Calcium Channel Antagonist Binding Research Articles

Comparative potencies of calcium channel antagonists and antischizophrenic drugs on central and peripheral calcium channel binding sites

Dihydropyridines are potent agents on [3H]nitrendipine binding sites in heart and brain membranes. Like the phenylalkylamines, they are slightly more active on heart than on brain [3H]nitrendipine binding sites. On the other hand, the diphenylalkylamines, the diphenylpiperazines and the antischizophrenic drugs of the diphenylbutylpiperdine type are more potent on brain [3H]nitrendipine binding sites. The findings suggest tissue heterogeneity of [3H]nitrendipine binding sites and the possible development of diphenylbutylpiperidine-diphenylbutylpiperazine analogues that could selectively act on brain calcium channel antagonist binding sites.

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.

Partial purification and characterization of the porcine brain enzyme hydrolyzing and synthesizing anandamide.

Anandamide (arachidonylethanolamide) is known as an endogenous agonist for cannabinoid receptors. An amidohydrolase, which hydrolyzed anandamide, was solubilized from the microsomal fraction of porcine brain with 1% Triton X-100. The enzyme was partially purified by Phenyl-5PW hydrophobic chromatography to a specific activity of approximately 0.37 mumol/min/mg of protein at 37 degrees C. As assayed with 14C-labeled substrates, the apparent Km value for anandamide was 60 microM, and anandamide was more active than ethanolamides of linoleic, oleic, and palmitic acids. Ceramidase and protease activities were not detected in our enzyme preparation. The purified enzyme also synthesized anandamide from free arachidonic acid in the presence of a high concentration of ethanolamine with a specific activity of about 0.16 mumol/min/mg of protein at 37 degrees C. On the basis of cochromatographies, pH dependence, heat inactivation, and effects of inhibitors such as arachidonyl trifluoromethyl ketone, p-chloromercuribenzoic acid, diisopropyl fluorophosphate, and phenylmethylsulfonyl fluoride, it was suggested that the anandamide amidohydrolase and synthase activities were attributable to a single enzyme protein.

In Vitro Binding of [ 3H]Nimodipine and [ 3H]CGS-19755 to Rat Brain in Focal Cerebral Ischemia

Focal cerebral ischemia results in increased in vivo binding of calcium channel antagonists to both the L-type voltage sensitive calcium channel (VSCC) and the N-methyl- d-aspartate (NMDA) receptor-linked calcium channel. It was the aim of this study to investigate the effect of focal cerebral ischemia on the in vitro binding of calcium channel antagonists to rat brain. Quantitative autoradiography was used to measure regional in vitro binding of the L-type VSCC antagonist [ 3H]nimodipine and the competitive NMDA receptor antagonist [ 3H]CGS-19755 to rat brain following 4 h of irreversible focal cerebral ischemia. [ 3H]Nimodipine binding to the nonischemic hemisphere was characterized by one binding site with regional binding affinity ( K D ) estimates ranging from 221 to 482 p M and maximal binding site densities ( B max ) ranging from 13.2 (9.6-17.5) pmol/g tissue (estimate and 95% confidence interval) in CA1 to 32.5 (26.5-39.9) pmol/g tissue in dentate. [ 3H]CGS-19755 binding to the nonischemic hemisphere was characterized by K D estimates ranging from 59 to 97 n M and B max values ranging from 143 (108-192) pmol/g tissue in cortex to 569 (515-641) pmol/g tissue in CA1. For [ 3H]CGS-19755 a model of two binding sites was applicable in several brain regions. No difference in binding site densities or binding affinities between ischemic and paired nonischemic structures (cortex and striatum) was observed with either ligand. In vitro binding of [ 3H]nimodipine and [ 3H]CGS-19755 to ischemic brain failed to identify ischemic-induced changes in calcium channel function previously reported by in vivo binding methods.

The effects of halothane on voltage-dependent calcium channels in isolated Langendorff-perfused rat heart.

Halothane has been previously shown in vitro to decrease both the inward calcium current in isolated cells and the density of calcium antagonist binding sites in cardiac sarcolemmal membranes prepared from several species, including humans, presumably contributing to the negative inotropic effects seen with volatile anesthetics. In this study we examined whether halothane produced similar changes in calcium channel antagonist binding characteristics ex vivo in an intact perfused heart by using isradipine, a dihydropyridine calcium channel blocker that binds specifically to the alpha 1 subunit of the L-type voltage-dependent calcium channel. The rat hearts were perfused by the Langendorff method in the presence of halothane and unlabeled isradipine. After the hearts were homogenized and prepared into membranes, a radioligand binding assay was performed and binding curves obtained. Data were analyzed by nonlinear regression analysis of a one-site binding equation and were evaluated by a paired t test. Halothane protected or inhibited the binding of unlabeled isradipine to calcium channels in a dose-dependent manner such that as the halothane is removed during the membrane preparation process, previously obscured sites were then available for specific binding of the radioligand. The sites that were protected by halothane had a lower affinity for [3H]-isradipine than controls. In both isolated membranes and the intact heart, halothane changes the availability of calcium channel antagonist binding sites, indicating a change in conformation of the voltage-dependent calcium channel in the presence of anesthetic. This change may result from a direct effect on the protein or from an indirect effect mediated through the membrane lipid bilayer. It also is demonstrated that halothane "protected" channels are probably a modified class of channels compared to those in control tissues as exemplified by the much lower affinity that the protected channels have for [3H]-isradipine. We conclude that a major mechanism by which halothane depresses contractility is mediated through the voltage-dependent calcium channel, and this process results from a conformational change in the channel.

Altered sarcolemmal calcium channel density and Ca 2+-pump ATPase activity in tachycardia heart failure

Whether sarcolemmal (SL) calcium handling is altered in endstage heart failure produced by chronic rapid pacing is not known. To investigate this we paced 7 rabbits at a rate of 400 beats/min for 35 ± 11 days. 6 animals served as non-paced controls. Purified left ventricular SL membranes were then prepared and tested for [ 3H]-nitrendipine binding and (Ca 2++Mg 2+)-dependent ATPase (Ca 2+-pump) activity. Results show a 50% reduction in calcium channel antagonist binding sites with B max values reduced from 450 ± 40 to 230 ± 8 fmoles/mg protein in response to chronic rapid pacing ( P < 0.01). This change was accompanied by a modest decrease in K d from 0.29 ± 0.09 to 0.22 ± 0.03 nM (not significant). V max values for the SL Ca 2+-pump ATPase were decreased from 387 to 164 nmoles/mg protein/min ( P < 0.01) with K Ca 2+ values reduced from 0.91 to 0.28 μM Ca 2+ ( P < 0.05) in response to tachycardia induced failure as compared to controls. ATPase activity in both groups was very sensitive to 25 μM calmidazolium and 5 μM vanadate. Results from this study indicate that both a reduction in SL calcium channel density and decrease in SL Ca 2+-pump ATPase activity are evident in tachycardia heart failure. We conclude that sarcolemmal calcium handling is altered in heart failure induced by chronic rapid pacing and that such changes may contribute to systolic dysfunction associated with this model of heart failure.

In vivo binding of [3H]nimodipine in rat brain after transient forebrain ischemia.

We report the regional variation in relative in vivo binding of the L-type voltage sensitive calcium channel (VSCC) antagonist [3H]nimodipine to brain following transient forebrain ischemia in the rat. At 30-min of reperfusion after 20 min of forebrain ischemia, [3H]nimodipine binding was significantly increased in striatum, CA3 and CA4, and dentate relative to binding in sham-operated rats, suggesting that VSCCs were responding to ischemic depolarization. Two h following ischemia, binding in all brain structures returned to normal levels indicating repolarization of cell membranes. At 24 h of recirculation, increased [3H]nimodipine binding was again observed in striatum and dentate. Binding remained elevated in the striatum and dentate, and increased binding became evident in the CA1 region of the hippocampus after 48 h of reperfusion. With the exception of the dentate gyrus, the second rise in [3H]nimodipine binding anticipated or coincided with the observed regional ischemic cell changes. These observations in global cerebral ischemia support previous work indicating that in vivo binding of [3H]nimodipine to the L-type VSCC may be an early and sensitive indicator of impending ischemic injury. Such measurements may be of use in identifying vulnerable brain regions and defining a therapeutic window of opportunity in models of cerebral ischemia.

The Role of L-Type Voltage-Dependent Calcium Channels in Anesthetic Depression of Contractility

In this chapter data are presented to show that not only halothane but also enflurane and isoflurane decrease the density of binding sites for the calcium antagonist [ 3 H]isradipine in adult and newborn rabbit myocardial membranes. The chapter provides an observation that halothane can interfere with the binding of calcium channel antagonist (CCA) to cardiac L-type voltage-dependent Ca 2+ channel (VDCC). It has been convincingly demonstrated that the other volatile anesthetics, enflurane and isoflurane, are both able to interfere with the binding of a radiolabeled dihydropyridine, [ 3 H]isradipine, to both cardiac and skeletal muscle membranes. This phenomenon is relevant to anesthetic depression of contractility in an intact organ. Halothane can actually protect VDCC from blockade by isradipine. However, the sites have been protected by halothane appear to have a markedly lower affinity for [ 3 H]isradipine than the sites that has been untouched by halothane. The decreased affinity that has been observed in the VDCC, “protected” by halothane suggests that halothane is either modifying the VDCC or selecting an independent group of VDCC. Isradipine is known to bind to inactive channels with high affinity, and it is possible that halothane “protects” channels that are in the “unavailable” state or that have been modified by phosphorylation or other mechanisms resulting in a change of the K d for isradipine. This is a distinctly different result from previous in vitro data and from the K d values obtained from control hearts and hearts perfused with isradipine alone.

O-222 - Effect of ethanol on calcium channel antagonist binding in chick embryo brain.

O-222 - Effect of ethanol on calcium channel antagonist binding in chick embryo brain.

Autoradiographic localization of the binding of calcium channel antagonist, [ 125I]ω-agatoxin IIIA, in rat brain

The calcium channel antagonists ω-agatoxin IIIA (ω-Aga-IIIA) and ω-conotoxin GVIA (ω-CgTx) were radioiodinated and used to locate binding sites in the rat brain by receptor autoradiography. While patterns of regional binding to sagittal sections of rat brain were generally similar for the 2 toxins, notable differences in the cerebellum and hippocampus were observed. Specific [ 125I]ω-Aga-IIIA binding was greatest in the granule cell layers of the cerebellum and of the dentate gyrus. In contrast, binding of [ 125I]ω-CgTx was most intense in the molecular layers of these structures. Less than one-third of [ 125I]ω-Aga-IIIA binding in rat brain slices was inhibited by pre-exposure to 250 nM ω-CgTx, while 40 nM ω-Aga-IIIA virtually eliminated the binding of [ 125I]ω-CgTx under the same conditions. The P-type calcium channel antagonist ω-Aga-IVA blocked only a small fraction of [ 125I]ω-Aga-IIIA and [ 125I]ω-CgTx binding. These autoradiographic data are consistent with membrane binding experiments and indicate that the combined use of agatoxins and conotoxins may be useful in the characterization of separate types of neuronal calcium channels.

A correlative study of calcium channel antagonist binding and local neuropathological features in the hippocampus in Alzheimer's disease

[ 3H]PN200-110 binding sites were studied by means of quantitative autoradiography in hippocampal sections of patients with Alzheimer's disease and age-matched control subjects. Choline acetyltransferase activity, plaque, tangle and cell densities were also determined in the same tissue samples used for autoradiographic studies. Quantitative autoradiographic analysis of [ 3H]PN200-110 binding in control hippocampus revealed a heterogeneous pattern similar to that described in rodents, being particularly high in the dentate gyrus. In Alzheimer's disease, [ 3H]PN200-110 binding was markedly reduced in the subiculum (control = 9.85 ± 1.41 pmol/g; Alzheimer = 3.41 ± 0.54 pmol/g, mean ± S.E.M., P < 0.001). In the subiculum there was a disproportionate reduction of [ 3H]PN200-110 binding in comparison to cell loss in Alzheimer's disease. The activity of choline acetyltransferase in the hippocampus was markedly reduced in Alzheimer's disease (controls 6.9 ± 1.0; Alzheimer 2.7 ± 0.9 nmol/h/mg protein, mean ± S.E.M., P < 0.01). There was a strong corrElation between choline acetyltransferase activity and [ 3H]PN200-110 binding in the subiculum. [ 3H]PN200-110 binding did not correlate with plaque density in the subiculum. The discrete reduction and preservation of [ 3H]PN200-110 binding in the present study is consistent with the pattern of selective cellular vulnerability in the hippocampal region in Alzheimer's disease.

Low-affinity binding sites for 1,4-dihydropyridines in skeletal muscle transverse tubule membranes revealed by changes in the fluorescence of felodipine.

The fluorescence changes accompanying the binding of the fluorescent calcium channel antagonist, felodipine, to transverse tubule membranes from rabbit skeletal muscle have been used to characterize low-affinity binding sites for 1,4-dihydropyridine derivatives in these preparations. In competition experiments, felodipine inhibited the high-affinity binding of (+)-[3H]PN200-110 to transverse tubule membranes with an apparent Ki of 5 +/- 2 nM. Binding of felodipine to additional low-affinity sites resulted in a large, saturable (Kd = 6 +/- 2 microM) increase in its fluorescence which could be excited either directly (380 nm) or indirectly via energy transfer from membrane protein (290 nm). The observed fluorescence enhancement was competitively inhibited by other 1,4-dihydropyridines with inhibition constants of 3-21 microM but was unaffected by the structurally unrelated calcium channel antagonists, diltiazem and verapamil, or by Ca2+, Cd2+, and La3+. Both high- and low-affinity binding sites appear to be localized in the transverse tubular system, since the magnitude of the observed fluorescence enhancement was higher in these membranes than in microsomal preparations and was directly proportional to the density of high-affinity sites for (+)-[3H]PN200-110. Furthermore, both high- and low-affinity sites appear to be conformationally coupled since, over the same concentration range that the fluorescence changes were observed, felodipine accelerated the rate of dissociation of [3H]PN200-110 previously bound to its high-affinity sites. Similar behavior has previously been reported for other 1,4-dihydropyridines [Dunn, S. M. J., & Bladen, C. (1991) Biochemistry 30, 5716-5721].(ABSTRACT TRUNCATED AT 250 WORDS)

Age-Related Changes in Calcium Antagonist Receptors in Rabbit Ureter

(+)-[3H]PN 200-110 (a dihydropyridine calcium channel antagonist) binding sites were studied in ureters of 1-day (neonatal), 6-week (premature), 6-month (young) and 4.5- to 5-year (old) female rabbits. Specific binding of (+)-[3H]PN 200-110 to ureteral membrane particulates was saturable, reversible and of high affinity. The densities (Bmax) of (+)-[3H]PN 200-110 binding sites were 46.7 +/- 2.5, 22.6 +/- 2.0, 12.7 +/- 1.8 and 11.9 +/- 1.6 fmol/mg protein in 1-day, 6-week, 6-month and 4.5- to 5-year rabbit ureters, respectively. The affinity constants (KD) of the binding sites for (+)-[3H]PN 200-110 were similar in all groups. Calcium agonists and antagonists inhibited (+)-[3H]PN 200-110 binding to 1-day and 6-week rabbit ureters with the following rank order of Ki values: nitrendipine < nifedipine < BAY K 8644 < verapamil. There were no significant differences in Ki values between the neonatal and premature groups. The data demonstrate the presence of an age-related down-regulation of (+)-[3H]PN 200-110 binding sites in rabbit ureteral membrane particulates.

Effects of chronic lithium, amitriptyline, and electroconvulsive shock, on calcium channel binding in a rat brain homogenate.

The effect of chronic lithium (0.1% in the diet for 28 days), amitriptyline (10 mg/kg SC daily for 14 days), and electroconvulsive shock (ECS) (given 5 x under halothane anaesthesia on alternate days) on the binding of the L-type calcium channel antagonist (+)-(3H)-PN 200-110 to rat cortical and hippocampal homogenates and the binding of the N-type calcium channel antagonist (125I)-omega-conotoxin to rat cortical homogenates was measured. These treatments did not alter the number or affinity of the binding sites for either ligand, suggesting that it is unlikely that the actions of chronic lithium, amitriptyline, and ECS affect calcium channel binding.

The interaction of BBR 2160 with calcium channel antagonist binding sites

The interaction of BBR 2160 with calcium channel antagonist binding sites

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 45Ca 2+-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 45Ca 2+-uptake. When combined at equimolar concentrations (10 −5 M), STR and nifedipine produced a potent (nM) inhibition (40%) of neuronal 45Ca 2+-uptake. In the spontaneously beating rat atria, STR produced a dose-dependent (10 −7−3 × 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 (EC 50 8 × 10 −6 M). Scatchard analysis revealed that STR increased the affinity (decreased the K d) of [ 3H]BAY K 8644 to a greater degree than that of [ 3H]nitrendipine for dihydropyridine binding sites. STR decreased the K d 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 Ca 2+ and partially inhibited by Na + in a concentration-dependent manner. Glycine (10 −2 M) did not affect the STR enhancement of [ 3H]nitrendipine binding. Extensive rinsing of brain membranes increased (∼two-fold) the affinity of STR for enhancement of [ 3H]nitrendipine binding. The effects of STR on [ 3H]nitrendipine binding both resembled and were unique to those of phencyclidine. These findings suggest that STR interacts specifically with calcium channels in brain and cardiac tissues at a site similar, but not identical to that of phencyclidine and which is distinct from the dihydropyridine calcium channel regulatory site.

Genetic up-regulation of calcium channels in a cellular model of ethanol dependence

Bovine adrenal chromaffin cells were maintained in culture medium containing ethanol (200mM) for 6 days. Cultures maintained in ethanol were viable and were morphologically similar to controls. There was a greater than 100% increase in the number of [ 3H]dihydropyridine calcium channel antagonist binding sites on the cell membranes from ethanol-treated cultures, which could be prevented by concomitant exposure to cycloheximide (5 μg·ml −1) and the mRNA synthesis inhibitor, anisomycin (10 μg·ml −1) implicating de novo synthesis of protein and genetic regulation, respectively. Release of catecholamines, induced by 18mM K +, from cultures grown in ethanol was enhanced. The increased release of catecholamines was inhibited by nM concentrations of dihydropyridine calcium channel antagonists, implying that an increase in the number of dihydropyridine-sensitive calcium channels accounts for this functional alteration.

Reduction of calcium channel antagonist binding sites by oxygen free radicals in rat heart

In view of the importance of Ca 2+-channels in controlling the entry of Ca 2+ into the myocardium, this study was undertaken to examine the effects of oxygen free radicals on the binding of Ca 2+-channel antagonists in rat heart by employing [ 3H]-nitrendipine as a ligand. Isolated heart membranes were incubated with xanthine + xanthine oxidase (a superoxide anion radicals generating system), hydrogen peroxide (an activated species of oxygen), or hydrogen peroxide + Fe 2+ (a hydroxyl radicals generating system). The assay of the [ 3H]-nitrendipine binding activity revealed that the maximal number of binding sites ( B max) were reduced in a time-dependent manner by superoxide radicals without any changes in the binding constant ( K d); a significant reduction of B max was seen after incubating membranes with xanthine + xanthine oxidase for a 10-min-period. Superoxide dismutase showed a protective effect on the superoxide radicals induced reduction in B max. Both hydrogen peroxide and hydroxyl radicals also depressed the B max for [ 3H]-nitrendipine binding without any significant change in K d; catalase and mannitol showed protective effects on hydrogen peroxide or hydroxyl radicals induced depression in B max, respectively. These results indicate that oxygen free radicals may reduce the number of Ca 2+-channels in the cell membrane and this change may contribute towards decreasing the voltage-dependent Ca 2+ influx in the cardiac cell.

Calcium Channel Agonist and Antagonist Binding in a Highly Enriched Sarcolemma Preparation Obtained from Canine Ventricle

The purpose of the present study was to characterize the binding of the Ca2+ channel agonist (+/-)[3H]Bay K 8644 in a highly enriched cardiac membrane preparation and to examine its interactions with other Ca2+ channel ligands. Scatchard analysis showed that (+/-)[3H]Bay K 8644 displayed some 265-fold less affinity for its receptor than did the Ca2+ channel antagonist (+)[3H]PN200-110. Furthermore, (+/-)[3H]Bay K 8644 binding site density was significantly less than that seen for (+)[3H]PN200-110. Kinetic analysis of (+/-)[3H]Bay K 8644 binding revealed biphasic association and dissociation rates. The Ca2+ channel antagonist MDL 12,330A stimulated both (+)[3H]PN200-110 and (+/-)[3H]Bay K 8644 binding by effects on Kd. In contrast, diltiazem inhibited (+)[3H]PN200-110 binding but had no effect on (+/-)[3H]Bay K 8644 binding. Both MDL 12,330A and diltiazem inhibited Ca2+-dependent contraction in rat aortic rings but the combination of these two drugs was less than additive in this regard. We conclude that the binding profile of (+/-)[3H]Bay K 8644 results from the racemic nature of this ligand and that MDL 12,330A and diltiazem interact at sites distinct from one another on or around the slow voltage-dependent Ca2+ channel.

Calcium Channel Agonist and Antagonist Binding in a Highly Enriched Sarcolemma Preparation Obtained from Canine Ventricle

Calcium Channel Agonist and Antagonist Binding in a Highly Enriched Sarcolemma Preparation Obtained from Canine Ventricle