Structural Basis of the Modulation of the Voltage-Gated Calcium Ion Channel Cav 1.1 by Dihydropyridine Compounds*.

1,4-Dihydropyridines (DHP), the most commonly used antihypertensives, function by inhibiting the L-type voltage-gated Ca2+ (Cav ) channels. DHP compounds exhibit chirality-specific antagonistic or agonistic effects. The structure of rabbit Cav 1.1 bound to an achiral drug nifedipine reveals the general binding mode for DHP drugs, but the molecular basis for chiral specificity remained elusive. Herein, we report five cryo-EM structures of nanodisc-embedded Cav 1.1 in the presence of the bestselling drug amlodipine, a DHP antagonist (R)-(+)-Bay K8644, and a titration of its agonistic enantiomer (S)-(-)-Bay K8644 at resolutions of 2.9-3.4 Å. The amlodipine-bound structure reveals the molecular basis for the high efficacy of the drug. All structures with the addition of the Bay K8644 enantiomers exhibit similar inactivated conformations, suggesting that (S)-(-)-Bay K8644, when acting as an agonist, is insufficient to lock the activated state of the channel for a prolonged duration.

Ritanserin blocks CaV1.2 channels in rat artery smooth muscles: electrophysiological, functional, and computational studies.

CaV1.2 channel blockers or 5-HT2 receptor antagonists constitute effective therapy for Raynaud's syndrome. A functional link between the inhibition of 5-HT2 receptors and CaV1.2 channel blockade in arterial smooth muscles has been hypothesized. Therefore, the effects of ritanserin, a nonselective 5-HT2 receptor antagonist, on vascular CaV1.2 channels were investigated through electrophysiological, functional, and computational studies. Ritanserin blocked CaV1.2 channel currents (ICa1.2) in a concentration-dependent manner (Kr = 3.61 µM); ICa1.2 inhibition was antagonized by Bay K 8644 and partially reverted upon washout. Conversely, the ritanserin analog ketanserin (100 µM) inhibited ICa1.2 by ~50%. Ritanserin concentration-dependently shifted the voltage dependence of the steady-state inactivation curve to more negative potentials (Ki = 1.58 µM) without affecting the slope of inactivation and the activation curve, and decreased ICa1.2 progressively during repetitive (1 Hz) step depolarizations (use-dependent block). The addition of ritanserin caused the contraction of single myocytes not yet dialyzed with the conventional method. Furthermore, in depolarized rings, ritanserin, and to a lesser extent, ketanserin, caused a concentration-dependent relaxation, which was antagonized by Bay K 8644. Ritanserin and ketanserin were docked at a region of the CaV1.2 α1C subunit nearby that of Bay K 8644; however, only ritanserin and Bay K 8644 formed a hydrogen bond with key residue Tyr-1489. In conclusion, ritanserin caused in vitro vasodilation, accomplished through the blockade of CaV1.2 channels, which was achieved preferentially in the inactivated and/or resting state of the channel. This novel activity encourages the development of ritanserin derivatives for their potential use in the treatment of Raynaud's syndrome.

Alteration in the phosphorylation status of NMDA receptor GluN2B subunit by activation of both NMDA receptor and L-type voltage gated calcium channel.

Calcium influx through N-methyl-D-aspartate receptors (NMDAR) and voltage-gated calcium channels (VGCC) play major roles in postsynaptic signaling mechanisms. NMDAR subunit GluN2B is phosphorylated at Ser1303. Phosphorylation at this site is a prominent event in cell culture systems as well as in vivo. However, the functional significance of phosphorylation at this site is not completely understood. In this study, we compared the effect of calcium signaling through NMDAR and VGCC on the phosphorylation status of GluN2B-Ser1303 in the rat in vivo. VGCC was activated by intraperitoneal (IP) injection of the activator, BayK8644 and NMDAR was activated by intracerebroventricular (ICV) injection of NMDA in separate experimental groups. We found that the level of phospho-GluN2B-Ser1303 in the cortex and in the hippocampus increased in response to activation of either channel. The effects could be prevented by prior ICV administration of the specific blockers of these channels such as MK-801 for NMDAR and nifedipine for VGCC. The effect was also blocked by pretreatment with ICV administration of KN-93 indicating that it is mediated through CaM kinase. Both during NMDAR activation and VGCC activation, cell survival associated signals such as phospho-AKT and phospho-CREB showed decrease, consistent with activation of cell death pathways during these treatments. We conclude that under in vivo conditions, calcium influx through either NMDAR or VGCC activates CaM kinase, which in turn phosphorylates GluN2B-Ser1303.

Hydroxyl Group and Vasorelaxant Effects of Perillyl Alcohol, Carveol, Limonene on Aorta Smooth Muscle of Rats.

The present study used isometric tension recording to investigate the vasorelaxant effect of limonene (LM), carveol (CV), and perillyl alcohol (POH) on contractility parameters of the rat aorta, focusing in particular on the structure-activity relationship. LM, CV, and POH showed a reversible inhibitory effect on the contraction induced by electromechanical and pharmacomechanical coupling. In the case of LM, but not CV and POH, this effect was influenced by preservation of the endothelium. POH and CV but not LM exhibited greater pharmacological potency on BayK-8644-induced contraction and on electromechanical coupling than on pharmacomechanical coupling. In endothelium-denuded preparations, the order of pharmacological potency on electrochemical coupling was LM < CV < POH. These compounds inhibited also, with grossly similar pharmacological potency, the contraction induced by phorbol ester dibutyrate. The present results suggest that LM, CV and POH induced relaxant effect on vascular smooth muscle by means of different mechanisms likely to include inhibition of PKC and IP3 pathway. For CV and POH, hydroxylated compounds, it was in electromechanical coupling that the greater pharmacological potency was observed, thus suggesting a relative specificity for a mechanism likely to be important in electromechanical coupling, for example, blockade of voltage-dependent calcium channel.

Cav1.2 channel current block by the PKA inhibitor H-89 in rat tail artery myocytes via a PKA-independent mechanism: Electrophysiological, functional, and molecular docking studies.

To characterize the role of cAMP-dependent protein kinase (PKA) in regulating vascular Ca2+ current through Cav1.2 channels [ICa1.2], we have documented a marked capacity of the isoquinoline H-89, widely used as a PKA inhibitor, to reduce current amplitude. We hypothesized that the ICa1.2 inhibitory activity of H-89 was mediated by mechanisms unrelated to PKA inhibition. To support this, an in-depth analysis of H-89 vascular effects on both ICa1.2 and contractility was undertaken by performing whole-cell patch-clamp recordings and functional experiments in rat tail main artery single myocytes and rings, respectively. H-89 inhibited ICa1.2 with a pIC50 (M) value of about 5.5, even under conditions where PKA activity was either abolished by both the PKA antagonists KT5720 and protein kinase inhibitor fragment 6-22 amide or enhanced by the PKA stimulators 6-Bnz-cAMP and 8-Br-cAMP. Inhibition of ICa1.2 by H-89 appeared almost irreversible upon washout, was charge carrier- and voltage-dependent, and antagonised by the Cav1.2 channel agonist (S)-(-)-Bay K 8644. H-89 did not alter both potency and efficacy of verapamil, did not affect current kinetics or voltage-dependent activation, while shifting to the left the 50% voltage of inactivation in a concentration-dependent manner. H-89 docked at the α1C subunit in a pocket region close to that of (S)-(-)-Bay K 8644 docking, forming a hydrogen bond with the same, key amino acid residue Tyr-1489. Finally, both high K+- and (S)-(-)-Bay K 8644-induced contractions of rings were fully reverted by H-89. In conclusion, these results indicate that H-89 inhibited vascular ICa1.2 and, consequently, the contractile function through a PKA-independent mechanism. Therefore, caution is recommended when interpreting experiments where H-89 is used to inhibit vascular smooth muscle PKA.

Functional expression of voltage-gated calcium channels in human melanoma.

The expression of voltage-gated calcium channels (VGCCs) has not been reported previously in melanoma cells in spite of increasing evidence of a role of VGCCs in tumorigenesis and tumour progression. To address this issue we have performed an extensive RT-PCR analysis of VGCC expression in human melanocytes and a range of melanoma cell lines and biopsies. In addition, we have tested the functional expression of these channels using Ca(2+) imaging techniques and examined their relevance for the viability and proliferation of the melanoma cells. Our results show that control melanocytes and melanoma cells express channel isoforms belonging to the Ca(v) 1 and Ca(v) 2 gene families. Importantly, the expression of low voltage-activated Ca(v) 3 (T-type) channels is restricted to melanoma. We have confirmed the function of T-type channels as mediators of constitutive Ca(2+) influx in melanoma cells. Finally, pharmacological and gene silencing approaches demonstrate a role for T-type channels in melanoma viability and proliferation. These results encourage the analysis of T-type VGCCs as targets for therapeutic intervention in melanoma tumorigenesis and/or tumour progression.

Dynamic interplay of excitatory and inhibitory coupling modes of neuronal L-type calcium channels.

L-type voltage-gated calcium channels (LTCCs) have long been considered as crucial regulators of neuronal excitability. This role is thought to rely largely on coupling of LTCC-mediated Ca(2+) influx to Ca(2+)-dependent conductances, namely Ca(2+)-dependent K(+) (K(Ca)) channels and nonspecific cation (CAN) channels, which mediate afterhyperpolarizations (AHPs) and afterdepolarizations (ADPs), respectively. However, in which manner LTCCs, K(Ca) channels, and CAN channels co-operate remained scarcely known. In this study, we examined how activation of LTCCs affects neuronal depolarizations and analyzed the contribution of Ca(2+)-dependent potassium- and cation-conductances. With the use of hippocampal neurons in primary culture, pulsed current-injections were applied in the presence of tetrodotoxin (TTX) for stepwise depolarization and the availability of LTCCs was modulated by BAY K 8644 and isradipine. By varying pulse length and current strength, we found that weak depolarizing stimuli tend to be enhanced by LTCC activation, whereas in the course of stronger depolarizations LTCCs counteract excitation. Both effect modes appear to involve the same channels that mediate ADP and AHP, respectively. Indeed, ADPs were activated at lower stimulation levels than AHPs. In the absence of TTX, activation of LTCCs prolonged or shortened burst firing, depending on the initial burst duration, and invariably augmented brief unprovoked (such as excitatory postsynaptic potentials) and provoked electrical events. Hence, regulation of membrane excitability by LTCCs involves synchronous activity of both excitatory and inhibitory Ca(2+)-activated ion channels. The overall enhancing or dampening effect of LTCC stimulation on excitability does not only depend on the relative abundance of the respective coupling partner but also on the stimulus intensity.

Sulfonylureas uncouple glucose-dependence for GPR40-mediated enhancement of insulin secretion from INS-1E cells.

Activation of GPR40 is reported to enhance insulin secretion in the presence of glucose. We determined whether sulfonylureas could replace glucose for GPR40-mediated enhancement of insulin secretion and investigated underlying mechanisms using INS-1E cells. GW9508, a specific agonist of GPR40, significantly enhanced insulin secretion in the presence of high concentrations of glucose. In contrast, sulfonylureas increased insulin secretion in the absence of glucose. In the presence of sulfonylureas, activation of GPR40 significantly enhanced insulin secretion. The L-type calcium channel (LTCC) activator S-(-)-Bay K8644 also concentration-dependently increased insulin secretion in the absence of glucose. In the presence of 10 micromol/L S-(-)-Bay K8644, GW9508 significantly increased insulin secretion. On the other hand, the LTCC blocker nifedipine significantly inhibited insulin secretion mediated by either glucose, glipizide or glucose plus GW9508. Thus, sulfonylureas could replace glucose to support GPR40-mediated enhancement of insulin secretion, whereas blockage of LTCC reduced both glucose and sulfonylurea-mediated insulin secretion.

Differences in the quantal release of catecholamines in chromaffin cells of rat embryos and their mothers.

Studies on the bulk catecholamine release from fetal and neonatal rat adrenals, adrenal slices, or isolated chromaffin cells stimulated with high K(+), hypoxia, hypercapnia, or acidosis are available. However, a study analyzing the kinetics of quantal secretion is lacking. We report here such a study in which we compare the quantal release of catecholamines from immature rat embryo chromaffin cells (ECCs) and their mothers' (MCCs). Cell challenging with a strong depolarizing stimulus (75 mM K(+)) caused spike bursts having the following characteristics. ECCs released more multispike events and wave envelopes than MCCs. This, together with narrower single-spike events, a faster decay, and a threefold smaller quantal size suggest a faster secretory machinery in ECCs. Furthermore, with a milder stimulus (25 mM K(+)) enhanced Ca(2+) entry by L-type Ca(2+) channel activator BAY K 8644 did not change the kinetic parameters of single spikes in ECCs; in contrast, augmentation of Ca(2+) entry increased spike amplitude and width, quantal size, and decay time in MCCs. This suggests that in mature MCCs, the last exocytotic steps are more tightly regulated than in immature ECCs. Finally, we found that quantal secretion was fully controlled by L-type voltage-dependent Ca(2+) channels (VDCCs) in ECCs, whereas both L- and non-L VDCCs (N and PQ) contributed equally to secretion control in MCCs. Our results have the following physiological, pharmacological, and clinical relevance: 1) they may help to better understand the regulation of adrenal catecholamine release in response to stress during fetal life and delivery; 2) if clinically used, L-type Ca(2+) channel blockers may augment the incidence of sudden infant death syndrome (SIDS); and 3) so-called Ca(2+) promotors or activators of Ca(2+) entry through L-type VDCCs may be useful to secure a healthy catecholamine surge upon violent stress during fetal life, at birth, or to prevent the SIDS in neonates at risk.

The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine.

In mouse intestine, caveolae and caveolin-1 (Cav-1) are present in smooth muscle (responsible for executing contractions) and in interstitial cells of Cajal (ICC; responsible for pacing contractions). We found that a number of calcium handling/dependent molecules are associated with caveolae, including L-type Ca(2+) channels, Na(+)-Ca(2+) exchanger type 1 (NCX1), plasma membrane Ca(2+) pumps and neural nitric oxide synthase (nNOS), and that caveolae are close to the peripheral endo-sarcoplasmic reticulum (ER-SR). Also we found that this assemblage may account for recycling of calcium from caveolar domains to SR through L-type Ca (+) channels to sustain pacing and contractions. Here we test this hypothesis further comparing pacing and contractions under various conditions in longitudinal muscle of Cav-1 knockout mice (lacking caveolae) and in their genetic controls. We used a procedure in which pacing frequencies (indicative of functioning of ICC) and contraction amplitudes (indicative of functioning of smooth muscle) were studied in calcium-free media with 100 mM ethylene glycol tetra-acetic acid (EGTA). The absence of caveolae in ICC inhibited the ability of ICC to maintain frequencies of contraction in the calcium-free medium by reducing recycling of calcium from caveolar plasma membrane to SR when the calcium stores were initially full. This recycling to ICC involved primarily L-type Ca(2+) channels; i.e. pacing frequencies were enhanced by opening and inhibited by closing these channels. However, when these stores were depleted by block of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump or calcium release was activated by carbachol, the absence of Cav-1 or caveolae had little or no effect. The absence of caveolae had little impact on contraction amplitudes, indicative of recycling of calcium to SR in smooth muscle. However, the absence of caveolae slowed the rate of loss of calcium from SR under some conditions in both ICC and smooth muscle, which may reflect the loss of proximity to store operated Ca channels. We found evidence that these channels were associated with Cav-1. These changes were all consistent with the hypothesis that a reduction of the extracellular calcium associated with caveolae in ICC of the myenteric plexus, the state of L-type Ca(2+) channels or an increase in the distance between caveolae and SR affected calcium handling.

Competitive and cooperative effects of Bay K8644 on the L-type calcium channel current inhibition by calcium channel antagonists.

Phenylalkylamines, benzothiazepines, and dihydropyridines bind noncompetitively to the L-type calcium channel. The molecular mechanisms of this interaction were investigated in enzymatically isolated rat ventricular myocytes using the whole-cell patch-clamp technique. When applied alone, felodipine, verapamil, and diltiazem inhibited the L-type calcium current with values of inhibitory constant (K(B)) of 11, 246, and 512 nM, respectively, whereas 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester (Bay K8644) activated I(Ca) with activation constant (K(A)) of 33 nM. Maximal activation of I(Ca) by 300 nM Bay K8644 strongly reduced the inhibitory potency of felodipine (apparent K(B) of 165 nM), significantly reduced the inhibitory potency of verapamil (apparent K(B) of 737 nM), but significantly increased the inhibitory potency of diltiazem (apparent K(B) of 310 nM). In terms of a new pseudoequilibrium two-drug binding model, the interaction between the dihydropyridine agonist Bay K8644 and the antagonist felodipine was found purely competitive. The interaction between Bay K8644 and verapamil or diltiazem was found noncompetitive, and it could be described only by inclusion of a negative interaction factor nu = -0.60 for verapamil and a positive interaction factor nu = +0.24 for diltiazem. These results suggest that at physiological membrane potentials, the L-type calcium channel cannot be simultaneously occupied by a dihydropyridine agonist and antagonist, whereas it can simultaneously bind a dihydropyridine agonist and a nondihydropyridine antagonist. Generally, the effects of the drugs on the L-type calcium channel support a concept of a channel domain responsible for binding of calcium channel antagonists and agonists changing dynamically with the membrane voltage and occupancy of individual binding sites.

TGF-beta1 calcium signaling in osteoblasts.

Transforming growth factor-beta1 (TGF-beta1) action is known to be initiated by its binding to multiple cell surface receptors containing serine/threonine kinase domains that act to stimulate a cascade of signaling events in a variety of cell types. We have previously shown that TGF-beta1 and BMP-2 treatment of primary human osteoblasts (HOBs) enhances cell-substrate adhesion. In this report, we demonstrate that TGF-beta1 elicits a rapid, transient, and oscillatory rise in the intracellular Ca(2+) concentration, [Ca(2+)](i), that is necessary for enhancement of cell adhesion in HOBs but does not alter the phosphorylation state of Smad proteins. This rise in [Ca(2+)](i) in HOB is not observed in the absence of extracellular calcium or when the cells are treated with the L-type Ca(2+) channel blocker, nifedipine, but is stimulated upon treatment with the L-type Ca(2+) channel agonist, Bay K 8644, or under high K(+) conditions. The rise in [Ca(2+)](i) is severely attenuated after treatment of the cells with thapsigargin, a selective endoplasmic reticulum Ca(2+) pump inhibitor. TGF-beta1 enhancement of HOB adhesion to tissue culture polystyrene is also inhibited in cells treated with nifedipine. These data suggest that intracellular Ca(2+) signaling is an important second messenger of the TGF-beta1 signal transduction pathway in osteoblast function.

Overexpression of regucalcin suppresses apoptotic cell death in cloned normal rat kidney proximal tubular epithelial NRK52E cells: change in apoptosis-related gene expression.

The effect of regucalcin, a regulatory protein in intracellular signaling pathway, on cell death and apoptosis was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 24-72 h in a medium without BS containing either vehicle, tumor necrosis factor-alpha (TNF-alpha; 0.1 or 1.0 ng/ml of medium), lipopolysaccharide (LPS; 0.1 or 1.0 microg/ml), Bay K 8644 (10(-9)-10(-7) M), or thapsigargin (10(-9)-10(-7) M). The number of wild-type cells was significantly decreased by culture for 42-72 h in the presence of TNF-alpha (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 microg/ml), Bay K 8644 (10(-7)-10(-5) M), or thapsigargin (10(-8) or 10(-7) M). The effect of TNF-alpha (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 microg/ml), Bay K 8644 (10(-7)-10(-6) M), or thapsigargin (10(-7) M) in decreasing the number of wild-type cells cultured for 24-72 h was significantly prevented in transfectants overexpressing regucalcin. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with LPS (1.0 microg/ml), Bay K 8644 (10(-7) M), or thapsigargin (10(-8) M) for 24 h, and this DNA fragmentation was significantly suppressed in transfectants. DNA fragmentation in adherent cells was not seen by culture with TNF-alpha (1.0 ng/ml). TNF-alpha-induced decrease in the number of wild-type cells was significantly prevented by culture with caspase-3 inhibitor (10(-8) M), while LPS- or Bay K 8644-induced decrease in cell number was significantly prevented by caspase-3 inhibitor or N omega-nitro-L-arginine methylester (NAME) (10(-5) M), an inhibitor of nitric oxide (NO) synthase. Thapsigargin-induced decrease in cell number was not prevented in the presence of two inhibitors. Bcl-2 and Akt-1 mRNA levels were significantly increased in transfectants cultured for 24 h as compared with those of wild-type cells, while Apaf-1, caspase-3, or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expressions were not significantly changed in transfectants. Culture with TNF-alpha (1.0 ng/ml), LPS (1.0 microg/ml), Bay K 8644 (l0(-7) M), or thapsigargin (10(-8) M) caused a significant increase in caspase-3 mRNA levels in wild-type cells. LPS (1.0 microg/ml) significantly decreased Bcl-2 mRNA expression in the cells. Their effects on the gene expression of apoptosis-related proteins were not significantly changed in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death and apoptosis induced by various factors which their action are mediated through many intracellular signaling pathways, and that it modulates the gene expression of apoptosis-related proteins.

Ceramide inhibits L-type calcium channel currents in GH3 cells.

In this study, we investigated the effect of ceramide on the L-type Ca2+ channel (L-channel) in GH3 cells. We found that C6-ceramide, but not C6-dihydroceramide, the inactive analogue, had an inhibitory effect on BayK 8644-stimulated GH release. Using patch clamp analysis, C6- and C2-ceramide, but not C6-dihydroceramide, were found to inhibit the L-channel current. Increasing intracellular ceramide level with sphingomyelinase also inhibited the L-channel current. The inhibitory effect of ceramide on the L-channel current was attenuated by calphostin C, a myristolated pseudosubstrate protein kinase C (PKC) inhibitor, and lavendustin A, a tyrosine kinase inhibitor. Combined treatment with lavendustin A and the myristolated PKC inhibitor blocked the effect of ceramide on the L-channel current. These results indicate that ceramide, a lipid messenger of the sphingomyelin pathway, is an important regulator of the L-channel in GH3 cells and both tyrosine kinase and PKC are involved in this effect of ceramide.

Magnesium sulfate inhibits the oxytocin-induced production of inositol 1,4,5-trisphosphate in cultured human myometrial cells.

OBJECTIVE: The purpose of this study was to determine the effects of magnesium sulfate on inositol trisphosphate production and the mechanism of these effects. STUDY DESIGN: Myometrium was obtained at the time of cesarean delivery from women before labor at term. Inositol trisphosphate was measured in the primary myometrial cell cultures after stimulation with oxytocin, sodium fluoride, or Bay K 8644 with or without preincubation with magnesium sulfate or nifedipine. Experiments were performed in either calcium-containing or calcium-free medium that contained egtazic acid and after preincubation with the intracellular calcium chelator BAPTA-acetoxymethylester. Inositol trisphosphate production was measured by radioreceptor assay. In separate experiments, changes in intracellular calcium concentrations ([Ca(2+)](i)) were measured with the use of Fura-2 and spectrophotofluorometry. RESULTS: Oxytocin, sodium fluoride, and Bay K 8644 increased inositol trisphosphate production 2- to 4-fold. Preincubation with magnesium sulfate (3 x 10(-3) mol/L) for > or = 5 minutes decreased oxytocin-, sodium fluoride-, and Bay K 8644-induced inositol trisphosphate production in either calcium-containing or calcium-free media. Preincubation with BAPTA-acetoxymethylester decreased oxytocin-stimulated inositol trisphosphate production by 78% in calcium-containing media and completely prevented the oxytocin response in calcium-free media. Magnesium sulfate decreased inositol trisphosphate production in calcium-containing media but had no additional effect in calcium-free media. Oxytocin and Bay K 8644 increased [Ca(2+)](i) in either calcium-containing or calcium-free media, and magnesium sulfate reduced this in both cases. CONCLUSION: Magnesium sulfate appears to inhibit phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activity and subsequent calcium release in cultured myometrial cells by a direct effect on phospholipase C.

Gestational changes in uterine L-type calcium channel function and expression in guinea pig.

Pregnancy can influence both the resting membrane potential and the ion channel composition of the uterine myometrium. Calcium flux is essential for excitation-contraction coupling in pregnant uterus. The uterine L-type calcium channel is an important component in mediating calcium flux and is purported to play a role in parturition. This study was undertaken to characterize gestational changes in 1) the uterine contractile response to the L-type calcium channel agonist, Bay K 8644; 2) the mRNA expression of channel subunits by semiquantitative reverse transcriptase-polymerase chain reaction; and 3) estimate channel protein levels by measuring (3)H-isradipine binding at the dihydropyridine binding site of the alpha(1c) subunit utilizing saturation binding methods. Sensitivity to Bay K 8644 increases beginning at 0.8 of gestation and persists through term. The change in sensitivity is coincident with an increased mRNA expression of the alpha(1c) and beta(2) subunits but with the least detectable amounts of isradipine binding. The expressed alpha(1c) transcript represents a novel structural variant with a 118-amino acid deletion in the III-IV linker and repeats IVS1-S3 of the protein sequence. The guinea pig uterine L-type calcium channel activity is highly regulated through gestation, but the regulation of mRNA expression may be different from regulation of protein levels, estimated by isradipine binding. The up-regulation of function, alpha(1c) subunit mRNA expression, and isradipine binding at term gestation are consistent with a role for this ion channel in parturition.

Bay K 8644 increases resting Ca2+ spark frequency in ferret ventricular myocytes independent of Ca influx: contrast with caffeine and ryanodine effects.

Bay K 8644, an L-type Ca2+ channel agonist, was shown previously to increase resting sarcoplasmic reticulum (SR) Ca2+ loss and convert post-rest potentiation to decay in dog and ferret ventricular muscle. Here, the effects of Bay K 8644 on local SR Ca2+ release events (Ca2+ sparks) were measured in isolated ferret ventricular myocytes, using laser scanning confocal microscopy and the fluorescent Ca2+ indicator fluo-3. The spark frequency under control conditions was fairly constant during 20 s of rest after interruption of electrical stimulation. Bay K 8644 (100 nmol/L) increased the spark frequency by 466+/-90% of control at constant SR Ca2+ load but did not change the spatial and temporal characteristics of individual sparks. The increase in spark frequency was maintained throughout the period of rest. The increase in Ca2+ spark frequency induced by Bay K 8644 was not affected by superfusion with Ca2+-free solution (with 10 mmol/L EGTA) but was suppressed by the addition of 10 micromol/L nifedipine (which by itself did not alter resting Ca2+ spark frequency). This suggests that the effect of Bay K 8644 on Ca2+ sparks is mediated by the sarcolemmal dihydropyridine receptor but is also independent of Ca2+ influx. Low concentrations of caffeine (0.5 mmol/L) increased both the average frequency and duration of sparks. Ryanodine (50 nmol/L) increased the spark frequency and also induced long-lasting Ca2+ signals. This may indicate long-lasting openings of SR Ca2+ release channels and a lack of local SR Ca2+ depletion. In lipid bilayers, Bay K 8644 had no effect on either single-channel current amplitude or open probability of the cardiac ryanodine receptor. It is concluded that Bay K 8644 activates SR Ca2+ release at rest, independent of Ca2+ influx and perhaps through a functional linkage between the sarcolemmal dihydropyridine receptor and the SR ryanodine receptor. In contrast, caffeine and ryanodine modulate Ca2+ sparks by a direct action on the SR Ca2+ release channels.

Calcium dependent positive inotropic effects of low phorbol ester concentrations in isolated rat hearts.

OBJECTIVE: The aim was to examine the cardiac effects of phorbol esters over a wide concentration range and to determine if the effects are related to Ca2+ availability. METHODS: Studies were carried out using isolated rat hearts exposed for 60 min either to phorbol 12-myristate 13-acetate (PMA, 10(-11) to 10(-6) M) or phorbol 12,13-dibutyrate (PDBu, 10(-12) to 10(-7) M) in the presence of either 1.25 or 2.50 mM CaCl2. Experiments were also done to assess the effect of BAY K8644, a Ca2+ agonist, on phorbol ester effects. After treatment, hearts were freeze clamped for later analysis of energy products. RESULTS: At the lowest concentrations studied, both PMA and PDBu produced positive inotropic effects, whereas higher concentrations resulted in a loss of contractile force in hearts perfused with 1.25 mM CaCl2. Doubling the CaCl2 concentration or the presence of BAY K8644 had little effect on the negative inotropic influence of either phorbol ester but reversed the positive inotropic effect to a negative inotropic one. Neither treatment had any effect on the coronary constricting effects of phorbol esters. Increases in resting tension and reductions in high energy phosphate content were evident only with the highest phorbol ester concentrations and were unaffected either by changes in CaCl2 concentrations or the presence of BAY K8644. CONCLUSIONS: At picomolar and nanomolar concentrations phorbol esters produce positive inotropic actions which are probably mediated by enhanced Ca2+ influx. Although higher concentrations produce negative inotropic effects, these are not influenced by [Ca2+]o nor are they related to disturbances in energy metabolism except at the highest concentrations. We conclude that phorbol esters produce complex concentration dependent cardiac effects which are not mediated by a single mechanism of action.

Absence of a direct coupling of a G protein to dihydropyridine binding sites in rat heart.

In rat heart membranes, the addition of guanine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), a stable GTP analogue, did not significantly modify the displacement of [3H]PN 200-110 binding by the 1,4-dihydropyridine (DHP) agonist Bay K 8644 and antagonists, nifedipine and nicardipine. These results are in agreement with some previously reported electrophysiological and pharmacological data, and they suggest that there is no direct involvement of a G protein in the modulation of DHP sensitive Ca channels in cardiac cells.

Activation of voltage-dependent calcium channels of mammalian sperm is required for zona pellucida-induced acrosomal exocytosis.

Previous work indicates that antagonists of the L-type voltage-dependent Ca2+ channel (VDCC) prevent the Ca(i) increase in mammalian sperm that is promoted by incubation in alkaline, K(+)-based media. Here, were provide additional evidence that sperm possess VDCC and show that their activation is required for the Ca2+ entry that mediates acrosomal exocytosis in both the presence and the absence of egg agonists. Specifically, we report that: (1) Sperm membrane potential changes, Ca(i) elevation, and acrosomal exocytosis have similar K+ dose dependencies, consistent with a characteristic requirement of a large depolarization for activation of the sperm VDCC; (2) High affinity binding sites (Kd approximately 0.35 +/- 0.03 and 0.45 +/- 0.06 nM; Bmax = 16.0 +/- 1.4 and 5.8 +/- 0.8 fmole/mg protein) for the VDCC antagonist, PN200-110, respectively, are present in membrane preparations from sperm of the ram and bull; (3) PN200-110 and the other VDCC antagonists nitrendipine, nisoldipine, verapamil, diltiazem, Ni2+, or Co2+ inhibit (IC50 = 0.1, 0.4, 0.6, 0.8, 1.0, 60, and 110 microM, respectively) the acrosomal exocytosis produced by combined elevation of pH0 and membrane depolarization; (4) Exocytosis induced by the ZP3 agonist of the mammalian egg also is inhibited by VDCC antagonists with similar dose dependencies; (5) Depolarizing treatments that presumably activate the sperm VDCC bypass the blockade of ZP3-induced exocytosis imposed by pertussis toxin. These results indicate that activation of the sperm VDCC is sufficient to induce sperm acrosomal exocytosis and that VDCC activation is necessary in the ZP3 signal transduction pathway. They also indicate that the presumed G-protein targets of pertussis toxin probably produce a required but indirect activation of the putative sperm VDCC. Possible intervening events include alteration of the voltage sensitivity of the VDCC, membrane depolarization, or both. We suggest that the depolarization-induced acrosome reaction may provide a useful system to investigate subsequent events in the exocytotic process.