Obtaining antibodies to 1,4-dihydropyridine calcium channel blockers

Immunization of rabbits with amlodipine conjugated with horseradish peroxidase resulted in raising polyclonal antibodies that allowed group determination of 1,4-dihydropyridine calcium channel blockers in aqueous solutions by ELISA with a sensitivity of 0.1 to 1.0 ng/ml for amlodipine, felodipine, nifedipine, and isradipine.     

Partitioning and location of Bay K 8644, 1,4-dihydropyridine calcium channel agonist, in model and biological membranes.

Several lines of evidence suggest that nonspecific drug interaction with the lipid bilayer plays an important role in subsequent recognition and binding to specific receptor sites in the membrane. The interaction of Bay K 8644, a 1,4-dihydropyridine (DHP) calcium channel agonist, with model and biological membranes was examined at the molecular level using small angle x-ray diffraction. Nonspecific drug partitioning into the membrane was examined by radiochemical assay. Nonspecific binding characteristics of [3H] Bay K 8644 were determined in both dipalmitoyl phosphatidylcholine (DPPC) vesicles above and below their thermal phase transition (Tm) and rabbit skeletal muscle light sarcoplasmic reticulum (LSR). In DPPC, the partition coefficient, Kp, was 14,000 above the Tm (55 degrees C) versus 160 in the gel phase (2 degrees C). The Kp determined in LSR membranes was 10,700. These values for both DPPC and LSR membranes can be compared with Kp = 290 in the traditional octanol/buffer system. Using small-angle x-ray diffraction, the equilibrium position of the electron-dense trifluoromethyl group of Bay K 8644 in DPPC (above Tm) and purified cardiac sarcolemmal (CSL) lipid bilayers was determined to be consistently located within the region of the first few methylene segments of the fatty acyl chains of these membranes. This position is similar to that observed for the DHP calcium channel antagonists nimodipine and Bay P 8857. We suggest this particular membrane location defines a region of local drug concentration and plane for lateral diffusion to a common receptor site. Below the DPPC membrane Tm, Bay K 8644 was shown to be excluded from this energetically favored position into the interbilayer water space. Heating the DPPC bilayer above the Tm (55 degrees C) showed that this exclusion was reversible and indicates that drug-membrane interaction is dependent on the bilayer physical state. The absence of any specific protein binding sites in these systems allows us to ascertain the potentially important role that the bulk lipid phase may play in the molecular mechanism of DHP binding to the specific receptor site associated with the calcium channel.     

Stereoselective inhibition of thromboxane-induced coronary vasoconstriction by 1,4-dihydropyridine calcium channel antagonists

The biological activity of the (+)-S- and (-)-R-enantiomers of niguldipine, of the (-)-S- and (+)-R-enantiomers of felodipine and nitrendipine, and of rac-nisoldipine and rac-nimodipine was investigated in vitro and in vivo. Inhibition of coronary vasoconstriction due to the thromboxane A2 (TxA2)-mimetic U-46619 in guinea pig Langendorff hearts, displacement of (+)-[3H]isradipine from calcium channel binding sites of guinea pig skeletal muscle T-tubule membranes, and blood pressure reduction in spontaneously hypertensive rats were determined. The enantiomers were obtained by stereoselective synthesis. Cross-contamination was less than 0.5% for both S- and R-enantiomers of niguldipine and nitrendipine and less than 1% for those of felodipine. From the doses necessary for a 50% inhibition of coronary vasoconstriction, stereoselectivity ratios for (+)-(S)-/(-)-(R)-niguldipine, (-)-(S)-/(+)-(R)-felodipine, and (-)-(S)-/(+)-(R)-nitrendipine of 28, 13, and 7, respectively, were calculated. The potency ratio rac-nisoldipine/rac-nimodipine was 3.5. Ratios obtained from binding experiments and antihypertensive activity were (+)-(S)-/(-)-(R)-niguldipine = 45 and 35, (-)-(S)-/(+)-(R)-felodipine = 12 and 13, (-)-(S)-/(+)-(R)-nitrendipine = 8 and 8, and rac-nisoldipine/rac-nimodipine = 8 and 7, respectively. Highly significant correlations were found between the in vitro potency of the substances to prevent U-46619-induced coronary vasoconstriction and their affinity for calcium channel binding sites as well as their antihypertensive activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The beta-adrenergic receptor-regulated 1,4-dihydropyridine calcium channel receptor sites in coronary artery smooth muscle.

The cross-regulatory communication from beta-adrenergic receptors to 1,4-dihydropyridine (DHP) Ca2+ channel agonist and antagonist binding sites and cooperativity between DHP binding sites were studied in microsomal membranes of canine coronary artery (purified to a factor 2.9 for DHPs). The maximal number of binding sites (Bmax) identified in coronary artery microsomal membranes (CAM) with Ca2+ channel agonist (-)-S-(3H)BAY K 8644 was two times higher than Bmax of sites labelled with Ca2+ channel antagonist (+)-(3H)PN 200-110. The exposure of CAM to isoprenaline was accompanied with down-regulation of beta-adrenergic receptors and with increase in binding capacity for DHPs. The increase in Bmax was proportional in both groups of experiments and was related to increased affinity of DHPs. The 1,4-DHP binding sites identified in vascular smooth muscle showed characteristics typical for classification of specific 1,4-DHP receptor on Ca2+ channels. The binding was of high affinity, saturable and reversible, it showed stereoselectivity and it was positively modulated by beta-adrenergic stimulation and its showed cAMP and GTP sensitivity. The results support the hypothesis that beta-receptors also regulate the mode of Ca2+ channels in coronary artery smooth muscle.     

Stereoselective photoaffinity labelling of the purified 1,4-dihydropyridine receptor of the voltage-dependent calcium channel

The voltage-dependent calcium channel from guinea-pig skeletal muscle T-tubules has been isolated with a rapid, two-step purification procedure. Reversible postlabelling of the channel-linked 1,4-dihydropyridine receptor and stereoselective photolabelling as a novel approach were employed to assess purity. A 135-fold purification to a specific activity of 1311 +/- 194 pmol/mg protein (determined by reversible equilibrium binding with (+)-[3H]PN200-110) was achieved. Three polypeptides of 155 kDa, 65 kDa and 32 kDa were identified in the purified preparation. The 155-kDa band is a glycoprotein. The arylazide photoaffinity probe (-)-[3H]azidopine bound with high affinity to solubilized membranes (Kd = 0.7 +/- 0.2 nM) and highly purified fractions (Kd = 3.1 +/- 2 nM), whereas the optical antipode (+)-azidopine was of much lower affinity. Irradiation of (-)-[3H]azidopine and (+)-[3H]azidopine receptor complexes with ultraviolet light led to preferential incorporation of the (-) enantiomer into the 155-kDa polypeptide in crude solubilized and purified preparations. The pharmacological profile of irreversible labelling of the 155-kDa glycoprotein by (-)-[3H]azidopine is identical to that found in reversible binding experiments. Specific photolabelling of the 155-kDa band by (-)-[3H]azidopine per milligram of protein increases 150-fold upon purification, whereas incorporation into non-specific bands in the crude solubilized material is identical for both, (-) and (+)-[3H]azidopine.     

Isolation, identification and synthesis of an endogenous arachidonic amide that inhibits calcium channel antagonist 1,4-dihydropyridine binding

This study was part of a broad search for endogenous regulators of L-type calcium channels. The screening for active fractions was done by measuring inhibition [³H]1,4-dihydropyridine (DHP) binding to rat cardiac and cortex membranes. An inhibitory fraction, termed lyophilized brain hexane-extractable inhibitor (LBHI), was isolated from hexane extracts of lyophilized calf brain. The active substance was purified by a series of chromatographic steps. ¹³C nuclear magnetic resonance (NMR), ¹H coherence spectroscopy (COSY) NMR and fast atom bombardment (FAB) mass spectroscopy suggested that LBHI was N-arachidonic acid-2-hydroxyethylamide. Synthesis of this substance and subsequent high performance liquid chromatography (HPLC) and NMR analysis confirmed this structure. Synthetic LBHI (SLBHI) inhibited [³H]DHP binding to rat cortex membranes with an IC₅₀ value of 15 μM and a Hill coefficient of 2. Saturation analysis in the presence of SLBHI showed a change in K_D (equilibrium dissociation constant), but not maximal binding capacity (B_max). SLBHI produced an increased dissociation rate, which, along with the Hill slope of > 1, suggested a non-competitive interacton with the DHP binding site. The results suggest that arachidonic acid derivatives may be endogenous modifiers of the DHP calcium antagonist binding site.     

Regulation of 1,4-dihydropyridine and beta-adrenergic receptor sites in coronary artery smooth muscle membranes.

The receptor sites for 1,4-dihydropyridine (DHP) calcium channel ligands were identified and pharmacologically characterized in partially purified canine coronary artery smooth muscle (CSM) membranes (purification factor for 1,4-DHPs 2.8 and 2.2 respectively) using Ca2+ channel agonist (-)-S-[3H]BAYK 8644 and antagonist (+)-[3H]PN 200-110 as radioligands. The beta-adrenergic receptors were identified with (-)-3-[125I]iodocyanopindolol (ICYP). Specific binding of 1,4-DHPs and ICYP to membrane fraction was saturable, reversible and of both high and low affinity. The Kd for 1,4-DHP Ca2+ channel agonist was 0.59 +/- 0.05 and for antagonist 0.35 +/- 0.06 nmol/l and for low affinity binding sites Kd = 9.0 +/- 0.18 and 18.0 +/- 1.1 nmol/l. The high affinity 1,4-DHP binding (Bmax = 265 +/- 21 and 492 +/- 12 fmol/mg protein), showed stereoselectivity, temperature-dependence as well as pharmacological specificity: isoprenaline- and GTP-sensitivity, positive modulation with dilthiazem and negative modulation with verapamil, that is, properties characteristic of 1,4-DHP receptor sites on L-type Ca2+ channels. The low affinity binding sites were characterized as nonselective, temperature independent, dipyridamol-sensitive and represented a nucleoside transporter. The proportion of high affinity binding sites identified in the CSM membranes was 1.85 : 1.0 in favour of the antagonist. Results obtained with [125I]omega Conotoxin GVI A demonstrated that CSM membrane fractions isolated from median layers of coronary artery were devoid of substantial contamination with fragments of neuronal cells.     

Photoaffinity labelling of a 33-35,000 dalton protein in cardiac, skeletal and smooth muscle membranes using a new 125I-labelled 1,4-dihydropyridine calcium channel antagonist

The binding sites for Ca2+ channel antagonists were probed using Bay P 8857 [2-iodoethyl isopropyl 1,4-dihydropyridine-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarbox ylate] that has been radiolabelled with 125I. This drug was shown to bind with high affinity to cardiac, smooth, and skeletal muscle membranes, with a KD approximately equal to 0.3 nM. A protein of molecular weight 33-35,000 daltons was specifically and irreversibly radiolabelled after irradiation of cardiac, skeletal and aortic smooth muscle membranes, incubated with the [125I]-Bay P 8857. The peptide labelled by 1,4-dihydropyridine binding therefore appears similar in size for cardiac, skeletal, and smooth muscle. This data suggests that of the three peptide subunits which reportedly comprise the skeletal and cardiac muscle 1,4-dihydropyridine receptor complex, the 33-35,000 dalton peptide contains the dihydropyridine binding site.     

The 1,4-dihydropyridine receptor associated with the skeletal muscle voltage-dependent Ca2+ channel. Purification and subunit composition

The dihydropyridine receptor associated with the voltage-dependent Ca2+ channel from rabbit skeletal muscle has been purified using the tritiated derivative of (+)-PN 200-110. The drug was used not only as a marker associated with the solubilized receptor but also in direct binding experiments performed after each purification step. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilization of a microsomal preparation resulted in an extract with a specific binding activity of 10 pmol/mg of protein. A combination of chromatographic steps utilizing anion exchange, lectin affinity, and gel filtration resulted in an 80-fold purification to a specific binding activity of 800 pmol/mg of protein. The affinity of (+)-[3H]PN 200-110 for the solubilized receptor was only slightly altered after the purification procedure. The KD values were 0.7 and 1.8 nM on the starting material and the most purified fractions, respectively. The subunit composition of the dihydropyridine receptor was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was consistent with three polypeptides of Mr 142,000, 33,000, and 32,000. The last two small components were not covalently associated with the larger one. In spite of a careful investigation of the conditions which improved the stability of the dihydropyridine receptor, a partial denaturation could not be prevented during purification. This resulted in an underestimation of receptor purity when calculated from the maximal specific binding activity as compared to the enrichment in the three polypeptides observed after polyacrylamide gel electrophoresis. Finally, application of the same purification procedure to solubilized microsomal preparations of chick and frog skeletal muscle demonstrated the presence of a large polypeptide component of Mr 135,000-141,000 associated with the Ca2+ channel from these sources. The doublet of small molecular weight was not found with the frog muscle.     

Binding of A 1,4-dihydropyridine calcium channel activator, (-) S Bay K 8644, to cardiac preparations

The 1,4-dihydropyridine Ca2+ channel activator, (-) [3H]Bay K 8644, binds to cardiac membranes and polarized [5 mM K+] and depolarized [50 mM K+] cardiac cells. Binding to microsomal membranes at 25 degrees C indicates a single set of binding sites, KD = 2.9 x 10(-9) M and a site density, 337 fmoles/mg protein, not different from that measured by antagonist 1,4-dihydropyridines. Binding to neonatal rat myocytes at 37 degrees C was independent of membrane potential with a KD value of 5 x 10(-8)M and a site density, 63 fmoles/mg protein, not significantly different from that measured by PN 200 110. These results indicate that 1,4-dihydropyridine activators and antagonists label the same number of binding sites in cardiac tissue, but that activator binding to intact myocytes is voltage-independent.     

Rat pre-prosomatostatin. Structure and processing by microsomal membranes

The tetradecapeptide hormone somatostatin arises from proteolytic processing of a large precursor, pre-prosomatostatin. Studies of other hormone precursors predict that the NH2 terminus of pre-prosomatostatin comprises a leader, or signal, region which is cleaved during its translation. Such co-translational cleavage would generate prosomatostatin. In these studies, we present the complete sequence of rat pre-prosomatostatin, deduced from the nucleotide sequence of cDNAs derived from a somatostatin-rich medullary thyroid carcinoma. These findings indicate that rat pre-prosomatostatin contains 116 amino acids (12,737 daltons). Cell-free translations of medullary thyroid carcinoma mRNA with dog pancreas microsomal membranes were performed to identify the cleavage point of the leader region from prosomatostatin. Partial microsequencing data indicates that the cleavage occurs between the glycine and alanine at positions 24 and 25 of pre-prosomatostatin. Thus, rat prosomatostatin contains 92 amino acids (10,388 daltons). Comparison of the amino acid sequences of the rat and human pre-prosomatostatins reveals only four amino acid substitutions. In view of the high degree of homology between rat and human pre-prosomatostatin, we expect a similar cleavage site and NH2-terminal structure for human prosomatostatin. The high level of conservation between rodents and humans of the entire pre-prosomatostatin molecule further suggests the possibility of biologic functions of the NH2-terminal portions of prosomatostatin.     

Separation of the optical isomers of a new 1,4-dihydropyridine calcium channel blocker (LF 2.0254) by liquid and supercritical fluid chromatography

The four optical isomers of a new calcium channel blocker LF 2.0254 (developed by Laboratoires Fournier) are resolved by chromatography using chiral stationary phases. Two methods are proposed: the first one involving two chiral stationary phases [(S)-N-(2-naphthyl)alanine, a Pirkle type CSP and Chiralcel OJ, a cellulose derived CSP] in the liquid chromatographic mode, the second one involving a single CSP (Chiralcel OJ) in the supercritical fluid chromatographic mode. © 1994 Wiley-Liss, Inc.     

Comparative aspects and temperature dependence of [3H]1,4-dihydropyridine Ca2+ channel antagonist and activator binding to neuronal and muscle membranes

Binding of [3H]nitrendipine, [3H]nimodipine, and (+)[3H]PN 200-110 to microsomal preparations of guinea pig smooth and cardiac muscle and brain synaptosomes revealed high affinity interaction with KD values in the sequence, (+)PN 200-110 greater than nitrendipine greater than nimodipine. Bmax values for a particular tissue were independent of the 1,4-dihydropyridine employed in radioligand binding at 25 degrees C. The temperature dependence of [3H]nitrendipine binding in cardiac and smooth muscle microsomal preparations and brain synaptosomes was measured from 0 degrees to 37 degrees C and for skeletal muscle preparations from 0 degrees to 30 degrees C. Bmax values increased with temperature for cardiac membranes, but did not vary in other tissues. van't Hoff plots were nonlinear in all tissues, enthalpy and entropy changes becoming increasingly negative with increasing temperature. Competition binding of the activator-antagonist enantiomeric 1,4-dihydropyridine pairs of Bay k 8644 and PN 202-791 for [3H]nitrendipine in smooth muscle did not reveal significant thermodynamic differences between activator and antagonist molecules.     

1,4-Dihydropyridines as calcium channel ligands and privileged structures

1. The 1,4-dihydropyridine nucleus serves as the scaffold for important cardiovascular drugs—calcium antagonists—including nifedipine, nitrendipine, amlodipine, and nisoldipine, which exert their antihypertensive and antianginal actions through actions at voltage-gated calcium channels of the Ca_V1 (L-type) class.2. These drugs act at a specific receptor site for which defined structure-activity relationships exist, including stereoselectivity.3. Despite the widespread occurrence of the Ca_V1 class of channel, the calcium antagonists exhibit significant selectivity of action in the cardiovascular system. This selectivity arises from a number of factors including subtype of channel, state-dependent interactions, pharmacokinetics, and mode of calcium mobilization.4. The 1,4-dihydropyridine nucleus is also a privileged structure or scaffold that can, when appropriately decorated substituents, interact at diverse receptors and ion channels, including potassium and sodium channels and receptors of the G-protein class.     

Multiple calcium channels in synaptosomes: voltage dependence of 1,4-dihydropyridine binding and effects on function

The voltage dependence of binding of the calcium channel antagonist, (+)-[3H]PN200-110, to rat brain synaptosomes and the effects of dihydropyridines on 45Ca2+ uptake have been investigated. Under nondepolarizing conditions (+)-[3H]PN200-110 binds to a single class of sites with a Kd of 0.07 nM and a binding capacity of 182 fmol/mg of protein. When the synaptosomal membrane potential was dissipated either by osmotic lysis of the synaptosomes or by depolarization induced by raising the external K+ concentration, there was a decrease in affinity (approximately 7-fold) with no change in the number of sites. The effects of calcium channel ligands on 45Ca2+ uptake by synaptosomes have been measured as a function of external potassium concentration, i.e., membrane potential. Depolarization led to a rapid influx of 45Ca2+ whose magnitude was voltage-dependent. Verapamil (100 microM) almost completely inhibited calcium uptake at all potassium concentrations studied. In contrast, the effects of dihydropyridines (2 microM) appear to be voltage-sensitive. At relatively low levels of depolarization (10-25 mM K+) nitrendipine and PN200-110 completely inhibited 45Ca2+ influx, whereas the agonist Bay K8644 slightly potentiated the response. At higher K+ concentrations an additional dihydropyridine-insensitive component of calcium uptake was observed. These results provide evidence for the presence of dihydropyridine-sensitive calcium channels in synaptosomes which may be activated under conditions of partial depolarization.     

Monoclonal antibodies that coimmunoprecipitate the 1,4-dihydropyridine and phenylalkylamine receptors and reveal the Ca2+ channel structure

Monoclonal hybridoma cell lines secreting antibodies against the (+)-PN 200-110 and the (-)-demethoxyverapamil binding components of the voltage-dependent calcium channel from rabbit transverse-tubule membranes have been isolated. The specificity of these monoclonal antibodies was established by their ability to coimmunoprecipitate (+)-[3H]PN 200-110 and (-)-[3H]demethoxyverapamil receptors. Monoclonal antibodies described in this work cross-reacted with rat, mouse, chicken, and frog skeletal muscle Ca2+ channels but not with crayfish muscle Ca2+ channels. Cross-reactivity was also detected with membranes prepared from rabbit heart, brain, and intestinal smooth muscle. These antibodies were used in immunoprecipitation experiments with 125I-labeled detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin] solubilized membranes. They revealed a single immunoprecipitating component of molecular weight (Mr) 170,000 in nonreducing conditions. After disulfide bridge reduction the CHAPS-solubilized (+)-PN 200-110-(-)-demethoxyverapamil binding component gave rise to a large peptide of Mr 140,000 and to smaller polypeptides of Mr 30,000 and 26,000 whereas the digitonin-solubilized receptor appeared with subunits at Mr 170,000, 140,000, 30,000, and 26,000. All these results taken together are interpreted as showing that both the 1,4-dihydropyridine and the phenylalkylamine receptors are part of a single polypeptide chain of Mr 170,000.     

Interaction of aluminum ions with phosphoinositide metabolism in rat cerebral cortical membranes

Aluminum (Al) is believed to exert a primary role in the neurotoxicity associated with dialysis encephalopathy and has been suggested to be involved in a number of other neurological disorders, including Alzheimer's disease. Al, complexed with fluoride to form fluoroaluminate (AlF4-), can activate the GTP-binding (G) proteins of the adenylate cyclase and retinal cyclic GMP phosphodiesterase systems. Since an involvement of G-proteins with cerebral phosphoinositide (PtdIns) metabolism has also been suggested, in this study we investigated the interaction of the stable GTP analogue GTP(S), Al salts and NaF with this system. In rat cerebral cortical membranes, GTP(S) dose-dependently stimulated [3H]inositol phosphates ([3H]InsPs) accumulation. This effect was potentiated by carbachol and was partially prevented by the GTP-binding antagonist GDP(S), indicating that CNS muscarinic receptor activation is coupled to PtdIns hydrolysis via putative G-protein(s). GTP(S) stimulation was also inhibited by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, which is known to exert a negative feedback control on agonist-stimulated PtdIns metabolism. Both Al salts and NaF mimicked the action of GTP(S) in stimulating PtdIns turnover. Their actions were highly synergistic, suggesting that AlF4- could be the active stimulatory species. However, the stimulatory effects of AlCl3 and/or NaF were not potentiated by carbachol and were not inhibited by GDP(S) and PMA, suggesting that separate sites of action might exist for GTP(S) and AlF4-. In the nervous tissue, activation of PtdIns hydrolysis by Al (probably as AlF4-) may be mediated by activating a regulatory G-protein at a location distinct from the GTP-binding site or by a direct stimulation of phospholipase C.