Diltiazem Binding Research Articles

Antinociceptive activity of NK1 receptor antagonists: non-specific effects of racemic RP67580.

1. Release of substance P in the dorsal horn is considered a primary event in the perception of pain. The profile of racemic RP67580, a non-peptide antagonist at the NK1 (substance P) receptor, was examined in a range of antinociception tests on rodents. 2. At doses up to 30 mg kg-1, s.c. racemic RP67580 exhibited antinociceptive activity in writhing and formalin paw tests in mice and gerbils. Acetic acid induced writhing and the licking response to formalin were reduced to 40-50% of the level observed in vehicle-treated animals (P < 0.05). However, this agent was not active in mouse tail flick, rat paw pressure or rat and guinea-pig formalin paw tests. 3. Like racemic RP67580, the calcium channel blockers nifedipine (30 mg kg-1, i.p.) and verapamil (10 or 20 mg kg-1, s.c.) inhibited the response to formalin by approximately 60% in gerbils (P < 0.05 compared with vehicle-treated animals). 4. Evidence for calcium channel antagonist activity of RP67580 was obtained in vitro. Racemic RP67580 inhibited calcium entry into depolarized strips of guinea-pig ileum longitudinal muscle myenteric plexus (apparent KB = 587 +/- 115 nM), inhibited [3H]-diltiazem binding to rabbit skeletal membranes (IC50 = 298 nM) and depressed high threshold calcium currents in neurones cultured from rat cortex (10% inhibition at 10 microM). 5. These findings indicate that the acute antinociceptive effects of RP67580 may not be attributable to a specific interaction with NK1 receptors and may be mediated via calcium channel blockade.

The interaction of the NK1 receptor antagonist CP‐96,345 with L‐type calcium channels and its functional consequences

1. We investigated the effects of the non-peptide NK1 receptor antagonist, CP-96,345, its inactive enantiomer CP-96,344, and the racemic mixture (+/-)-CP-96,345, on the binding of [3H]-nimodipine and [3H]-diltiazem to L-type calcium channels in rat cerebral cortex membranes. In isolated peripheral tissues containing tachykinin receptors, the effects of (+/-)-CP-96,345 have been compared with those of diltiazem. 2. In guinea-pig trachea, (+/-)-CP-96,345 produced antagonism of responses to the selective NK1 agonists [Sar9, Met(O2)11]SP and substance P-methyl ester that was apparently competitive in nature (pKB 7.0-7.5), while in guinea-pig ileum the antagonism was not surmountable. 3. The reduction of maximum responses by (+/-)-CP-96,345 in the guinea-pig ileum was not selective; it was obtained with muscarinic agonists and other agents, and was also observed in the portal vein of the rat where NK1 receptors are not present. 4. The tissue-specific reduction of maximum responses by (+/-)-CP-96,345 in ileum was reproduced by diltiazem. 5. (+/-)-CP-96,345 produced a concentration-dependent enhancement of [3H]-nimodipine binding to rat cerebral cortex membranes with a maximal stimulation of 186 +/- 29% above control (EC50 83.2 nM). Scatchard analysis revealed that (+/-)-CP-96,345 increased the affinity of [3H]-nimodipine for its binding sites without affecting Bmax (control: KD = 0.32 nM; with 100 nM (+/-)-CP-96,345: KD = 0.074 nM). 6. CP-96,345, CP-96,344, and the racemate all inhibited [3H]-diltiazem binding in rat cerebral cortex membranes with Ki values of 22.5 nM, 34.5 nM and 29.9 nM respectively; a similar value was obtained for diltiazem itself (33.6 nM). In comparison, CP-96,345 and ( +/- )-CP-96,345 inhibited the binding of[125I]-Bolton-Hunter-conjugated substance P in this tissue with Ki values of 59.6 nM and 82.0 nM respectively, while CP-96,344 had no measurable affinity (IC50> 10 microM).7. Substance P and a range of ligands selective for NK1, NK2, or NK3 receptors had no significant effect at 10 microM on either [3H]-diltiazem or [3H]-nimodipine binding.8. The results indicate that in addition to possessing affinity for the NK1 receptor, the non-peptide antagonist, CP-96,345, displays high affinity for [3H]-diltiazem binding sites on L-type calcium channels.The functional effect that may be observed in integrated models will be a consequence of either property, or be a composite effect of NK1 receptor antagonism and L-channel blockade.

The benzazepine/benzothiazepine binding domain of the cardiac L-type Ca2+ channel is accessible only from the extracellular side.

The whole-cell tight seal recording technique was used to investigate location of benzothiazepine binding site of the cardiac L-type Ca2+ channel. For this we utilized a permanently charged compound, SQ 32.428, out of a series of benzazepine drugs which have been characterized as competitive inhibitors of diltiazem binding. The non-permanently charged derivative SQ 32.910 was initially tested to electrophysiologically establish Ca2+ antagonistic properties of benzazepines. Upon extracellular application, either compound was able to completely block Ca2+ currents. At a stimulating frequency of 0.2 Hz IC50 concentrations of SQ 32.910 and SQ 32.428 were determined as 35 nM and 15 microM, respectively. Intracellular application of SQ 32.428 was then compared to control experiments in the absence of drug. Initially, adequate drug dialysis was confirmed with 100 microM D890, which produced a progressive inhibition of Ca2+ currents within 10 min after whole-cell access. In contrast, internal application of 100 microM SQ 32.428 did not change time-course of Ca2+ currents compared to control run-down. These results show that the benzazepine/benzothiazepine binding domain of the cardiac L-type Ca2+ channel is accessible only from the extracellular side and therefore suggest an extracellular location on the alpha 1-subunit of the Ca2+ channel protein.

In vitro characterization of a novel Ca 2+ entry blocker: SR 33805

In this study, SR 33805 was shown to inhibit competitively [ 3H]fantofarone binding to cardiac sarcolemmal membranes. In contrast, SR 33805 was shown to inhibit allosterically [ 3H](+)-PN200-110, [ 3H](−)-D888 and cis-(+)-[ 3H]diltiazem binding. In isolated rabbit atrial preparations, SR 33805 was shown to be the least potent of fantofarone, nifedipine, verapamil and diltiazem in terms of both negative chronotropic and inotropic responses (IC 50's 6 and 12 μM, respectively). In superfused rat aortic strips, SR 33805 like other Ca 2+ channel antagonists, caused a significant inhibition of both K +-induced 45Ca 2+ influx and contractile responses. In addition this agent was shown to antagonize Ca 2+-induced contractions in K +-depolarized aorta with a pA 2 value of 8.39±0.02. In femoral, renal and basilar arteries, SR 33805 was equiactive to the other Ca 2+ channel antagonists studied in antagonizing K +-induced contractions (IC 50 ∼ 40 nM), but unlike the reference Ca 2+ channel antagonists, was equiactive in antagonizing serotonin-induced contractions (IC 50 ∼ 250 nM). This suggests that the effects of SR 33805 depend mainly on membrane potential. In conclusion, SR 33805 is a potent Ca 2+ channel antagonist which, unlike fantofarone, verapamil and diltiazem, is highly selective for vascular smooth muscle and devoid of any potent negative inotropic actions.

Bis(benzylisoquinoline) analogs of tetrandrine block L-type calcium channels: evidence for interaction at the diltiazem-binding site.

Bis(benzylisoquinoline) alkaloids block Ca2+ uptake through the L-type Ca2+ channel and modulate binding of ligands to four distinct sites (dihydropyridine, benzothiazepine, aralkylamine, and (diphenylbutyl)piperidine) in the Ca2+ entry blocker receptor complex of the channel. These alkaloids are structural analogs of tetrandrine, which has previously been demonstrated to block the L-type Ca2+ channel through interaction at the benzothiazepine (diltiazem) site (King et al., 1988). Different alkaloid conformational classes display either alpha-beta, beta-alpha, alpha-alpha, or beta-beta stereochemistry at the two chiral isoquinoline carbons. Compounds from all four classes were tested for their ability to interact with Ca2+ entry blocker ligands. All analogs completely inhibit diltiazem binding, but many only partially inhibit D-600 and fluspirilene binding. For dihydropyridine binding, the compounds show either stimulation or inhibition or exhibit no effect. This profile is quite different from the interaction displayed by diltiazem or tetrandrine. Scatchard analyses show effects predominantly on Kd for diltiazem, D-600, and PN200-110 binding. Representative conformers do not effect diltiazem dissociation rates but alter dissociation kinetics of ligands which bind to the other three sites. A correlation of the ability of these compounds to inhibit Ca2+ uptake through the L-type Ca2+ channel in GH3 cells exists only with their inhibition of diltiazem binding but not with inhibition of binding of ligands representing other classes of Ca2+ entry blockers. These data, taken together, indicate that a variety of bis(benzylisoquinoline) congeners act to block the L-type Ca2+ channel by binding to the benzothiazepine site on the channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyamines allosterically modulate [ 3H]nitrendipine binding to the voltage-sensitive calcium channel in rat brain

The effects of polyamines on radioligand binding to the slow voltage-dependent Ca 2+ channel were studied using membranes from the rat cerebral cortex. [ 3H]Diltiazem binding was inhibited by arcaine (IC 50 = 55 μM) and, in decreasing order of potency, by agmatine, spermidine, spermine and putrescine. Under control conditions, only spermidine and spermine allosterically inhibited [ 3H]nitrendipine binding while arcaine, agmatine and putrescine were inactive. Nevertheless, putrescine antagonized the effect of spermine as well as the allosteric effects of diltiazem and verapamil on the binding of [ 3H]nitrendipine, in a manner analogous to that shown previously for Ca 2+. Thus, polyamines may function as endogenous modulators of the voltage-dependent Ca 2+ channel.

Interaction of pinaverium (a quaternary ammonium compound) with 1,4-dihydropyridine binding sites in rat ileum smooth muscle.

1. The interaction of pinaverium bromide, a quaternary ammonium compound, with binding sites for (L-type) calcium channel blockers was investigated in rat ileum smooth muscle. 2. Pinaverium inhibited [3H]-(+)-PN200-110 ([3H]-(+)-isradipine) specific binding to tissue homogenates incompletely (Ki 0.38 microM; maximal inhibition 80%). In contrast, binding to single cell preparations (obtained by collagenase treatment) and to saponin-treated homogenates was completely inhibited. These data are compatible with the view that, in untreated homogenates, 20% of [3H]-(+)-isradipine binding sites are not accessible to pinaverium because it is associated with sealed inside-out vesicles. 3. Pinaverium bromide increased the apparent KD of [3H]-(+)-isradipine binding to saponin-treated homogenates but did not significantly affect the Bmax value. Moreover, the dissociation rate constant of [3H]-(+)-isradipine binding was not changed by pinaverium. These data suggest that pinaverium interacts with the dihydropyridine binding site in a competitive manner. However, in contrast to uncharged dihydropyridine calcium antagonists, pinaverium inhibited, rather than stimulated, [3H]-diltiazem binding to rat brain membranes (at 30-37 degrees C). 4. Although Bmax values of [3H]-(+)-isradipine were similar in homogenates prepared from tissue and cells (collagenase-treated), the KD value was significantly higher in cell homogenates (166 vs 95 pM). Similarly, the Ki value of pinaverium was higher in cell preparations than in tissue homogenates (0.77 vs 0.38 microM). Thus, collagenase can significantly modify the dihydropyridine recognition site.5. The competitive interaction of pinaverium, a permanently charged drug, with [3H]-(+)-isradipine bound to intact cells and its absence of interaction with [3H]-(+)-isradipine bound to sealed inside-out vesicles imply that the dihydropyridine receptor lies near the external surface of the plasma membrane.

Binding characteristics of a new 1,5-benzothiazepine, clentiazem, to rat cerebral cortex and skeletal muscle membranes

The binding properties of a new 1,5-benzothiazepine, clentiazem (TA-3090), were investigated in rat cerebral cortex and skeletal muscle membranes with [ 3H]diltiazem and [ 3H]nitrendipine as radioligands. Clentiazem inhibited [ 3H]diltiazem binding to cerebral cortex membranes at the same concentrations as diltiazem at 2°C. However, at 37°C clentiazem was 3 times more potent to inhibit binding than diltiazem. [ 3H]Nitrendipine binding was modulated by clentiazem in a temperature-dependent manner. At 37°C clentiazem significantly enhanced [ 3H]nitrendipine binding to rat cerebral cortex membranes, whereas it has an inhibitory effect on [ 3H]nitrendipine binding at 0°C and no effect at 25°C. Of two optical isomers of clentiazem and four of diltiazem, only d-cis isomers (clentiazem and diltiazem) increased [ 3H]nitrendipine binding, indicating that both compounds have the same stereoselectivity for increasing [ 3H]nitrendipine binding. These results suggest that clentiazem binds to the same 1,5-benzothiazepine binding sites as diltiazem but with greater affinity.

Binding of Diltiazem to Albumin, α1‐Acid Glycoprotein and to Serum in Man

Binding of drugs can vary considerably. Therefore, binding of the calcium antagonist diltiazem was studied in protein solutions and in serum of healthy persons, patients with renal failure, patients with cirrhosis, patients with rheumatoid arthritis, patients with myocardial infarction, and intensive care patients. The effect of in vitro addition of some cardiovascular drugs (lidocaine, disopyramide, quinidine, and bupivacaine) on the binding of diltiazem in serum of healthy volunteers was also investigated. Diltiazem is bound as well to alpha 1-acid glycoprotein (AAG) as to albumin. In patients with renal failure, myocardial infarction, and rheumatoid arthritis and in intensive care patients, AAG concentrations are increased, and in the patients with myocardial infarction an increased binding of diltiazem is found. In patients with cirrhosis, AAG concentrations and diltiazem binding are decreased. In vitro addition of lidocaine, disopyramide, bupivacaine, or quinidine in concentrations between 5 and 100 micrograms/mL, before dialysis, decreases the binding of diltiazem; the displacing effect is most pronounced with bupivacaine.

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

Abstract 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.

Lactamimides: A novel chemical class of calcium antagonists with diltiazem-like properties

The effects of a series of lactamimides on [ 3 H]d-cis- diltiazem binding to rat brain membranes, on [ 3H]nitrendipine binding to cardiac membranes, and on calcium-induced contractions in depolarized guinea pig taenia and ileum preparations were examined. Several of the lactamimides examined displaced [ 3 H]d-cis- diltiazem binding and antagonized, in a competitive fashion, calcium-induced contractions. Over the series of lactamimides, there was a highly significant, positive linear correlation ( r = 0.87, P < 0.001) between their potency to displace [ 3 H]d-cis- diltiazem and their potency to antagonize calcium-induced contractions in the depolarized taenia and ileum preparations. Of the lactamimides examined, MDL 16,582A [ N-(2,2-diphenylpentyl)azacyclotridecan-2-imine · hydrochloride] had potency equivalent to d- cis-diltiazem with p A 2 values of 7.27 and 7.38, respectively, against calcium-induced contractions in the guinea pig ileum. These lactamimides are a novel chemical class displaying diltiazem-like calcium antagonist properties.

Substituted Diphenylbutylpiperidines Bind to a Unique High Affinity Site on the L-type Calcium Channel: Evidence for a fourth site in the cardiac calcium entry blocker receptor complex

Fluspirilene binds with high affinity to a single class of sites in purified porcine cardiac sarcolemmal membrane vesicles at a Kd of 0.6 nM and a Bmax that is in approximately 1:1 stoichiometry with other Ca2+ entry blocker receptors. Fluspirilene binding is modulated by various classes of L-type Ca2+ channel effectors. Metal ion channel inhibitors (e.g. Cd2+) stimulate binding primarily by increasing ligand affinity, whereas channel substrates (e.g. Ca2+) inhibit binding. Dihydropyridine, aralkylamine, and benzothiazepine Ca2+ entry blockers partially inhibit binding with Ki values equivalent to their respective Kd values, indicating close coupling between binding sites for the former agents and the diphenylbutylpiperidine site. All of these agents function as mixed inhibitors and affect both Kd and Bmax of fluspirilene binding. Only other substituted diphenylbutylpiperidines (e.g. pimozide) inhibit binding competitively. Diphenylbutylpiperidines, on the other hand, block nitrendipine, D-600, and diltiazem binding through a noncompetitive mechanism with Ki values much reduced from their measured Kd values, suggesting that coupling between the diphenylbutylpiperidine site and receptors for diverse Ca2+ entry blockers is more indirect. In addition, high affinity sites have been detected for fluspirilene in bovine aortic sarcolemmal vesicles, rat brain synaptic membranes, and GH3 rat anterior pituitary cell plasma membranes. Fluspirilene also effectively blocks Ca2+ flux through L-type Ca2+ channels in GH3 cells. Together, these results suggest that fluspirilene binds with high affinity to a unique fourth site in the Ca2+ entry blocker receptor complex and that substituted diphenylbutylpiperidines represent a new structural class of potent L-type Ca2+ channel inhibitors.

Native and detergent-solubilized membrane extracts from Drosophila heads contain binding sites for phenylalkylamine calcium channel blockers

Membrane extracts from Drosophila melanogaster heads contain binding sites for [ 3H]D-600, a verapamil-like calcium channel blocker. Scatchard analysis suggested a two binding site model with a dissociation constant ( K d) of 3.3 ± 0.28 nM and a B max of 1.3 ± 0.14 pmol/mg protein for the high affinity site and a K d of 150 ± 28 nM and a B max of 29 ± 10 pmol/mg protein for the low affinity site. The rank order of affinity of phenylalkylamine binding to the high affinity site was (−)-D-888 > (±)-verapamil > (±)-D-600. Displacement of (−)-[ 3H])D-888 binding by phenylalkylamines is stereoselective. The diphenylpiperazine cinnarizine is an effective inhibitor of [ 3H]D-600 binding. However, [ 3H]D-600 binding is only partially inhibited by diltiazem and bepridil at 10 −4M concentrations, and is not affected by dihydropyridine calcium channel drugs at concentrations < 10 −5M. Moreover, only extremely low specific [ 3H]nitrendipine, (+)-[ 3H]PN200-110, or d-(cis)- [ 3H]diltiazem binding was detectable. The phenylalkylamine photoaffinity probe, [N-methyl- 3H]LU 49888, labeled two Proteinase-K sensitive proteins of 136,000 and 27,000 Da. The high molecular weight, trypsin-sensitive band behaves like the high affinity phenylalkylamine receptor, while the low molecular weight, trypsin-insensitive band has properties of the low affinity receptor. Digitonin-solubilized binding activity has a sedimentation coefficient of 12 S on a sucrose gradient.

Interactions of DPI 201-106, a novel cardiotonic agent, with cardiac calcium channels.

The interaction of DPI 201-106, a novel cardiotonic agent, with the calcium entry blocker receptor complex was studied using porcine cardiac sarcolemmal membranes. DPI 201-106 and the chemically-related calcium antagonist, cinnarizine, produce concentration-dependent inhibition of nitrendipine, gallopamil and diltiazem binding to their respective sites in these vesicles. This effect of DPI 201-106 is not stereoselective since resolved stereoisomers of this compound display equal potency in inhibiting each of the binding reactions. Equilibrium ligand binding studies revealed that DPI 201-106 and cinnarizine cause mixed inhibitory patterns at the aralkylamine and benzothiazepine sites (i.e. both Kd and Bmax values were affected) while mainly increasing Kd at the dihydropyridine site. The kinetics of ligand dissociation from the three calcium entry blocker receptors, together with measurements of dihydropyridine association kinetics, further demonstrate that DPI 201-106 interacts at a unique site in the receptor complex and allosterically modulates binding of nitrendipine, gallopamil and diltiazem. The functional consequences of the above interactions with the calcium channel were studied in isolated cardiac preparations. In guinea-pig atria, DPI 201-106 increased force of contraction. This inotropic effect is seen only with the S(-) enantiomer and is unaltered by nitrendipine-, verapamil- or diltiazem-pretreatment, indicating DPI 201-106 does not act as a stimulant of this channel. Furthermore, DPI 201-106 did not alter the inotropic action of Bay K 8644, a calcium channel stimulant. Spontaneous rate of guinea-pig right atria is decreased by both DPI 201-106 and cinnarizine. In addition, potassium-induced contractures in cat papillary muscles are reduced by both agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of D-[3H]cis-diltiazem binding to membrane fractions and specific binding of calcium channel blockers to isolated flagellar membranes of Chlamydomonas reinhardtii

ABSTRACT Plasma membranes were separated from the intracellular membranes by using an aqueous two-polymer phase system. D-[3H]cis-diltiazem was employed to characterize benzothiazepine-selective receptors in these different membrane fractions of Chlamydomonas reinhardtii. The separation revealed that one type of binding site with higher affinity (KD = 33 nM) can be attributed to the intracellular membrane fraction and a second type with lower affinity (KD = 313nM) to the plasma membrane fraction. The apparent dissociation constants determined from the association and dissociation rate constants in kinetic experiments are comparable to those determined by saturation experiments. The maximum numbers of binding sites of the intracellular mem-brane fraction and the plasma membrane fraction are Bmax = 6·4pmolmg-1 protein and Bmax= 19pmolmg-1 protein, respectively. D-[3H]cis-diltiazem binding is inhibited by (±)ver-apamil and calcium chloride in both fractions. Moreover, nifedipine stimulates D-[3H]cis-diltia-zem binding by the intracellular membrane fraction, but shows no effect on the plasma membrane fraction. Ligand displacement binding studies with isolated flagella revealed the occurrence of a D-cis- diltiazem binding site with about the same affinity to this drug (KD = 400 nM) as the one found in the plasma membrane fraction. The maximum number of binding sites is 4-5pmolmg-1 protein. The apparent dissociation constants for specific [3H]nimodipine and [3H]verapamil binding to the flagella were calculated to be 8nM and 38 nM, respectively. The corresponding Bmax values are 345fmolmg-1 protein and 1’Spmolmg-1 protein, respectively.

Verapamil, diltiazem and nifedipine interactions with calmodulin stimulated (Ca 2+ + Mg 2+)-ATPase

The functional interactions of the three prototype Ca 2+ antagonists, verapamil, diltiazem and nifedipine, were examined in relation to the calmodulin regulated plasma membrane Ca 2+ pump ATPase. For this we used low ionic strength derived, calmodulin depleted, human red cell ghost membranes. Exogenously added calmodulin activated basal (Ca 2+ + Mg 2+)-ATPase in a concentration-dependent manner. Half-maximal activation by 6 nM calmodulin was antagonized by 10 −3 M verapamil and 10 −3 M diltiazem 25.1 and 12.1% respectively. The inhibition appeared to be specific for calmodulin activation since basal activity was not affected by these agents. Nifedipine had no effects on basal or calmodulin stimulated (Ca 2+ + Mg 2+)-ATPase activity. Unlike dihydropyridine modulation of verapamil and diltiazem binding at high affinity channel sites, nifedipine in this system did not alter the inhibitory responses of verapamil and diltiazem. The calmodulin directed antagonism of the two drugs was shown to be strictly additive over a full range of calmodulin concentrations and appeared to change predominantly the V max and, to a lesser degree, the affinity of calmodulin for the (Ca 2+ + Mg 2+)-ATPase. It is concluded that this model system provides evidence for additional functional discrepancies among the various classes of Ca 2+ antagonists.

Evidence for a distinct Ca2+ antagonist receptor for the novel benzothiazinone compound HOE 166.

The pharmacological and binding properties of the novel enantiomerically pure benzothiazinone (R)-(+)-3,4-dihydro-2-isopropyl-4-methyl-2-[2-[4-[4-[2-(3,4,5-tri- methoxyphenyl)-ethyl]-piperazinyl]-butoxyl-phenyl]-2H-1,4- benzothiazine-3-one dihydrochloride (HOE 166), are described. HOE 166 stereoselectively inhibited KCl-but not noradrenaline-induced contractions of guinea-pig pulmonary arteries, rabbit aorta, rat mesenteric artery preparations and k-strophantin-induced enhancement of guinea-pig papillary muscle contraction in a dose-dependent manner. KCl-induced smooth muscle contraction was inhibited by HOE 166 with IC50-values of approximately 70 nM (5-11 times less potent than nifedipine, 2-16 times more potent than verapamil), the respective S-(-)-enantiomer being approximately 10-fold less potent. HOE 166 decreased the upstroke velocity of the slow action potential in partially depolarized guinea-pig papillary muscle at similar concentrations than nifedipine. To investigate possible interactions with the calcium channel, HOE 166 and its S-(-)-enantiomer were characterized by radioligand binding studies in heart, brain and skeletal muscle transverse-tubule membranes. HOE 166 was a 4-15 times more potent inhibitor of reversible (+)-[3H]PN200-110, (-)-[3H]desmethoxyverapamil and d-cis [3H]diltiazem binding compared to its pharmacologically less active (S)-(-)-enantiomer, with IC50 values in the low nanomolar range. Extensive equilibrium and kinetic studies suggest that HOE 166 exerts its Ca2+-antagonistic effect by binding to a Ca2+-channel-associated drug receptor which is distinct from the 1,4-dihydropyridine, phenylalkylamine or benzothiazepine-selective domain. This HOE 166-selective site is, however, allosterically linked to the other sites of the Ca2+ antagonist receptor complex. We conclude that HOE 166 is a novel calcium antagonist.

Interaction of tetrandrine with slowly inactivating calcium channels. Characterization of calcium channel modulation by an alkaloid of Chinese medicinal herb origin.

Tetrandrine, a bis-benzylisoquinoline alkaloid derived from the Chinese medicinal herb Stephania tetrandra, is a putative Ca2+ entry blocker whose mechanism of action is unknown. To investigate this mechanism, the effects of tetrandrine were characterized on binding of three chemical classes of Ca2+ entry blockers in cardiac sarcolemmal membrane vesicles. In the range 25-37 degrees C, tetrandrine completely blocks diltiazem binding, partially inhibits D-600 binding, and markedly stimulates nitrendipine binding, with greatest enhancement occurring at 37 degrees C. The potency of tetrandrine is increased 10-fold as temperature is raised from 25 to 37 degrees C. Scatchard analyses indicate that inhibition of diltiazem binding and stimulation of nitrendipine binding result from changes in ligand affinities while inhibition of D-600 binding is due to both an increase in KD and decrease in Bmax of aralkylamine receptors. Ligand dissociation studies reveal that tetrandrine increases D-600 off-rates, decreases nitrendipine off-rates, but has no effect on diltiazem dissociation kinetics. In addition, tetrandrine reversibly blocks inward Ca2+ currents through L-type Ca2+ channels in GH3 anterior pituitary cells. These results indicate that tetrandrine interacts directly at the benzothiazepine-binding site of the Ca2+ entry blocker receptor complex and allosterically modulates ligand binding at other receptors in this complex. These findings suggest that tetrandrine is a structurally unique natural product Ca2+ entry blocker and provide a rationale explanation for the therapeutic effectiveness of this agent.

Interaction of Calmodulin and Calcium Antagonists with [3H]Diltiazem and [3H]Nitrendipine Binding Sites

The interaction of calmodulin antagonists and hydrophobic calcium antagonists with calmodulin and calcium antagonist ( [3H]nitrendipine and [3H]diltiazem) binding sites was investigated. The classical calmodulin antagonists calmidazolium, trifluorperazine, and W-7 were active at similar concentrations in the three experimental systems. The hydrophobic calcium antagonists prenylamine and bepridil, however, interacted with [3H]diltiazem binding at concentrations up to 50 times lower than their calmodulin inhibiting concentrations. The structural requirements for binding to calmodulin and to calcium channels are thus not identical for these hydrophobic drugs, suggesting that the calcium channel interacting properties of these antagonists are not a direct consequence of their calmodulin binding properties.

Direct activation of Ca2+ channels by palmitoyl carnitine, a putative endogenous ligand

1 Palmitoyl carnitine, a lipid metabolite which accumulates in cytoplasmic membranes during ischaemia, has been shown to resemble the Ca2+ channel activator, Bay K 8644, in K+-depolarized smooth muscle. Palmitoyl carnitine caused concentration-dependent (1-1000 mumol l-1) augmentations in the sensitivity to Ca2+ of K+-depolarized taenia preparations from the guinea-pig caecum. The (+/-)-isomer was equieffective with the (-)-isomer, whereas carnitine was ineffective and palmitic acid relaxed the tissues. The shift to the left of Ca2+ concentration-response curves induced by palmitoyl carnitine (100 mumol l-1) was additive with that of Bay K 8644 (1 mumol l-1). 2 The interactions of palmitoyl carnitine with the different classes of calcium-antagonist were similar to those seen with Bay K 8644. Schild plots of the calcium-antagonist effects of nifedipine were shifted to the right following preincubation of the taenia with palmitoyl carnitine (30-300 mumol l-1). The inhibitory effects of verapamil were especially sensitive to palmitoyl carnitine (100 mumol l-1). Whereas the potency of diltiazem as a calcium-antagonist was reduced by palmitoyl carnitine (100 mumol l-1), the inhibitory effects of the lipophilic class III calcium-antagonists, cinnarizine and flunarizine, were entirely resistant to palmitoyl carnitine (100 mumol l-1). 3 Although palmitoyl carnitine has detergent properties in high concentrations and lyses red blood cells, these effects were not Ca2+-dependent, nor were they modified by calcium-antagonists. Other detergents did not have selective interactions with Ca2+ channels. 4 Palmitoyl carnitine inhibited [3H]-nitrendipine, [3H]-verapamil and [3H]-diltiazem binding to rat cortical membranes with IC50 values (mumol l-1) of 120 +/- 1, 95 +/- 17 and 120 +/- 15 mumol l-1 respectively. The inhibition showed little temperature-dependence, in contrast to that of Bay K 8644, except for a small reduction in the IC50 value for [3H]-verapamil binding at 37 degrees C (42 +/- 5 mumol l-1). Palmitoyl carnitine interacted selectively with the Ca2+ channel, in that effects on ligand binding to alpha-adrenoceptors, beta-adrenoceptors and 5-HT1A receptors occurred only at 5-10 fold higher concentrations. 5 It is concluded that palmitoyl carnitine, at concentrations which have previously been shown to occur in the cytoplasm during myocardial ischaemia, may interact directly with Ca2+ channels and may therefore be considered as an endogenous modulator of channel function. The site of action differs from that of other agents.