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.

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.

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.

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.

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.

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.

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.

Effects of the calcium channel activator Bay K 8644 on general anaesthetic potency in mice.

1. The effects of the calcium channel activator, Bay K 8644, on the anaesthetic potencies of ethanol, argon and pentobarbitone were examined in mice. 2. Bay K 8644, at 1 mg kg-1 i.p., significantly antagonized the general anaesthetic potencies of ethanol and argon, but at 5 and 10 mg kg-1 this compound increased the general anaesthetic potency of these drugs. 3. At doses of 1, 5 and 10 mg kg-1 Bay K 8644 antagonized the anaesthetic effects of pentobarbitone. Bay K 8644, at the highest dose used, did not alter the brain concentrations of pentobarbitone or the blood concentrations of ethanol. 5. The effects of the different doses of Bay K 8644 on the actions of ethanol and of argon are compatible with the known partial agonist properties of this compound on calcium channels in vitro. 6. The actions of Bay K 8644 on the anaesthetic effects of pentobarbitone suggests that specific interactions may be involved in the anaesthetic actions of this compound.

The effects of chronic treatment with the dihydropyridine, Bay K 8644, on hyperexcitability due to ethanol withdrawal, in vivo and in vitro.

1. The effects of chronic treatment with the dihydropyridine, Bay K 8644, were studied on the ethanol withdrawal syndrome, in vivo and in vitro. 2. Addition of racemic Bay K 8644 to the drinking mixture, throughout the chronic ethanol treatment, decreased the behavioural excitability seen during ethanol withdrawal in vivo. 3. All the signs of hyperexcitability in field potentials in the isolated hippocampal slice, caused by ethanol withdrawal, were decreased by the chronic administration of Bay K 8644. 4. These effects resembled those previously reported for chronic administration of calcium channel antagonists; racemic Bay K 8644 has both calcium channel activating and antagonist properties. 5. Measurement of brain levels of Bay K 8644 at the end of the chronic treatment showed that the compound reached micromolar concentrations during the treatment, but none could be detected in the tissues at the time of the above measurements. 6. It is possible that the results might be explained by predominance of the calcium channel antagonist properties of this compound, owing to the high central concentrations achieved during the treatment. Tolerance to the calcium channel activating properties of Bay K 8644 may also have occurred during the chronic treatment.

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.

Regional distribution of calcium channel ligand (1,4-dihydropyridine) binding sites and 45Ca2+ uptake processes in rat brain.

The binding of nimodipine, a 1,4-dihydropyridine Ca2+ channel antagonist, and of Bay K 8644, a Ca2+ channel activator, was measured in several regions of rat brain and compared to the distribution of K+ depolarization-induced 45Ca2+ uptake into synaptosomes. The maximum binding densities (Bmax) of [3H]nimodipine and [3H]Bay K 8644 were not significantly different one from the other, but differed according to brain region with binding being highest in the olfactory bulb and hippocampus, intermediate in the caudate nucleus and cerebral cortex (various regions), and lowest in the cerebellum [563 to 107 fmol/mg protein (mean)]. The KD values, [3H]nimodipine = 1.8 X 10(-10) M (mean) and [3H]Bay K 8644 = 1.4 X 10(-9) M (mean), did not differ according to region. Depolarization-induced 45Ca2+ uptake in synaptosomes occurred as fast (1 sec) and slow (10 sec) components distinguished by their selective occurrence in choline-containing and pre-depolarized preparations respectively. Distribution of the fast component of uptake paralleled that of [3H]nimodipine binding, being least in the cerebellum and greatest in the hippocampus and cortex, but the magnitude of the slow phase of 45Ca2+ uptake did not vary in the three brain regions studied.

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.

Photoaffinity labeling with dihydropyridine derivatives of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain.

The characteristics of photoaffinity labeling with the calcium agonist [3H]Bay K 8644 (Bay) and the calcium antagonists [3H]nitrendipine (Nit) and (+)PN200-110 (PN) of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain were investigated. In all these crude membranes, [3H](+)PN (20 nM) was mainly photoincorporated into one protein band with a molecular weight of 30,000 - 41,000 Da. It was also incorporated into some other bands of all these crude membranes. The photoincorporation of [3H](+)PN into these crude membranes was inhibited by the presence of 20 microM unlabeled (+)PN. The photoincorporation of [3H](+)PN into these crude membranes depended on its dose and on the time of UV irradiation. No incorporation of [3H](+)PN was observed in the absence of UV irradiation. The incorporation was not affected by the presence of 1 mM CaCl2 and/or 0.15 M NaCl, but was significantly decreased by 20 microM (+)PN and slightly decreased by 20 microM (-)PN, 20 microM Bay, 1 mM diltiazem, or 1 mM verapamil. Namely, enantiomers of PN caused various extents of stereoselective inhibition of photoaffinity labeling by [3H](+)PN of specific protein bands in these crude membranes. [3H]Nit was photoincorporated into these crude membranes in the same way as [3H](+)PN, but [3H]Bay was not photoincorporated. However, 20 microM unlabeled Nit did not consistently inhibit photoaffinity labeling with [3H]Nit. These findings suggested that measurement of photoaffinity of crude membranes from rat skeletal, cardiac, and uterine muscles and whole brain with [3H](+)PN by UV irradiation is a useful method for investigating the characteristics of the voltage-dependent calcium channels that are affected by 1,4-dihydropyridine derivatives.

Modulation of neurotransmitter metabolism by dihydropyridine calcium channel ligands in mouse brain.

The regional concentrations of dopamine, serotonin, dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindole acetic acid were measured in mouse brain following administration of the dihydropyridine calcium channel activator BAY K 8644, and antagonist, nifedipine. BAY K 8644 (1-8 mg/kg) produced dose- and time-dependent increases in dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid concentrations in the caudate, without altering dopamine and serotonin levels. No changes in 5-hydroxyindoleacetic acid concentration were observed in the raphe nuclei, hypothalamus, hippocampus and frontal cortex. Nifedipine (4 mg/kg) blocked BAY K 8644- (2 mg/kg) elicited increases in dihydroxyphenylacetic acid in the caudate. Furthermore, a higher dose of nifedipine (8 mg/kg) decreased dihydroxyphenylacetic acid and homovanillic acid, but did not affect dopamine, serotonin or 5-hydroxyindoleacetic acid concentrations, while a lower dose of nifedipine (2 mg/kg) significantly increased serotonin, 5-hydroxyindoleacetic acid and homovanillic acid, but did not affect dopamine and dihydroxyphenylacetic acid concentrations. The findings that both BAY K 8644 and nifedipine affect neurotransmitter metabolism in vivo in a dose-, time- and brain region-dependent manner, suggest that high-affinity dihydropyridine calcium channel binding sites play an important role in regulating neurotransmitter turnover in the central nervous system.

Thermodynamic and kinetic aspects of agonist and antagonist binding to 1,4-dihydropyridine receptors.

The kinetic and equilibrium binding properties of the 1,4-dihydropyridine activator [3H](-)-S-Bay K 8644 and the antagonist [3H](+)-PN 200-110 were determined in rat heart membrane particulate preparations at temperatures between 4 and 37 degrees C. The binding of [3H](-)-S-Bay K 8644 was temperature-dependent with a single binding site with KD = 3.57 nM and Bmax = 330 fmol/mg.protein at 25 degrees C. The association and dissociation rate constants were 3.4 x 10(7) min-1 M-1 and 0.095 min-1 respectively at 25 degrees C and decreased slightly at lower temperatures. In contrast, [3H](+)-PN 200-110 bound to high (KD(H) = 0.032 nM, Bmax(H) = 316 fmol/mg.protein) and low affinity sites (KD(L) = 27.6 nM and Bmax(L) = 6432 fmol/mg.protein) at 25 degrees C in rat heart preparation. A similar two-site binding of [3H](+)-PN 200-110 was found in rat brain preparation, but only a single binding site was detected in rat skeletal muscle. Binding of [3H](+)-PN 200-110 to the high and low affinity sites in cardiac membranes was sensitive and insensitive respectively to temperature. Association and dissociation rates of [3H](+)-PN 200-110 at the high affinity binding sites were best fitted as mono-exponential functions. Association and dissociation rates of [3H](+)-PN 200-110 were 3.94 x 10(8) min-1 M-1 and 7.86 x 10(-3) min-1 at 25 degrees C. The association rate varied only slightly (3-fold), but the rate of dissociation decreased significantly (200-fold) with temperature from 37 to 4 degrees C. Thermodynamic analysis of equilibrium binding showed that the binding of activator was enthalpy driven, whereas the binding of antagonist to the high affinity site was both entropy- and enthalpy-driven and to the low affinity site was totally entropy-driven.

Functional dihydropyridine binding site associated with slow calcium channel in rat cultured neurones.

There is a high affinity binding of [3H]PN 200-110 (Kd = 0.21 nM) to slow calcium channels in cultured neurones. Several calcium antagonists, which recognize the [3H]PN 200-110 binding site, did not affect the K+-induced calcium uptake. The calcium channel activator BAY K 8644 increased the calcium uptake in depolarizing conditions and this effect was antagonized by pharmacological concentrations of calcium entry blockers. We conclude that the dihydropyridine binding site is involved in the modulation of calcium entry through the voltage-sensitive channel in depolarized cultured neurones.

The effects of strychnine on the regulation of voltage-dependent calcium channels by dihydropyridines in brain and heart.

The effects of strychnine (STR) were investigated on K(+)-stimulated 45Ca2(+)-uptake into mouse brain neurons, the contractile activity of spontaneously beating rat atria and on [3H]nitrendipine and [3H]BAY K 8644 binding to dihydropyridine calcium channel antagonist and agonist binding sites on brain and cardiac membranes. STR (10(-6)-10(-4) M) had no effect on neuronal 45Ca2(+)-uptake. When combined at equimolar concentrations (10(-5) M), STR and nifedipine produced a potent (nM) inhibition (40%) of neuronal 45Ca2(+)-uptake. In the spontaneously beating rat atria, STR produced a dose-dependent (10(-7)-3 x 10(-4) M) decrease in chronotropy but did not affect inotropy. STR (10(-4) M) completely inhibited the positive chronotropic, but did not affect the positive inotropic effects of (-)-S-BAY K 8644. [3H]Nitrendipine and [3H]BAY K 8644 binding to brain and cardiac membranes was enhanced by STR in a concentration-dependent manner (EC50 8 X 10(-6) M). Scatchard analysis revealed that STR increased the affinity (decreased the Kd) of [3H]BAY K 8644 to a greater degree than that of [3H]nitrendipine for dihydropyridine binding sites. STR decreased the Kd of [3H]nitrendipine binding by increasing and decreasing the microassociation and microdissociation constants respectively. STR enhanced [3H]nitrendipine binding to the same extent in the cerebral cortex, striatum, hippocampus, cerebellum, brainstem and spinal cord. The enhancement of [3H]nitrendipine binding in brain was completely inhibited by Ca2+ and partially inhibited by Na+ in a concentration-dependent manner. Glycine (10(-2) M) did not affect the STR enhancement of [3H]nitrendipine binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of calcium channel agonist binding sites in the coronary artery.

The binding properties (3H) BAY k 8644 a 1,4-dihydropyridine calcium channel agonist were studied in the subcellular membrane fraction isolated from the coronary artery by differential centrifugation. The specific binding of (3H) BAY k 8644 to microsomal membranes of the coronary smooth muscle was rapid, saturable, reversible and of both high and low affinity. The dissociation constants obtained from Scatchard analysis with (3H) BAY k 8644 and nitrendipine were 0.60 +/- 0.02 nmol.l-1 and 9.1 +/- 0.1 nmol.l-1 for the high and low affinity binding site respectively and the estimated maximal numbers of binding sites in the plasma membrane fraction were 0.76 +/- 0.02 and 3.15 +/- 0.18 pmol.mg-1 of protein respectively. The substituted dihydropyridine calcium channel antagonists nitrendipine and nifedipine competitively inhibited specific (3H)BAY k 8644 binding suggesting a common high affinity 1,4-dihydropyridine binding site in the coronary microsomal fraction for calcium channel activator and antagonists. The low affinity agonist binding sites were significantly inhibited by adding nucleoside carrier inhibitors, 2-deoxyadenosine and dipyridamole, and by -SH alkylating agent N-ethylmaleimide. The results suggests that the coronary artery contains both high and low affinity calcium channel binding sites (in a 1:5 ratio) with the low affinity calcium channel agonist binding sites being associated with nucleoside carrier and/or with-SH groups.