Verapamil Binding Sites Research Articles

Protecting the vasculature: An eye toward the future

Although calcium antagonists were originally developed for use in the management of patients with angina pectoris, they are now used in the management of other cardiovascular disorders, including hypertension. More recently, the calcium antagonists have been under investigation for their potential protective role in atherosclerosis. Coupled with these new possibilities for therapeutic use are the development of new, long-acting, tissue-specific calcium antagonists. Amlodipine belongs to this group, and although it is a dihydropyridine-based calcium antagonist, its pharmacologic profile differs from that of other dihydropyridine-based calcium antagonists. Differences include: different pH optimum for receptor binding, different rates of association and dissociation, and differences in allosteric interaction with the diltiazem and verapamil binding sites. Amlodipine, when given orally to rabbits receiving a high-cholesterol diet, reduces atheroma formation. Evidence of its ability to protect the vasculature is provided by its ability to significantly increase (p < 0.001) survival in stroke-prone hypertensive rats.

Effects of Ro 40-5967, a Novel Calcium Antagonist, on Myocardial Function During Ischemia Induced by Lowering Coronary Perfusion Pressure in Dogs: Comparison with Verapamil

Ro 40-5967 is a structurally novel calcium antagonist that binds to the verapamil binding site but has very weak negative inotropic effects compared to verapamil. The goals of the present study were to assess the effects of Ro 40-5967 on myocardial function during ischemia, and to compare them to those of verapamil. For this purpose, in anesthetized dogs where the circumflex coronary artery was cannulated and perfused at controlled pressure, myocardial function was measured using piezo electric crystals implanted into the endocardium. Myocardial distribution of coronary blood flow was assessed using radioactive microspheres. Ischemia was produced by lowering perfusion pressure from 100 to 15 mm Hg in steps. During ischemia, Ro 40-5967 partially prevented the decrease of segmental shortening (p less than 0.01) and increased coronary flow, especially in the endocardium (p less than 0.01). In contrast, verapamil did not improve myocardial function during ischemia. The protective effect of Ro 40-5967 could be partially explained by the decrease of arterial pressure and heart rate induced by Ro 40-5967. However, Ro 40-5967 injected directly inside the coronary artery did not induce systemic effects, but still had protective effects (p less than 0.05). Verapamil injected intracoronary produced severe cardiac depression. We conclude that in the present model during ischemia Ro 40-5967 has no negative inotropic effects, and in contrast to verapamil can improve the myocardial function.

Stereoselective protein binding of verapamil enantiomers

The binding of the (+)- and (−)-enantiomers of verapamil (V) to purified albumin (40 g/L), α 1-acid glycoprotein (0.55 g/L) and fresh serum has been studied over a wide range of verapamil concentrations (0.055 to 22 μM). The free fraction of the pharmacologically more potent (−)-V was always greater than that of (+)-V. Similar free fractions were observed in solutions of α 1-acid glycoprotein ((+)- V 0.079 ± 0.016; (−)- V 0.142 ± 0.020) and fresh serum ((+)- V 0.096 ± 0.009; (−)-V 0.136 ± 0.006), however the free fraction was higher in a solution of albumin ((+)- V 0.400 ± 0.030; (−)- V 0.572 ± 0.029). Saturation of verapamil binding sites was observed for α 1-acid glycoprotein only. Enantioselective verapamil serum binding was also noted in samples collected from five healthy volunteers following oral and intravenous verapamil administration. The free fraction of the individual isomers in vitro when added to predose serum as the pseudoracemic drug ((+)- V 0.06 ± 0.01, (−)-V 0.12 ± 0.02) was similar to that observed for the enantiomers when studied separately in vitro, indicating that the binding of each enantiomer is independent of the other optical isomer. The free fraction ex vivo after intravenous therapy ((+)- V 0.06 ± 0.01, (−)- V 0.12 ± 0.02) was similar to that observed in vitro in that subjects pre-dose serum. The free fraction of both enantiomers, however, was higher after oral drug therapy ((+)- V 0.13 ± 0.02, (−)- V 0.23 ± 0.03). The lower binding noted may be a result of competition for serum binding sites by verapamil metabolites, which attain higher concentrations following oral dosing.

Rabbit skeletal muscle microsomes contain two distinct phenylalkylamine‐binding sites

Lu49888, a photoaffinity analog of verapamil, was used to identify specific binding sites for phenylalkylamines of calcium channels present in rabbit skeletal muscle microsomes. Direct binding equilibrium measurements and displacement curves of Lu49888 by its non-radioactive analog yielded an apparent single class of binding sites with Kd and Bmax values of 16.5 nM and 7.5 pmol/mg respectively. Lu49888 was specifically incorporated into three proteins of apparently 165 kDa, and 33 kDa. Incorporation into the 55-kDa protein was blocked by 10--50-fold higher concentrations of unlabeled phenylalkylamines compared to incorporation into the 165-kDa protein, suggesting that the 165-kDa and 55-kDa proteins contain a high and a low-affinity verapamil-binding site respectively. The photoaffinity-labeled proteins were solubilized by 1% digitonin or 1% Chaps in roughly equal amounts. The 165-kDa protein bound to wheat-germ-agglutinin(WGA)--Sepharose and sedimented in sucrose density gradients with the same constant as the purified dihydropyridine receptor, which has been reconstituted to a functional calcium channel. The 55-kDa membrane protein did not bind to the WGA-Sepharose column and sedimented in sucrose density gradients with a lower s value than the 165-kDa protein. The 165-kDa but not the 55-kDa membrane protein was specifically labeled by azidopine, the photoaffinity analogue of dihydropyridines. The 55-kDa protein of the purified dihydropyridine receptor was not significantly labeled by Lu49888 showing that the 55-kDa protein of the membrane is unrelated to the purified high-affinity dihydropyridine receptor.

3H]-verapamil binding to rat cardiac sarcolemmal membrane fragments; an effect of ischaemia.

The [3H]-verapamil binding activity of rat cardiac sarcolemmal fragments was studied, using membranes harvested from non-perfused, aerobically-perfused and ischaemic hearts. Glass-fibre filters were found to contain specific, high affinity--(KD 38 +/- 3.1 nM) [3H]-verapamil binding sites--making them unsuitable for use in [3H]-verapamil binding studies. Incubation of membranes from non-perfused hearts in a medium containing 150 mM NaCl, 1 mM CaCl2 and 50 mM Tris revealed two populations of [3H]-verapamil binding sites. When centrifugation instead of filtration was used to separate bound and free [3H]-verapamil, high affinity sites with a KD of 0.57 +/- 0.19 microM and a Bmax of 38 +/- 5.2 pmol mg-1 protein, and low affinity sites with a KD of 78 +/- 27.5 microM and a Bmax of 2.9 +/- 1.3 nmol mg-1 protein were detected. However, only low affinity binding sites could be detected in membranes which had been incubated in a cation-free medium containing 50 mM Tris. [3H]-verapamil binding to the low and high affinity sites was saturable, reversible, stereospecific and displaceable by D600 greater than diltiazem greater than Ca2+ but not by nifedipine, nitrendipine, nisoldipine or prazosin. The two populations of binding sites survived aerobic perfusion and 60 min ischaemia at 37 degrees C. Ischaemia reduced the Bmax and KD but selectivity was maintained.

3H]verapamil binding sites in cardiac membrane fragments

3H]verapamil binding sites in cardiac membrane fragments

Specific binding of the calcium antagonist [3H]verapamil to membrane fractions from plants.

Specific binding of the Ca2+ channel blocker [3H] verapamil to a membrane fraction from plants has been characterized. Binding to zucchini membranes was saturable and reversible. The apparent equilibrium dissociation constant is KD = 102 nM and the maximum number of binding sites is Bmax = 60 pmol/mg of protein. The KD determined from the association and dissociation rate constants is 130 nM. [3H]Verapamil binding to zucchini membranes could not be inhibited by the Ca2+ antagonists nifedipine and diltiazem. However, [3H]verapamil could be displaced by diltiazem but not by nifedipine from corn membranes. Sucrose density fractionation of zucchini membrane preparations revealed that [3H]verapamil binding sites are located primarily at the plasma membrane.

Characterization of verapamil binding sites in cardiac membrane vesicles.

Specific, saturable, and reversible binding of verapamil has been demonstrated in crude cardiac sarcolemmal membranes. These receptors possess a Kd of approximately 50 nM for verapamil as determined by either equilibrium binding studies, competition binding analysis, or kinetic analysis of on and off rates and display an average density of 1.25 pmol/mg of protein. Specificity of binding is indicated by several criteria. Competition studies with the verapamil analog D-600 indicate that (-)D-600 is 200-fold more potent than the (+)-isomer in displacing bound verapamil. Likewise, several other aryl alkyl amine Ca2+ entry blockers effectively displace bound ligand. In addition, dihydropyridines and diltiazem promote partial (25-35%) displacement of bound verapamil with Ki values similar to the Kd values for their respective receptors. Characterization of nitrendipine binding in this preparation indicates an average density of 0.3 pmol of receptors/mg of protein suggesting that the verapamil:nitrendipine binding site ratio is approximately 4:1. Binding characteristics of verapamil and nitrendipine receptors in highly purified sarcolemmal vesicles are similar to those in the crude preparation except that the ratio of verapamil:nitrendipine sites approaches 1 and nitrendipine and diltiazem promote almost complete displacement of bound verapamil. Fractionation studies of crude sarcolemmal membranes indicate that excess verapamil receptors, insensitive to the action of dihydropyridines or diltiazem, are located in a high-density, nonmitochondrial, non-sarcolemmal membrane fraction. Thus, verapamil receptors exist in two locations in cardiac tissue but only in the sarcolemmal membrane are these receptors coupled to the dihydropyridine receptor.

Properties of receptors for the Ca2+-channel blocker verapamil in transverse-tubule membranes of skeletal muscle. Stereospecificity, effect of Ca2+ and other inorganic cations, evidence for two categories of sites and effect of nucleoside triphosphates.

The verapamil receptor associated with the voltage-dependent calcium channel of rabbit skeletal muscle transverse tubule membranes has the following properties. (i) This receptor is stereospecific and discriminates between the different stereoisomers of verapamil, gallopamil and diltiazem. (ii) Inorganic divalent cations inhibit the binding of [3H]verapamil to its receptor in an apparently non-competitive fashion. The rank order of potency is: Ca2+ = Mn2+ greater than Mg2+ greater than Sr2+ greater than Ba2+ much greater than Co2+ much greater than Ni2+. Ca2+ and Mn2+ have inhibition constants of 0.3 mM. Binding of [3H]verapamil is also sensitive to monovalent cations such as Cs+, K+, Li+ and Na+. The most active of these cations (Cs+ and K+) have inhibition constants in the range of 30 mM. (iii) Binding of [3H]verapamil is pH-dependent and reveals the presence on the verapamil receptor of an essential ionizable group with a pKa of 6.5. (iv) A low-affinity binding site for verapamil and for some other Ca2+ channel blockers is detected by studies of dissociation kinetics of the [3H]verapamil receptor in the presence of high concentrations of verapamil, gallopamil, bepridil and diltiazem. (v) GTP and nucleoside analogs change the properties of [3H]verapamil binding to verapamil binding sites. High-affinity binding sites seem to be transferred into low-affinity sites. Dissociation constants obtained from inhibition studies of [3H]verapamil binding are in the range of 0.1-0.3 mM for GTP, ATP and Gpp(NH)p.

3H]verapamil binding sites in brain and skeletal muscle: Regulation by calcium

3H]verapamil binding sites in brain and skeletal muscle: Regulation by calcium

Competitive effects of verapamil and calcium ion as regulators of myocardial enzyme leakage.

Verapamil (Vp) has been found to augment the creatine kinase (CK) and lactate dehydrogenase (LDH) leakage from isolated mouse heart incubated in vitro at 25 degrees C. This leakage was concentration-dependent, and in the second hour followed the Henri-Michaelis-Menten kinetics model. Least-squares estimates obtained with this model for the Km values of verapamil were 0.38 +/- 0.16 mM (CK leakage) and 0.51 +/- 0.21 mM (LDH leakage). Calcium ion inhibited this effect of verapamil, completely abolishing it when the [Ca2+]:[Vp] greater than or equal to 2. At lower [Ca2+], the effect of verapamil also followed hyperbolic kinetics; the Km of verapamil was increased, but the asymptomatic leakage rate at high [Vp] was not changed significantly. These features indicate that the calcium ion is a competitive inhibitor of verapamil-augmented enzyme leakage. These observations suggest that there may exist a common calcium ion and verapamil binding site, or two allosterically related binding sites, which represent a prime determinant of myocardial leakage. The concept of a vulnerable site that binds calcium ions and verapamil competitively and initiates or vitiates processes that influence myocardial enzyme leakage is attractive because it suggests new approaches to the problem of myocardial preservation. As verapamil is thought not to enter the cell, the site of the competitive inhibition may be the sarcolemmal coat.