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.     

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)     

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.     

Regulation of beta-adrenergic receptors and calcium channel agonist binding sites in cultured human embryonal smooth muscle cells

Recent electrophysiological studies with cell membrane patches of cardiac myocytes and an electrically excitable cell line derived from rat pituitary tumor suggested that voltage activated calcium channels must be phosphorylated to respond to membrane depolarization (Armstrong and Eckert 1986; Trautwein and Kameyama 1986). In view of the "phosphorylation hypothesis" we investigated the adenylate-cyclase activity, the characteristics of beta-adrenergic and calcium channel agonist binding sites in control and desensitized (exposure to isoproterenol) human embryonal cells (HEC), and in fragmented membrane preparations of canine coronary smooth muscle. Our results suggest that down-regulation of the membrane-bound beta-adrenergic receptors, induced by isoproterenol in human embryonal cells and also in adult canine vascular tissue, results in physical translocation of beta-adrenergic binding sites into the light membrane fraction. This phenomenon is accompanied with an increased intracellular concentration of cAMP in and an increased binding of the calcium channel agonist (3H) BAYK 8644 to both HEC and canine smooth muscle membrane preparations. It could be concluded that phosphorylation of beta-adrenergic receptors regulates not only the beta subcellular distribution of the beta receptors but also the availability of calcium channel agonist binding sites in the cellular membrane.     

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.     

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.     

An ATP, calcium and voltage sensitive potassium channel in porcine coronary artery smooth muscle cells

There is increasing interest in the roles played by potassium channels of smooth muscle in protecting against ischemic and anoxic insults. Hence, potassium-selective channels were studied in freshly dispersed porcine coronary artery smooth muscle cells using the inside-out variant of the patch-clamp technique. The most abundant potassium channel had a conductance of 148 pS in a 5.4/140 mM K+ gradient, at 0 mV, and was regulated by cytoplasmic ATP (0.05-3.0 mM), cytoplasmic Ca2+ (0.1-10 microM) and voltage. ATP and AMP-PNP (0.5 mM) reduced the probability of channel opening (Po) by 87 and 92%, respectively. This inhibition was partially reversed by the addition of 0.5 mM ADP. ADP on its own (2 mM) reduced Po by 46%. It appears, therefore, that this channel shares properties with both the ATP-sensitive and the calcium-regulated potassium channels, raising the possibility that it plays a central role in the regulation of coronary blood flow.     

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.     

The subcellular calcium distribution in the smooth muscle cells of the pig coronary artery

In order to complete preliminary investigations on the subcellular calcium localisation in smooth muscle cells, further experiments are presented using smooth muscle cells from the coronary artery of the pig. The methods used were a precipitation technique using potassium oxalate and autoradiography using ⁴⁵Ca. In all cases we were able to reproduce the results obtained in our preliminary study. The preparations clearly show calcium oxalate precipitates in the cell membrane, the sarcoplasmic reticulum, the microvesicles, mitochondria and the nucleus membrane. These findings were supported by silver grain distributions in autoradiograms obtained by means of ⁴⁵Ca. The qualitative results obtained histochemically are in good agreement with estimations of the calcium distribution in subcellular fractions obtained by atomic absorption spectrophotometry.     

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.     

Muscarinic receptor reserve and beta-adrenergic sensitivity in tracheal smooth muscle

The possibility that differences in beta-adrenergic sensitivity among canine trachealis muscles contracted with different contractile agonists are related to differences in the receptor-occupancy characteristics of the contractile agonists was investigated. Relaxation to isoproterenol was compared in muscles contracted with the muscarinic agonists McN-A-343 and acetylcholine (ACh). The apparent dissociation constant (pKB) values for the M1-antagonist, pirenzepine, against ACh (6.96 +/- 0.18) and McN-A-343 (6.84 +/- 0.08) were similar. The pKB values for the M3-antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) against ACh (8.76 +/- 0.13) and McN-A-343 (8.71 +/- 0.10) were also similar, suggesting that these agonists were activating the same subtype of muscarinic receptor, probably M3. However, the contractile response to ACh was associated with a greater receptor reserve than that for McN-A-343. Isoproterenol relaxed muscles contracted with McN-A-343 much more effectively than those contracted with an equieffective concentration of ACh. The results suggest that the relative resistance of ACh-induced contractions to relaxation by isoproterenol may not be an inherent quality of muscarinic receptor stimulation. The large receptor reserve available to ACh may act to buffer the contractile response from the inhibitory effects of beta-adrenergic stimulation. Alternatively, ACh may be able to initiate subcellular mechanisms that are unavailable to agonists of lower efficacy.     

Calcium uptake studies of 1,4-dihydropyridine agonists into rabbit aortic smooth muscle cells in culture

The effects of the three dihydropyridine calcium channel agonists (+/-)BAY K 8644, (+)202-791 and (+/-)CGP 28392 on 45Ca++ uptake were studied in cultures of rabbit aortic smooth muscle cells. At 10(-7) M each agonist enhanced 45Ca++ uptake in 15-50 mM K+ but had no effect on the basal 45Ca++ uptake at 5 mM K+. At the uptake threshold of 15 mM K+ each agonist potentiated 45Ca++ uptake in a dose-dependent manner with half maximal effects at 2.4 nM for (+/-)BAY K 8644, 22 nM for (+)202-791 and 18 nM for (+/-)CGP 28392. The agonists showed no significant antagonistic activity. Responses were antagonized competitively by nifedipine and non-competitively by (+/-)D-600. The 45Ca++ uptake dose-response curves and the half maximal effects of the three agonists were over the same range of concentrations as their inhibition of [3H]nitrendipine binding to rat ventricular receptor membrane preparations. The data suggest that these cells mimic the calcium uptake by the intact aorta better than commercial vascular smooth muscle lines or cardiac cells.     

Rat cerebral cortical synaptoneurosomal membranes. Structure and interactions with imidazobenzodiazepine and 1,4-dihydropyridine calcium channel drugs.

Small angle x-ray scattering has been used to investigate the structure of synaptoneurosomal (SNM) membranes from rat cerebral cortex. Electron micrographs of the preparation showed SNM with classical synaptic appositions intact, other vesicles, occasional mitochondria, and some myelin. An immunoassay for myelin basic protein placed the myelin content of normal rat SNM at less than 2% by weight of the total membrane present. X-Ray diffraction patterns showed five diffraction orders with a unit cell repeat for the membrane of 71 to 78 A at higher hydration states. At lower hydration, 11 orders appeared; the unit cell repeat was 130 A, indicating that the unit cell contained two membranes. Electron density profiles for the 130-A unit cell were determined; they clearly showed the two opposed asymmetrical membranes of the SNM vesicles. SNM membrane/buffer partition coefficients (Kp) of imidazobenzodiazepine and 1,4-dihydropyridine (DHP) calcium channel drugs were measured; Kp's for DHP drugs were approximately five times higher in rabbit light sarcoplasmic reticulum than in SNM. Ro 15-1788 and the DHP BAY K 8644 bind primarily to the outer monolayer of vesicles of intact SNM membranes. Nonspecific equilibrium binding of Ro 15-1788 occurs mainly in the upper acyl chain of the bilayer in lipid extracts of SNM membrane.     

Exercise training depletes sarcoplasmic reticulum calcium in coronary smooth muscle.

We examined the effects of chronic exercise training on sarcoplasmic reticulum (SR) Ca uptake, spontaneous SR Ca release, and whole-cell currents in coronary smooth muscle cells. Single coronary artery smooth muscle cells demonstrated increases in intracellular free Ca (Cai) during depolarization (measured with fura-2) that were abolished by diltiazem (10(-4) M). Diltiazem significantly inhibited (80%) refilling of the SR Ca store. The SR Ca store of exercise-trained pigs was 64% less after 11 min vs. 2 min of recovery, whereas cells from sedentary pigs showed no depletion. Exercise-training-induced depletion of the SR Ca store was abolished when ryanodine (10(-5) M) was applied during the recovery, but depletion was enhanced by low concentrations of ryanodine (10(-8) M). In smooth muscle from sedentary pigs, 10(-8) M ryanodine mimicked the effects of exercise training by depleting the SR Ca store during 11 min of recovery (54% depletion). When allowed a longer recovery without ryanodine (14 min or without prior depolarization), the SR Ca store in cells from exercise-trained pigs returned toward peak levels. The outward K current vs. voltage relationship did not differ in cells from exercise-trained or sedentary pigs. Exercise training reduced the number of spontaneous transient outward currents normally found in cells from sedentary pigs. We introduce a model that provides a rational basis to explain the results obtained in this study.     

IP3 decreases coronary artery tone via activating the BKCa channel of coronary artery smooth muscle cells in pigs

Large conductance Ca²⁺-activated K⁺ channel (BK_Ca) is a potential target for coronary artery-relaxing medication, but its functional regulation is largely unknown. Here, we report that inositol trisphosphate (IP3) activated BK_Ca channels in isolated porcine coronary artery smooth muscle cells and by which decreased the coronary artery tone. Both endogenous and exogenous IP3 increased the spontaneous transient outward K⁺ currents (STOC, a component pattern of BK_Ca currents) in perforated and regular whole-cell recordings, which was dependent on the activity of IP3 receptors. IP3 also increased the macroscopic currents (MC, another component pattern of BK_Ca currents) via an IP3 receptor- and sarcoplasmic Ca²⁺ mobilization-independent pathway. In inside-out patch recordings, direct application of IP3 to the cytosolic side increased the open probability of single BK_Ca channel in an IP3 receptor-independent manner. We conclude that IP3 is an activator of BK_Ca channels in porcine coronary smooth muscle cells and exerts a coronary artery-relaxing effect. The activation of BK_Ca channels by IP3 involves the enhancement of STOCs via IP3 receptors and stimulation of MC by increasing the Ca²⁺ sensitivity of the channels.