(+)-PN200-l 10 and Ouabain Binding Sites in Purified Bovine Adrenomedullary Plasma Membranes and Chromaffin Cells

Bovine adrenal medulla plasma membranes were purified by a differential centrifugation procedure using sucrose and Urografin discontinuous density gradients; the membranes were enriched 10-12-fold in acetylcholinesterase activity and [3H]ouabain binding sites. Specific (+)-[3H]PN200-110 binding to these membranes amounted to 90% of total binding and was saturable and of high affinity (KD = 41 pM; Bmax = 119 fmol/mg of protein) with a Hill coefficient close to 1, a result suggesting the presence of a single, homogeneous population of dihydropyridine receptors. The association and dissociation rate constants were, respectively, 7.5 X 108 M-1 min-1 and 0.023 min-1. Unlabeled (+)-PN200-110 displaced (+)-[3H]PN200-110 binding with a potency 100-fold higher than (-)-PN200-110 (IC50,0.5 and 45nM, respectively). Although the two enantiomers of BAY K 8644 completely displaced (+)-[3H]PN200-110 binding, they exhibited no stereoselectivity (IC50, 69 and 83 nM,respectively). Whereas ( +/- )-nitrendipine very potently displaced (+)-[3H]PN200-110 binding (IC50 = 1.3 nM) verapamil and cinnarizine displaced the binding by only 30 and 40% at 1 microM, and diltiazem increased it by 20% at 10 microM. [3H]Ouabain bound to plasma membranes with a KD of 34 nM and a Bmax of 9.75 pmol/mg of protein, a figure 80-fold higher than the Bmax for (+)-PN200-110. [3H]Ouabain also bound to intact chromaffin cells with a Bmax of 244 fmol/10(6) cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of depolarization stimulated 45Ca2+ uptake in cultured neuronal cells by calcium channel activators and antagonists

The effect of dihydropyridine calcium agonists and antagonists on 45Ca2+ uptake into primary neuronal cell cultures was investigated. K+ stimulated neuronal 45Ca2+ accumulation in a concentration dependent manner. This effect was further enhanced by the calcium agonists Bay K 8644 and (+)-(S)-202-791 with EC50 values of 21 nM and 67 nM respectively. The calcium antagonists PN 200-110 and (-)-(R)-202-791 inhibited Bay K 8644 (1 microM) stimulated uptake with IC50 values of 20 nM and 130 nM respectively. 45Ca2+ efflux from neuronal cells was measured in the presence and absence of Na+. Efflux occurred at a much greater rate from cells incubated in the presence of Na+, indicating the existence of an active Na+/Ca2+ exchanger in these neurons. The data suggests that voltage sensitive calcium channels of these neurons are sensitive to dihydropyridines and thus that dihydropyridine binding sites have a functional role in these neuronal cultures.     

A new class of calcium entry blockers defined by 1,3-diphosphonates. Interactions of SR-7037 (belfosdil) with receptors for calcium channel ligands

Tetrabutyl-2(2-phenoxyethyl)-1,3-propylidene diphosphonate (SR-7037) completely displaced dihydropyridine [( 3H]PN200-110), phenylalkylamine [( 3H]D888), and benzothiazepine [( 3H]diltiazem) ligands from brain L-type calcium channels. Half-maximal inhibition of [3H]PN200-110 binding occurred at 19 nM with a Hill coefficient of 0.96. SR-7037 primarily decreased the affinity for [3H]PN200-110 with a small, but significantly, effect on the maximal binding capacity. Kinetic studies showed that this was due to an increased radioligand dissociation rate from 0.04 min-1 to 0.43 min-1 in the presence of the diphosphonate. Displacement of [3H]D888 by SR-7037 was biphasic with respective IC50 of 44 and 8400 nM. Likewise, unlabeled (-)-D888 identified two sites with IC50 values of 0.9 and 27 nM. Both SR-7037 (1000 nM) and D888 (200 nM) accelerated radioligand dissociation about 2-fold. [3H]Diltiazem binding was inhibited by SR-7037 with an IC50 value of 29 nM. The inhibition of dihydropyridine binding by SR-7037 is enhanced by most divalent cations at millimolar concentrations with the following potency: Mn2+ greater than Mg2+ greater than Ca2+ greater than Co2+. Barium has the opposite effect. The half-maximal effect of calcium occurred at 6 microM free ion. Specific binding of [3H]D888 was antagonized in the presence of 1 mM CaCl2. It is concluded that SR-7037 has allosteric interactions with the dihydropyridine receptor of the L-type calcium channel. The differential effect of Ca2+ on the potency of D888 and diltiazem relative to that of SR-7037 indicates that the three drugs may bind to nonequivalent sites. These results support specific calcium channel inhibition, possibly at a novel site, as the primary mechanism of the diphosphonate's pharmacological actions.     

Calcium binding to extracellular sites of skeletal muscle calcium channels regulates dihydropyridine binding

The binding of dihydropyridine (PN200-110) to skeletal muscle microsomes (which were 84% sealed inside-out vesicles) was not influenced by the addition of calcium or magnesium nor by addition of their chelators (EDTA or EGTA) unless the vesicles were pretreated with the calcium-magnesium ionophore A23187 and EDTA to remove entrapped cations. Separation of inside-out vesicles from right-side-out vesicles by wheat germ agglutinin chromatography revealed that only the right-side-out vesicles exhibited a calcium-, magnesium-, and chelator-dependent binding of PN200-110. Dihydropyridine binding to cardiac sarcolemma membranes (which were 46% inside-out) and to solubilized skeletal muscle membranes was inhibited by EDTA and could be fully restored by 10 microM calcium or 1 mM magnesium. Calcium increased PN200-110 binding to partially purified rabbit skeletal muscle calcium channels from 3.9 pmol/mg protein to 25.5 pmol/mg protein with a pK0.5 = 6.57 +/- 0.059 and a Hill coefficient of 0.56 +/- 0.04. Magnesium increased binding from 0.7 pmol/mg protein to 16.8 pmol/mg protein with a pK0.5 = 3.88 +/- 0.085 and a Hill coefficient of 0.68 +/- 0.074. These studies suggest that calcium binding to high affinity sites or magnesium binding to low affinity sites on the extracellular side of skeletal muscle T-tubule calcium channels regulates dihydropyridine binding. Further, similar calcium and magnesium binding sites exist on the cardiac calcium channel and serve to allosterically regulate dihydropyridine binding.     

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.     

Purified calcium channels have three allosterically coupled drug receptors

(-)-[3H]Desmethoxyverapamil and (+)-[3H]PN 200-110 were employed to characterize phenylalkylamine-selective and 1,4-dihydropyridine-selective receptors on purified Ca2+ channels from guinea-pig skeletal muscle t-tubules. In contrast to the membrane-bound Ca2+ channel, d-cis-diltiazem (EC50 = 4.5 +/- 1.7 microM) markedly stimulated the binding of (+)-[3H]PN 200-110 to the purified ionic pore. In the presence of 100 microM d-cis-diltiazem (which binds to the benzothiazepine-selective receptors) the Bmax for (+)-[3H]PN 200-110 increased from 497 +/- 81 to 1557 +/- 43 pmol per mg protein, whereas the Kd decreased from 8.8 +/- 1.7 to 4.7 +/- 1.8 nM at 25 degrees C. P-cis-Diltiazem was inactive. (-)-Desmethoxyverapamil, which is a negative heterotropic allosteric inhibitor of (+)-[3H]IN 200-110 binding to membrane-bound channels, stimulated 1,4-dihydropyridine binding to the isolated channel. (-)-[3H]Desmethoxyverapamil binding was stimulated by antagonistic 1,4-dihydropyridines [(+)-PN 200-110 greater than (-)(R)-202-791 greater than (+)(4R)-Bay K 8644] whereas the agonistic enantiomers (+)(S)-202-791 and (-)(4S)-Bay K 8644 were inhibitory and (-)-PN 200-110 was inactive. The results indicate that three distinct drug-receptor sites exist on the purified Ca2+ channel, two of which are shown by direct labelling to be reciprocally allosterically coupled.     

Effects of isoflurane on voltage-dependent calcium fluxes in rabbit T-tubule membranes: comparison with alcohols

The effects of racemic (+/-) and (+)- and (-)-stereoisomers of isoflurane on depolarization-induced (45)Ca(2+) fluxes mediated by voltage-dependent Ca(2+) channels were investigated in transverse tubule membrane vesicles from rabbit skeletal muscle. In the concentration range 0.5 to 2 mM, (+/-)-isoflurane inhibited (45)Ca(2+) fluxes and functionally modulated the effects of the Ca(2+) channel antagonist nifedipine (1-10 microM). Isoflurane-induced inhibition of (45)Ca(2+) fluxes was not significantly affected by pretreatment with either pertussis toxin (5 microg/ml) or phorbol 12-myristate 13-acetate (50 nM). Further experiments indicated that there were no significant differences between (+)- and (-)-stereoisomers of isoflurane with respect to the extent of inhibition of (45)Ca(2+) fluxes. Radioligand binding studies indicated that racemic and (+)- and (-)-isoflurane were equally effective in displacing the specific binding of [(3)H]PN 200-110 to transverse tubule membranes. There were no apparent differences between the effects of (+)- and (-)-isoflurane on the characteristics of [(3)H]PN 200-110 binding. Although the concentrations of isoflurane for the inhibitions of (45)Ca(2+) fluxes and radioligand bindings were similar, the concentrations of n-alcohols required for the inhibition of (45)Ca(2+) fluxes were lower than those for the displacement of radioligand. Comparison of the data for the displacement of [(3)H]PN 200-110 binding and the inhibition of (45)Ca(2+) fluxes by isoflurane and by n-alcohols suggested that both isoflurane and n-alcohols may have more than a single binding site. In conclusion, results indicate that isoflurane, independent of intracellular Ca(2+) levels, nonstereospecifically inhibits the function of voltage-dependent Ca(2+) channels and this effect is mediated through multiple binding sites.     

Insulin Enhances Development of Functional Voltage-Dependent Ca2+ Channels in Aneurally Cultured Human Muscle

Voltage-dependent Ca2+ channels were studied by the binding of the potent Ca2+ channel antagonist PN200-110 and by the K+-induced 45Ca2+ uptake in human muscle cultured aneurally in the presence of insulin, fibroblast growth factor, and epidermal growth factor, added in combination or individually. Compared to the muscle grown in medium without growth factors, 14-15 days of treatment with insulin (10 micrograms/ml) alone or in combination with two other growth factors caused a 3.4- and 3.8-fold increase per culture dish in the number of PN200-110 binding sites, respectively. There was no change in the affinity of the ligand-receptor complex. Under the same conditions, there was also fourfold increase of the K+-induced 45Ca2+ uptake in cultured human muscle. Neither fibroblast growth factor nor epidermal growth factor alone influenced PN200-110 binding sites. Our study demonstrates that insulin enhances the development of functional voltage-dependent Ca2+ channels in cultured human muscle.     

Dihydropyridine-sensitive Ca channel in aneurally cultured human muscles Relationship between high-affinity binding site and inhibition of calcium uptake

Dihydropyridine-sensitive Ca²⁺ channels and the relationship between binding of dihydropyridine derivatives and depolarization-induced Ca²⁺ uptake have been studied in aneurally cultured human muscle. Analysis of the equilibrium binding of the 1,4-dihydropyridine derivative (+)-PN200-110 revealed a single high-affinity binding site with a K_d of 0.15±0.05 nM and a B_max of 87±12 fmol/mg protein. Inhibition of (+)-[³H]PN200-110 binding by nitrendipine revealed a K_i of 0.8 nM for the nitrendipine-receptor complex. Depolarization of cultured human muscle achieved by elevating the K⁺ concentration increased the uptake ⁴⁵Ca²⁺ which was inhibited by nitrendipine with an IC₅₀ of 1.1 nM. This study demonstrates that aneurally cultured human muscle has dihydropyridine-sensitive voltage-dependent Ca²⁺ channels which are functional when the fibers are depolarized.     

Inhibition of dihydropyridine-sensitive calcium channels by the plant alkaloid ryanodine

At micromolar concentrations, ryanodine interacts with the dihydropyridine receptor of rabbit skeletal muscle transverse tubules. Ryanodine displaces specifically bound [3H]PN200-110 with an apparent inhibition constant of approx. 95 microM and inhibits dihydropyridine-sensitive calcium channels in the same preparation with an IC50 of approx. 45 microM. These concentrations of ryanodine are approximately three orders of magnitude higher than those required to saturate binding of the alkaloid to the ryanodine receptor of sarcoplasmic reticulum and to open the calcium release channel of sarcoplasmic reticulum (i.e. 20 nM (1988) J. Gen. Physiol. 92, 1-26). Thus at sufficiently high dose, ryanodine may affect SR as well as plasma membrane Ca permeabilities.     

Purification of the dihydropyridine receptor of the voltage-dependent Ca2+ channel from skeletal muscle transverse tubules using (+) [3H]PN 200-110

The rabbit skeletal muscle T-tubule membranes preparation is the richest source of organic Ca2+ blocker receptor associated with the voltage-dependent Ca2+ channel. Solubilization by 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (CHAPS) in the presence of glycerol leads to a 52% recovery of active receptors as determined by (+)[3H]PN 200-110 binding experiments. The dissociation constant of the (+) [3H]PN 200-110 solubilized-receptor complex was 0.4 +/- 0.2 nM by equilibrium binding and 0.13 nM from the rate constants of association (k1 = 0.116 nM-1 min-1) and dissociation (k-1 = 1.5 10(-2) min-1). The (+) [3H]PN 200-110 receptor has been substantially purified by a combination of filtration of Ultrogel A2 column and lectin affinity chromatography in the presence of trace amount of specifically bound (+) [3H]PN 200-110. The purified material contained polypeptides of apparent molecular weights of 142 000, 32 000 and 33 000. These three components copurified with (+)[3H]PN 200-110 binding activity.     

Dihydropyridine [methyl-3H]PN 200–110 Binding and Myogenesis in Intact Muscle Cells In Vitro

The radioligand dihydropyridine [methyl-3H]PN 200-110 binds to contracting myotubes in culture derived from chick embryo pectoralis muscle. [methyl-3H]PN 200-110 binds specifically to high-affinity sites, with nonspecific binding only between 15 and 30% of the total binding. A Scatchard plot of the specific binding revealed a single high-affinity binding site with a KD (dissociation constant) of 0.5 nM +/- 0.2 nM and Bmax (number of binding sites) of 100 fmol/10(6) nuclei. We employed this sensitive assay to probe the appearance of high-affinity [methyl-3H]PN 200-110 binding sites during myogenesis. The time course of appearance of high-affinity binding sites lags behind that of fusion. Low-calcium media prevented the differentiation of myoblasts and blocked the appearance of high-affinity sites. Chelation of intracellular calcium before or after fusion of myoblasts with the calcium indicator Quin 2 prevented the appearance of dihydropyridine binding sites. These findings are consistent with the view that the expression of dihydropyridine receptors is modulated by the intracellular calcium.     

Kinetics of binding of dihydropyridine calcium channel ligands to skeletal muscle membranes: evidence for low-affinity sites and for the involvement of G proteins.

Detailed kinetic studies of the binding of the calcium channel antagonist (+)-[3H]PN200-110 to membrane preparations from rabbit skeletal muscle have demonstrated that, in addition to the high-affinity sites (Kd = 0.30 +/- 0.05 nM) that are readily measured in equilibrium and kinetic experiments, there are also dihydropyridine binding sites with much lower affinities. These sites were detected by the ability of micromolar concentrations of several dihydropyridines to accelerate the rate of dissociation of (+)-[3H]-PN200-110 from its high-affinity sites. The observed increase in rate was dependent on the concentration of competing ligand, and half-maximal effects occurred at approximately 10 microM for the agonist (+/-)-Bay K8644 and for the antagonists nifedipine, (+/-)-nitrendipine, and (+)-PN200-110. The low-affinity sites appear to be stereospecific since (-)-PN200-110 (1-200 microM) did not affect the dissociation rate. The possible involvement of guanine nucleotide binding proteins in dihydropyridine binding has been investigated by studying the effects of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) on binding parameters. At a concentration of 10 microM, neither GTP gamma S nor GDP beta S significantly affected the binding of dihydropyridines to their high-affinity sites. GTP gamma S did, however, increase the ability of (+/-)-Bay K8644, but not of (+/-)-nitrendipine, to accelerate the rate of dissociation of tightly bound (+)-[3H]PN200-110. GDP beta S did not affect the dose dependence of either the agonist or the antagonist.(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.     

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.     

Internal and external effects of dihydropyridines in the calcium channel of skeletal muscle

The agonist effect of the dihydropyridine (DHP) (-)Bay K 8644 and the inhibitory effects of nine antagonist DHPs were studied at a constant membrane potential of 0 mV in Ca channels of skeletal muscle transverse tubules incorporated into planar lipid bilayers. Four phenylalkylamines (verapamil, D600, D575, and D890) and d-cis-diltiazem were also tested. In Ca channels activated by 1 microM Bay K 8644, the antagonists nifedipine, nitrendipine, PN200-110, nimodipine, and pure enantiomer antagonists (+)nimodipine, (-)nimodipine, (+)Bay K 8644, inhibited activity in the concentration range of 10 nM to 10 microM. Effective doses (ED50) were 2 to 10 times higher when HDPs were added to the internal side than when added to the external side. This sidedness arises from different structure-activity relationships for DHPs on both sides of the Ca channel since the ranking potency of DHPs is PN200-110 greater than (-)nimodipine greater than nifedipine approximately S207-180 on the external side while PN200-110 greater than S207-180 greater than nifedipine approximately (-)nimodipine on the internal side. A comparison of ED50's for inhibition of single channels by DHPs added to the external side and ED50's for displacement of [3H]PN200-110 bound to the DHP receptor, revealed a good quantitative agreement. However, internal ED50's of channels were consistently higher than radioligand binding affinities by up to two orders of magnitude. Evidently, Ca channels of skeletal muscle are functionally coupled to two DHP receptor sites on opposite sides of the membrane.     

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)     

Rapid incorporation of the solubilized dihydropyridine receptor into phospholipid vesicles

We describe the rapid incorporation of the CHAPS solubilized dihydropyridine receptor into phospholipid vesicles. A series of sucrose gradient sedimentation experiments demonstrate that the (+)-[3H]PN200-110-labeled dihydropyridine receptor is associated with lipid vesicles following detergent removal by Extracti-gel chromatography. Solubilization of the receptor results in a loss of (+)-[3H]PN200-110 binding affinity relative to that observed in native membranes; the high affinity binding of (+)-[3H]PN200-110 can be restored upon reincorporation of the receptor into phospholipid vesicles. Similarly, the incorporation of the receptor restores its stability to incubation at 37 degrees C relative to that of the detergent solubilized receptor, thereby mimicking the properties of the membrane bound form of the receptor. The dissociation rate of (+)-[3H]PN200-110 from the reconstituted receptor is shown to be allosterically regulated by verapamil and diltiazem, indicating that the binding sites for these calcium antagonists have been inserted along with the dihydropyridine receptor into phospholipid vesicles. The results presented in this report, thus demonstrate the successful reconstitution of the dihydropyridine receptor into phospholipid vesicles by a variety of criteria. The reconstitution method described here is rapid and efficient, and should now facilitate structure-function studies of this receptor and its interrelationships with other regulatory components of the voltage-sensitive calcium channel system.     

Mechanism of vascular relaxation by thaligrisine: functional and binding assays

In the present study we examine the mechanism by which thaligrisine, a bisbenzyltetrahydroisoquinoline alkaloid, inhibits the contractile response of vascular smooth muscle. The work includes functional studies on rat isolated aorta and tail artery precontracted with noradrenaline or KCl. In other experiments rat aorta was precontracted by caffeine in the presence or absence of extracellular Ca2+. In order to assess whether thaligrisine interacts directly with calcium channel binding sites or with alpha-adrenoceptors we examined the effect of the alkaloid on [3H]-(+)-cis diltiazem, [3H]-nitrendipine and [3H]-prazosin binding to cerebral cortical membranes. The functional studies showed that the alkaloid inhibited in a concentration-dependent manner the contractile response induced by depolarization in rat aorta (IC50 = 8.9+/-2.9 microM, n=5) and in tail artery (IC50 = 3.04+/-0.3 microM, n=6) or noradrenaline induced contraction in rat aorta (IC50 = 23.0+/-0.39 microM, n=9) and in tail artery (IC50 = 3.8+/-0.9 microM, n=7). In rat aorta, thaligrisine concentration-dependently inhibited noradrenaline-induced contraction in Ca2+-free solution (IC50 = 13.3 microM, n=18). The alkaloid also relaxed the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (IC50 = 7.7 microM, n=4). The radioligand receptor-binding study showed that thaligrisine has higher affinity for [3H]-prazosin than for [3H]-(+)-cis-diltiazem binding sites, with Ki values of 0.048+/-0.007 microM and 1.5+/-1.1 microM respectively. [3H]-nitrendipine binding was not affected by thaligrisine. The present work provides evidence that thaligrisine shows higher affinity for [3H]-prazosin binding site than [3H]-(+)-cis-diltiazem binding sites, in contrast with tetrandrine and isotetrandrine that present similar affinity for both receptors. In functional studies thaligrisine, acted as an alpha1-adrenoceptor antagonist and as a Ca2+ channel blocker, relaxing noradrenaline or KCl-induced contractions in vascular smooth muscle. This compound specifically inhibits the refilling of internal Ca2+-stores sensitive to noradrenaline, by blocking Ca2+-entry through voltage-dependent Ca2+-channels.     

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 l 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 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. [25]. 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.