (pimozide), and store-operated channels (SKF 96365 and econazole) did not block the transient. mRNA and protein for the alpha1-C subunit of L-type channels was expressed in the 2-cell embryo. The L-type calcium channel agonist (+/-) BAY K 8644 induced [Ca2+]i transients and, PAF and BAY K 8644 each caused mutual heterologous desensitization of each other's responses. Depolarization of the embryo (75 mM KCl) induced a [Ca2+]i transient that was inhibited by diltiazem and verapamil. Whole-cell patch-clamp measurements detected a voltage-gated channel (blocked by diltiazem, verapamil, and nifedipine) that was desensitized by prior responses of embryos to exogenous or embryo-derived PAF. Replacement of media Ca2+ with Mn2+ allowed Mn2+ influx to be observed directly; activation of a diltiazem-sensitive influx channel was an early response to PAF. The activation of a voltage-gated L-type calcium channel in the 2-cell embryo is required for normal signal transduction to an embryonic trophic factor.

Mechanism of angiotensin II-induced phospholipase D activation in bovine adrenal glomerulosa cells

Based on previous data demonstrating activation of phospholipase D (PLD) in response to angiotensin II (AngII), we have hypothesized a role for PLD in mediating aldosterone secretion from bovine adrenal glomerulosa cells. In this study we demonstrate that a PLD-generated signal(s) is required for the AngII-elicited secretory response, since interfering with lipid second messenger formation using a primary alcohol inhibited AngII-induced aldosterone secretion, but not that elicited by incubation with a hydrophilic cholesterol analog, 22( R)-hydroxycholesterol, which bypasses signaling pathways. Three mechanisms for hormonal activation of PLD have been described in other systems: direct receptor coupling, activation through protein kinase C (PKC) and a combination of these two mechanisms. Our results indicate that the PKC activator, phorbol 12-myristic 13-acetate (PMA), is able to activate PLD, and that receptor engagement is apparently not necessary for PLD activation in response to this agent. Maximal doses of AngII and PMA produced no additive effect on PLD activation, suggesting that these two agents function through a common PKC pathway. This interpretation was confirmed by the ability of a PKC inhibitor, Gö 6976, to inhibit partially AngII-induced PLD activation. Finally, treatment with the calcium ionophores A23187 or ionomycin or the calcium channel agonist BAY K8644 had no effect on PLD activity. Likewise, inhibiting calcium influx with high-dose nitrendipine affected neither basal PLD activity nor that stimulated by AngII. Thus, our results suggest a role for PKC, independent of calcium influx, in mediating AngII-induced PLD activation in glomerulosa cells.

Effects of dextromethorphan on the seizures induced by kainate and the calcium channel agonist BAY k-8644: comparison with the effects of dextrorphan

BAY k-8644 (an L-type Ca 2+ channel agonist of the dihydropyridine class) is recognized as a potent convulsant agent. In this study, we used BAY k-8644 to explore the effects of dextromethorphan (DM) and its major metabolite, dextrorphan (DX), on the (pro)convulsant activity regulated by calcium channels. BAY k-8644 (2 mg/kg, s.c) potentiated seizures induced in rats by kainic acid (KA) (10 mg/kg, i.p.). DM appears more efficacious than DX in attenuation of KA-induced seizures. The anticonvulsant effect of a low dose (12.5 mg/kg, s.c.) of DM was reversed by BAY k-8644 (2 mg/kg) challenge. In contrast, BAY k-8644 (1 or 2 mg/kg) did not significantly affect an anticonvulsant effect from a higher dose (25 mg/kg) of either DM or DX. Intracerebroventricular injection of BAY k-8644 (37.5 μg) significantly induced seizures in mice. DM (12.5 or 25 mg/kg) pretreatment more significantly attenuated seizures evoked by BAY k-8644 than did DX (12.5 or 25 mg/kg). Furthermore, seizure activity induced by KA or BAY k-8644 was consistent with respective activator protein-1 DNA binding activity of the hippocampus. Therefore, our results suggest that the anticonvulsant effects of the morphinans involve, at least in part, the L-type calcium channel. They also suggest that DM is a more potent anticonvulsant than DX in the KA and BAY k-8644 seizure models.

Amlodipine increases nitric oxide synthesis in cytokine-stimulated cultured vascualar smooth muscle cells.

If calcium channel blockers affect nitric oxide synthesis in the vascular tissue, they could influence disease progression in coronary arteries. We investigated the effects of the calcium channel blocker amlodipine on nitric oxide synthesis by measuring the production of nitrite, a stable metabolite of nitric oxide, in vascular smooth muscle cells. We measured the production of nitrate in cultured rat vascular smooth muscle cells with the Griess reagent Inducible nitric oxide synthase protein and mRNA expression were assayed by Western blotting and reverse transcription-polymerase chain reaction, respectively. The levels of NF-kappaB proteins in nuclear extracts were analyzed by gel retardation assay. Incubation of cultures with interleukin-1 , (10 ng/ ml) for 24 h caused a significant increase in nitrite generation. Interleukin-1 l-induced nitrite production by vascular smooth muscle cells was significantly increased by amlodipine in a dose-dependent manner. This augmentative effect of amlodipine was completely abolished in the presence of N(G)-monomethyl-L-arginine or actinomycin D. Amlodipine-induced nitrite production was accompanied by increased inducible nitric oxide synthase mRNA and protein accumulation. Interleukin-1 , induced NF-kappaB activation in vascular smooth muscle cells, and addition of amlodipine further increased this NF-kappaB activation. The effect of amlodipine on nitrite production was maintained in the presence of the calcium channel agonist Bay K 8644. Amlodipine enhances nitric oxide synthesis in cytokine-stimulated cultured vascular smooth muscle cells by L-type calcium channel-independent mechanisms.

Intracellular calcium and the relationship to contractility in an avian model of heart failure.

Global contractile heart failure was induced in turkey poults by furazolidone feeding (700 ppm). Abnormal calcium regulation appears to be a key factor in the pathophysiology of heart failure, but the cellular mechanisms contributing to changes in calcium fluxes have not been clearly defined. Isolated ventricular myocytes from non-failing and failing hearts were therefore used to determine whether the whole heart and ventricular muscle contractile dysfunctions were realized at the single cell level. Whole cell current- and voltage-clamp techniques were used to evaluate action potential configurations and L-type calcium currents, respectively. Intracellular calcium transients were evaluated in isolated myocytes with fura-2 and in isolated left ventricular muscles using aequorin. Action potential durations were prolonged in failing myocytes, which correspond to slowed cytosolic calcium clearing. Calcium current-voltage relationships were normal in failing myocytes; preliminary evidence suggests that depressed transient outward potassium currents contribute to prolonged action potential durations. The number of calcium channels (as measured by radioligand binding) were also similar in non-failing and failing hearts. Isolated ventricular muscles from failing hearts had enhanced inotropic responses, in a dose-dependent fashion, to a calcium channel agonist (Bay K 8644). These data suggest that changes in intracellular calcium mobilization kinetics and longer calcium-myofilament interaction may be able to compensate for contractile failure. We conclude that the relationship between calcium current density and sarcoplasmic reticulum calcium release is a dynamic process that may be altered in the setting of heart failure at higher contraction rates.