Calcium Channel Binding Research Articles

Long-term effects of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate and 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione in the rat basal ganglia: calcification, changes in glutamate receptors and glial reactions

Previous data from our laboratory indicate that 25 mM ibotenic acid induces intracellular calcifications in the rat basal forebrain. Because of the lack of specificity of ibotenic acid for a glutamate receptor subtype, a dose–response study with α-amino-3-hydroxy-5-methyl-4-isoxazole propionate was undertaken and calcified areas (identified with Alizarin Red staining) as well as astro- and microglial reactions (by autoradiography with [ 3H]lazabemide and [ 3H]Ro 5-4864) were quantified at one month post-lesion. α-Amino-3-hydroxy-5-methyl-4-isoxazole propionate administered into the globus pallidus induced, in a dose-dependent manner, the formation of calcium deposits and the activation of both glial cells, the microglial reaction being particularly robust. From this study, a dose of 5.4 mM α-amino-3-hydroxy-5-methyl-4-isoxazole propionate was selected for further experiments. [ 3H]α-amino-3-hydroxy-5-methyl-4-isoxazole propionate, [ 3H]dizocilpine maleate and [ 3H]PN 200-110 binding in vitro were performed to assess autoradiographically whether the tissue damage was associated with changes in glutamate receptors and calcium channel binding sites. In the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate-treated animals, the specific binding of [ 3H]α-amino-3-hydroxy-5-methyl-4-isoxazole propionate was significantly reduced by 28% in the lesioned ventral pallidum, whereas it was unchanged in the globus pallidus and substantia innominata. In these three nuclei, calcifications developed and an increase in both glial markers was measured. In contrast, the binding of [ 3H]PN 200-110 and [ 3H]dizocilpine maleate were unaffected. Co-injection of 15 mM 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione, a selective α-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor antagonist, prevented the formation of calcium concretions, the microglial reaction and the decrease in [ 3H]α-amino-3-hydroxy-5-methyl-4-isoxazole propionate binding but it failed to inhibit totally the astroglial reaction induced by α-amino-3-hydroxy-5-methyl-4-isoxazole propionate. This may suggest that the microglial reaction and calcification take place through different mechanisms from the astrogliosis associated with the neuronal loss. In conclusion, acute administration of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate in the rat globus pallidus elicits a dose-dependent calcification process associated with a chronic reaction of astrocytes and microglia. α-amino-3-hydroxy-5-methyl-4-isoxazole propionate-induced injury is accompanied by a slight reduction of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors in the ventral pallidum, whereas the binding of N-methyl- d-aspartate and L-type calcium channels receptors remains unchanged in any lesioned nucleus.

Refined solution structure of omega-conotoxin GVIA: implications for calcium channel binding.

The polypeptide omega-conotoxin GVIA (GVIA) is an N-type calcium channel blocker from the venom of Conus geographus, a fish-hunting cone shell. Here we describe a high-resolution solution structure of this member of the 'inhibitor cystine knot' protein family. The structure, based on NMR data acquired at 600 MHz, has mean pairwise RMS differences of 0.25 +/- 0.06 and 1.07 +/- 0.14 A over the backbone heavy atoms and all heavy atoms, respectively. The solvent-accessible side chains are better defined than in previously published structures and provide an improved basis for docking GVIA with models of the calcium channel. Moreover, some side chain interactions important in GVIA folding in vitro and in stabilizing the native structure are defined clearly in the refined structure. Two qualitatively different backbone conformations in the segment from Thr11 to Asn14 persisted in the restrained simulated annealing calculations until a small number of lower bound constraints was included to prevent close contacts from occurring that did not correspond with peaks in the NOESY spectrum. It is possible that GVIA is genuinely flexible at this segment, spending a finite time in the alternative conformation, and this may influence its interaction with the calcium channel.

Polypeptide ?-conotoxin GVIA as a basis for new analgesic and neuroprotective agents

The ω-conotoxins are small polypeptides (of around 25 residues) cross-linked by three disulfide bonds. At least two of these, ω-conotoxins GVIA and MVIIA, are potent and selective blockers of N-type voltage-gated calcium channels. Administered intravenously, GVIA causes postural hypotension and blocks cardiac sympathetic and vagal reflexes, homeostatic cardiovascular effects that should normally be preserved. Administered intrathecally, MVIIA and GVIA are analgesic in acute, chronic, and neuropathic pain models, and protective following ischaemia-induced neuronal injury. We have determined the three-dimensional structure of GVIA and mapped onto that structure its calcium channel binding surface. This information is now being used in the structure-based design of truncated and stabilised peptidic analogues of GVIA and in the development of peptidomimetic analogues. This article summarizes these data and outlines the strategies being pursued in the development of low molecular weight analogues for therapeutic applications. Drug Dev. Res. 46:206–218, 1999. © 1999 Wiley-Liss, Inc.

Structure-Function Relationships of ω-Conotoxin GVIA: SYNTHESIS, STRUCTURE, CALCIUM CHANNEL BINDING, AND FUNCTIONAL ASSAY OF ALANINE-SUBSTITUTED ANALOGUES

The structure-function relationships of the N-type calcium channel blocker, omega-conotoxin GVIA (GVIA), have been elucidated by structural, binding and in vitro and in vivo functional studies of alanine-substituted analogues of the native molecule. Alanine was substituted at all non-bridging positions in the sequence. In most cases the structure of the analogues in aqueous solution was shown to be native-like by 1H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Replacement of Lys2 and Tyr13 with Ala caused reductions in potency of more than 2 orders of magnitude in three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) and a rat brain membrane binding assay. Replacement of several other residues with Ala (particularly Arg17, Tyr22 and Lys24) resulted in significant reductions in potency (<100-fold) in the functional assays, but not the binding assay. The potencies of the analogues were strongly correlated between the different functional assays but not between the functional assays and the binding assay. Thus, the physiologically relevant assays employed in this study have shown that the high affinity of GVIA for the N-type calcium channel is the result of interactions between the channel binding site and the toxin at more sites than the previously identified Lys2 and Tyr13.

Binding and distribution of three prototype calcium channel blockers in perfused rat liver.

This work represents a study of the binding and distribution of three different calcium channel blockers in the Sprague-Dawley rat liver, using an in situ perfusion technique. For this purpose, [3H] desmethoxyverapamil, [3H] PN200-110 (isradipine) and [3H] azidopine were used as binding probes interacting with calcium channels. The perfusion steps of the liver involved both portal vein and thoracic inferior vena cava cannulations as inlet and outlet respectively. The subhepatic inferior vena cava was ligated to prevent leakage of the perfusate. Buffer, containing the tracer drug, was administered via the portal vein at a rate of 1 mL/min and perfusate collected at the same rate within specified time intervals during 50 min. The concentration of the tracer solutes in the perfusate's outlet increased with time, and steady state was observed for all tracers at > or = 40 min. The effect of adding cold isradipine to tracer desmethoxyverapamil, or cold verapamil to tracer PN200-110 were also assessed. First order rate constants for hepatocellular influx, efflux and calcium channel binding of the tracer substances were obtained using a simplified model from Goresky et al. These constants were mathematically manipulated and changed into permeability constants, second order binding constants, and residency times. Tracer solute influx across hepatocellular membranes is solubility-diffusion controlled, is inversely related to the molecular weights and is different in value from the efflux constants. Cold isradipine reduced the binding constant of desmethoxyverapamil by 36%, while cold verapamil reduced the binding constant of PN200-110 by 23%. Azidopine cellular distribution was low, however, binding to its receptor was analogous to desmethoxyverapamil and PN200-110. Moreover, PN200-110 had the highest residency time with no effect of cold verapamil on its receptor binding, while desmethoxyverapamil had the lowest residency time which significantly increased in the presence of cold isradipine.

Binding Sites for Ca2+‐Channel Effectors and Ryanodine inPeriplaneta americana—Possible Targets for New Insecticides

The calcium channel and the 'calcium release channel' of muscle membrane of the cockroach Periplaneta americana have been characterized. Biological assays with calcium channel blockers and ryanodine on different insects and acari revealed pronounced insecticidal effects with ryanodine, but not with calcium channel blockers, at concentrations between 0.1 and 300 μg ml -1 . Skeletal muscle membranes derived either from the tubular network or from the sarcoplasmatic reticulum of P. americana were characterized with respect to the binding of the dihydropyridine (DHP) [ 3 H]isradipine (PN 200-110), the phenylalkylamine [ 3 H]verapamil and the alkaloid [ 3 H]ryanodine. Preliminary binding studies with the benzothiazepine [ 3 H]diltiazem suggest a low-affinity binding site with a IC 50 value of 3.3 μM. All binding sites tested were sensitive to treatment with proteinase K. Optimal conditions for binding of the radioligand ryanodine revealed the highest specific binding at pH 8 and at calcium chloride concentrations between 100 and 500 μM. EGTA at 10 μM abolished 95% of the ryanodine binding. Binding studies with calcium channel binding sites revealed a pronounced effect of low Ca 2+ concentrations on specific isradipine binding, whereas verapamil and diltiazem binding were only reduced by the presence of 200 μM EGTA. With respect to high Ca 2+ concentrations, specific binding of diltiazem, isradipine and verapamil was reduced by 73, 40 and 20%, respectively, at 5 mM Ca 2+ . Radioligand binding experiments showed high-affinity binding sites for ryanodine and isradipine. K D values of 0.95 nM (B max = 550 fmol mg -1 protein) and 0.75 nM (B max = 213 fmol mg -1 protein) were determined respectively. A lower-affinity binding site was identified in binding studies with verapamil (K D = 7.4 nM and B max = 27 fmol mg -1 protein). [ 3 H]isradipine displacement studies with several dihydropyridines revealed the following ranking of affinity : nitrendipine > isradipine > Bay K8664 >> nicardipine. Displacement of [ 3 H]verapamil binding by effectors of the phenylalkylamine binding site showed that bepridil and S(-)verapamil had the highest affinities of the compounds tested followed by (±)verapamil, nor-methylverapamil and R(+)verapamil.

Solution structure of ω-conotoxin MVIIA using 2D NMR spectroscopy

The solution structure of ω-conotoxin MVIIA (SNX-111), a peptide toxin from the fish hunting cone snail Conus magus and a high-affinity blocker of N-type calcium channels, was determined by 2D NMR spectroscopy. The backbones of the best 44 structures match with an average pairwise RMSD of 0.59 angstroms. The structures contain a short segment of triple-stranded β-sheet involving residues 6–8, 20–21, and 24–25. The structure of this toxin is very similar to that of ω-conotoxin GVIA with which is has only 40% sequence homology, but very similar calcium channel binding affinity and selectivity.

Altered muscle calcium channel binding kinetics in autoimmune motoneuron disease.

While skeletal muscle is not apparently affected directly in amyotrophic lateral sclerosis (ALS), immunoglobulin G fractions purified from patients with ALS (ALS IgG) bind dihydropyridine (DHP)-sensitive L-type voltage-gated calcium channel (VGCC) antigen isolated from skeletal muscle in ELISA and Western immunoblot, and alter VGCC function in vitro. To determine whether muscle VGCC properties are altered in ALS, VGCC-enriched subsarcolemmal membrane fractions were prepared from biopsied quadriceps muscle of patients with ALS, with other neurologic diseases, or without apparent muscle disease, and tested for DHP binding with [3H]PN200-110. ALS muscle VGCCs possessed eightfold higher binding affinities for [3H]PN200-110 than did VGCCs from muscle fractions of most other patients, independent of denervation-induced increases in DHP binding site number. Similarly elevated DHP binding affinities were observed in specimens from patients with autoimmune motor neuropathies, suggesting that ALS and immune mediated motoneuron disease share skeletal muscle L-type VGCC alterations.

Why is nisoldipine a specific agent in ischemic left ventricular dysfunction?

Nisoldipine is a dihydropyridine calcium entry blocker that inhibits contraction of vascular smooth muscle with a potency that is 2–3 times greater than its impact on myocardial contractility. In isolated human coronary arteries, tonic contractions induced by serotonin are inhibited by nisoldipine with a potency 10 times greater than that in internal mammary arteries and 1,000 times greater than that in electrically driven myocardium. In contrast, nifedipine had little effect and verapamil and diltiazem had none. In this article an hypothesis is reviewed that relates vascular smooth muscle selectivity to membrane potential sensitivity. Nisoldipine's effect on calcium channel binding and blocking is enhanced by the degree of depolarization of the cell membrane. Verapamil and diltiazem are not membrane-potential sensitive. Vascular smooth muscle cells are more depolarized than myocardial cells, and human coronary arteries have a particularly small membrane potential. Thus, the potency of nisoldipine in these organs parallels the degree of membrane depolarization. This may then suggest ischemia selectivity, since membrane depolarization occurs in ischemic tissue. Nisoldipine might therefore have a potent negative inotropic effect and an enhanced vasodilator action in ischemic myocardium, yet leave normoxic regions functionally intact. Some experimental evidence is discussed.

Pharmacological profile of a novel neuronal calcium channel blocker includes reduced cerebral damage and neurological deficits in rat focal ischemia

Excessive calcium entry into depolarized neurons contributes significantly to cerebral tissue damage following ischemia. Therefore, blocking voltage-operated calcium channels on nerve cells should provide significant neuroprotection in ischemia. We now report on a novel neuronal calcium channel blocker, NNC 09-0026, in terms of its selective effects on neuronal calcium current and its efficacy in reducing infarct size and neurological deficits in a rat model of focal stroke. In the present studies, the effects of NNC 09-0026 on neuronal calcium influx, calcium channel binding, and cardiovascular parameters were determined. Also, phencyclidine, NNC 09-0026, or vehicle were administered IV to rats subjected to permanent middle cerebral and common carotid artery occlusions. Infarct volumes and contralateral forepaw and hindlimb neurological deficits were assessed at 24 and 48 h after onset of stroke. NNC 09-0026 exhibited a pharmacological profile suggesting selectivity at neuronal calcium channels. It inhibited potassium-stimulated calcium uptake into rat synaptosomes with an IC 50 of 13 μM. Voltage-operated calcium currents measured from cultured rat dorsal root ganglion cells using the patch clamp technique were blocked by 43% at 10 μM ( p < 0.05). The compound showed only weak effects on smooth muscle from the guinea pig taenia coli and was relatively inactive at displacing nitrendipine and ω-conotoxin in receptor-binding studies. Single, bolus injections of NNC 09-0026 as high as 10 mg/kg IV produced only 12% reduction in heart rate and a 28% decrease in blood pressure. Phencyclidine, 1.5 or 3 mg/kg, IV bolus 30 min pre- and 24 h postocclusion, reduced infarct volume by 46% and 52% ( p < 0.05) and decreased neurological deficits in a parallel manner. Phencyclidine did produce significant neurobehavioral side effects in all animals. NNC 09-0026, 30 mg/kg, IV administered slowly over a 1-h period beginning 30 min postischemia, reduced infarct volume by 45% and neurological deficits ( p < 0.05). No obvious neurobehavioral side effects were produced by NNC 09-0026. These data indicate that NNC 09-0026 is a relatively selective neuronal calcium channel blocker that can provide significant neuroprotection in a rat model of focal ischemia.

Synthesis and in vitro pharmacology of new 1,4-dihydropyridines. 1. 2-(ω-Aminoalkylthiomethyl)-1,4-dihydropyridines as potent calcium channel blockers

The synthesis and in vitro calcium channel blocking activities and binding of 2-(ω- aminoalkylthiomethyl)-4-(substituted)phenyl-1,4-dihydropyridines, by determination of the displacement of [ 3H]nitrendipine from the calcium channel binding sites on rat cortex have been discussed. It has been shown that increasing the alkyl chain length on the 2-position of the 1,4-dihydropyridine ring from ethyl to pentyl does not affect the calcium channel blocking activity of 3-nitrophenyl substituted dihydropyridines, measured on K +-depolarisation induced contractile responses in rat aorta strips. It did not seem to be important whether the 1,4-dihydropyridines bore 2 identical or different ester moieties on the 3- and 5-position of the 1,4-dihydropyridine ring.

Measures of Skeletal Muscle Calcium Channels and Acetylcholine Receptors in Thyroidectomized Rats

Hypothyroidism frequently presents with muscle complaints. No consistent histopathology nor electrophysiology explains these symptoms and signs. As well, no previous study shows specific changes in components of the nerve-muscle synapse nor excitation-contraction coupling in adult muscles, but changes are seen in hormone-treated embryonic myoblasts. In this study, adult male Holtzman rats underwent thyroidectomy and their age-matched euthyroid controls were simultaneously subjected to sham operation. Thirty days post-operative, animals were sacrificed for anterior tibialis muscles harvest. Muscle dihydropyridine type calcium channel (isradipine) and acetylcholine receptor (alpha-bungarotoxin) binding were measured and compared between experimental treatment groups. There were no significant differences in either the affinity or density of isradipine binding. However, hypothyroid muscles showed a nearly 50% reduction in acetylcholine receptor density when compared to control muscles. Thyroidectomy is associated with specific effects on components of neuromuscular transmission in adult fast twitch muscle.

Preserved neurotransmitter receptor binding following ischemia in preconditioned gerbil brain

Preconditioning the brain with sublethal ischemia induces tolerance to subsequent ischemie insult. Using [ 3H]quinuclidinyl benzilate (QNB), [ 3H]MK 801, [ 3H]cyclohexyladenosine, [ 3H]muscimol, and [ 3H]PN200-110, we investigated the alterations in neurotransmitter receptor and calcium channel binding in the gerbil hippocampus following ischemia with or without preconditioning. Two-minute forebrain ischemia, which produced no neuronal damage, resulted in no alterations in binding except for a slight reduction in [ 3H]QNB binding in the CA1 subfield. Three-minute ischemia destroyed the majority of CA1 pyramidal cells and caused, in CA1, reductions in binding of all ligands used. Preconditioning with 2-min ischemia followed by 4 days of reperfusion protected against CA1 neuronal damage and prevented the reductions in binding although [ 3H]QNB and [ 3H]PN200-110 binding transiently decreased in the early reperfusion period, suggesting downregulation. Thus, preconditioning protects against damage to the neurotransmission system as well as histopathological neuronal death.

Multiple actions of glaucine on cyclic nucleotide phosphodiesterases, alpha 1-adrenoceptor and benzothiazepine binding site at the calcium channel.

1. In the present study, the properties of glaucine (an aporphine structurally related to papaverine) were compared with those of papaverine, diltiazem, nifedipine and prazosin. The work includes functional studies on rat isolated aorta contracted with noradrenaline, caffeine or KCl, and a determination of the affinity of glaucine at calcium channel binding sites of alpha-adrenoceptors, by use of [3H]-(+)-cis-diltiazem, [3H]-nitrendipine and [3H]-prazosin binding to cerebral cortical membranes. The effects of glaucine on the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta were also determined. 2. Contraction evoked by noradrenaline (1 microM) or depolarizing solution (60 mM KCl) were inhibited in a concentration-dependent manner by all the compounds tested. As expected, prazosin showed a greater selectivity of action on NA-induced contraction, whereas nifedipine and diltiazem appeared more potent on KCl-induced contraction. Glaucine had a greater potency on the contraction elicited by noradrenaline whereas papaverine acted non specifically. 3. In Ca(2+)-free solution, prazosin (0.1 microM) and glaucine (0.1 mM) inhibited the contraction evoked by NA; diltiazem (0.1 mM) diminished this contraction whereas nifedipine (1 microM) had no effect. Preincubation of tissues with glaucine, diltiazem, nifedipine and prazosin did not modify the contractile response induced by caffeine. In contrast, papaverine (0.1 mM) significantly inhibited the contractions evoked by NA or caffeine in Ca(2+)-free medium. 4. Glaucine and papaverine show affinity at the [3H]-prazosin binding site and at the benzothiazepine binding site of the Ca(2+)-channel receptor complex, but have no effect at the dihydropyridine binding site in rat cerebral cortex. Glaucine exerts some selectivity as an inhibitor of [3H]-prazosin binding as opposed to [3H]-(+ )-cis-diltiazem binding while papaverine appears to have approximately equal affinity in this respect.5. This study confirms the presence of four phosphodiesterase (PDE) activities in bovine aorta: a calmodulin-activated PDE (CaM-PDE type I) which hydrolyzed preferentially guanosine 3':5'-cyclic monophosphate (cyclic GMP); a cyclic GMP selective form (cGMP-PDE type V); and two low Km adenosine 3':5'-cyclic monophosphate (cyclic AMP) PDEs that are insensitive to the stimulatory effect of CaM, one of which was inhibited by cyclic GMP (CGI-PDE, type III) and the other by rolipram (cAMP-PDE, type IV). Glaucine selectively inhibits one of the two forms of Ca2+-independent low Km cAMP-PDE, the type IV. In contrast, papaverine exerts a non-selective inhibitory effect upon all PDE forms.6. The present work provides evidence that glaucine, a benzyltetrahydroisoquinoline alkaloid, has interesting properties as an alpha l-adrenoceptor antagonist, calcium entry blocker (through the benzothiazepine recognition site in the calcium channel) and as a selective inhibitor of the rolipram-sensitive cAMP-PDE, type IV PDE.

Evidence for the presence of regional differences in the calcium antagonist receptors in lower urinary tract smooth muscle.

(+)-[3H]PN 200-100 (a dihydropyridine calcium channel antagonist) was utilized to characterize calcium channel binding sites in rabbit bladder dome, bladder base, and urethra. Specific binding of (+)-[3H]PN 200-110 to membrane particulates was saturable, reversible, linear to protein concentration, and of high affinity. The density of (+)-[3H]PN 200-110 binding sites (Bmax values in fmol/mg of protein) and the affinity constants for (+)-[3H]PN 200-110 (KD value in pM) in urethra, bladder dome and bladder base were 64.1 +/- 7.8 and 179 +/- 31; 21.9 +/- 3.0 and 213 +/- 36; and 18.8 +/- 4.2 and 140 +/- 28, respectively. Agonists and antagonists inhibited (+)-[3H]PN 200-110 binding with Ki values in the following rank order: nitrendipine less than nifedipine less than niguldipine much less than Bay K 8644 much less than verapamil. Although carbachol-induced contractile responses were 20-30 times smaller in muscle strips from urethra than from bladder base or bladder dome, KCl-induced contractions were only 3-4 times smaller in urethra than in bladder tissues. Nifedipine inhibited carbachol-induced contractions in urethra, bladder dome, and bladder base by 76%, 64%, and 60%, respectively, and completely inhibited KCl-induced contractions in all three tissues. IC50 values for nifedipine inhibition of both carbachol- and KCl-induced contractions were significantly smaller in urethra than in bladder base or bladder dome. Nitrendipine, niguldipine and verapamil inhibited urethral contractions induced by carbachol and KCl to the same degree as did nifedipine. The IC50 values, obtained from functional studies, for calcium channel antagonists were in good agreement with Ki values obtained from binding studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Stereoisomers of calcium antagonists which differ markedly in their potencies as calcium blockers are equally effective in modulating drug transport by P-glycoprotein

The (−)-isomer of verapamil is 10-fold more potent as a calcium antagonist than the (+)-isomer. However, both enantiomers are equally effective in increasing cellular accumulation of anticancer drugs [Gruber et al., Int J Cancer 41: 224–226, 1988]. In addition to verapamil, there exists a wide variety of stereoisomers with phenylalkylamines and dihydropyrdine structures which markediy differ in their potency as calcium antagonists. We have tested these drugs for their ability to increase intracellular accumulation of [ 3H]vinblastine ([ 3H]VBL) in a doxorubicin-resistant cell line (F4-6RADR) derived from the Friend mouse leukemia cell line (F4-6P) and in COS-7 monkey kidney cells. Both cell types express substantial amounts of multidrug resistance gene 1 mRNA and P-glycoprotein as revealed by RNA and immuno blot analysis. The enantiomers with phenylalkylamine structures [(±)-verapamil; (±)-devapamil; (±)-emopamil)] and with dihydropyridine structures [(±)-isradipine; (±)-nimodipine; (±)-felodipine; (±)-nitrendipine; (±)-niguldipine] increased [ 3H]VBL accumulation in both cell lines at micromolar concentrations. Although the stereoisomers of these drugs differ markedly in their potency as calcium channel blockers they were about equally effective in increasing VBL levels in the cells. There was no substantial difference in the potencies of the phenylalkylamine drugs in affecting cellular [ 3H]VBL transport. Major potency differences, however, were observed in the dihydropyridine drug series with the niguldipine isomers as the most effective drugs. Moreover, the niguldipine enantiomers were equally as effective in reversing VBL resistance in F4-6RADR cells as were the verapamil enantiomers. Since (-)-niguldipine (B859-35) displays a 45-fold lower affinity for calcium channel binding sites than (+)-niguldipine, but is equally potent in inhibiting drug transport by P-glycoprotein and in reversing drug resistance, it may be, in addition to (+)-verapamil, another usefui candidate drug for the treatment of multidrug resistance in cancer patients.

Calcium antagonists

In addition to the recent publications on the pharmacological properties of such new calcium-channel blockers (CCBs) as amlodipine, felodipine, falipamil, clentiazem, lacidipine, levopamil (with 5-hydroxytryptamine2 antagonistic properties), KB-2796, SR 33557 and S-312-d, we believe that the most important contributions of the past years have been in the areas of hypertension, cardiac failure and atherosclerosis. Evidence suggests that calcium-channel binding sites play a major role in the mechanisms of inhalational anesthetic properties and that abnormality in sarcoplasmic reticulum calcium channel-binding sites are also involved in malignant hyperthermia (MH).

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.

Urinary Bladder Function 6 Months after the Onset of Diabetes in the Spontaneously Diabetic BB Rat

Urinary bladder function was examined in the spontaneously diabetic BB rat six months after the onset of diabetes. Diabetes caused significant decreases in rat weight and increases in bladder body weights and in vivo bladder capacities compared to age-matched controls, but no changes in the weights of bladder bases. The absolute contractile responses of urinary bladder body and base strips to nerve stimulation, carbachol, 5-hydroxytryptamine, ATP, phenylephrine, and KCl were unaltered by diabetes. However, when the data were corrected for tissue mass, there were slight but not significant decreases in contractile responses of strips from diabetic rats. There were increases in total muscarinic receptor numbers and calcium channel binding sites in bladder bodies from BB rats as a result of the increases in tissue mass. The data indicate that the six month-diabetic BB rat differs from the streptozotocin-diabetic rat in the sensitivity of the urinary bladder to the complications of diabetes, probably as a result of the insulin treatment required to keep BB rats alive.

Characterization of 1,4, Dihydropyridine Calcium Channel Binding Sites in the Human Prostate

The binding and functional properties of calcium channel receptors have not been previously characterized in the normal or hyperplastic prostate. Dihydropyridine (DHP) binding sites have been characterized in other tissues using the ligands 3H-nitrendipene and (+)3H-PN200-110. Saturation experiments were performed on homogenates obtained from five human prostate adenomas using these ligands. The binding of 3H-nitrendipine and (+)3H-PN200-110 in the prostate was saturable and of high affinity. The mean Kd of 3H-nitrendipine and (+)3H-PN200-110 was 0.92 ± 0.11nM and 0.14 ± 0.02nM, respectively. The mean Bmax of 3H-nitrendipine and (+)3H-PN200-110 was 0.57 ± 0.06 and 0.19 ± 0.02fmol/mg. wet wt., respectively. The percent specific binding of 3H-nitrendipene and (+)3H-PN200-110 was 18 ± 1% and 38 ± 4%, respectively. The pharmacology of (+)3H-PN200-110 binding sites was further characterized using competition displacement experiments. The IC50 corrected values for Bay K 8644, nifedipine, verapamil, and diltiazem in the human prostate and other tissues are of the same order of magnitude. The prostate contains an abundance of high affinity DHP binding sites. The physiologic significance of the DHP binding sites in the prostate requires further investigation.