Modulation of [3H]glibenclamide binding to cardiac and insulinoma membranes.

The existence of a single or of multiple populations of glibenclamide binding sites is a subject of controversy. In the present study, radioligand binding techniques were employed to determine whether multiple populations of [3H]glibenclamide binding sites exist in pancreatic tumor (insulinoma) cells. Additional studies were performed to further characterize the binding of [3H]glibenclamide to insulinoma and cardiac membranes. [3H]Glibenclamide bound to high (0.1 nM) and low (240 nM) affinity binding sites in insulinoma membranes. The physiological relevance of multiple populations of sites is unknown. The binding of glibenclamide to insulinoma and cardiac membranes was altered by guanine nucleotides and not adenine nucleotides. This suggests glibenclamide binding can be modulated by G-proteins. Glibenclamide binding was also modulated by divalent cations. The divalent cations, Ca2+ and Zn2+, stimulated specific glibenclamide binding to cardiac and insulinoma membranes, while Mg2+ and Mn2+ enhanced cardiac binding only. Moreover, the lowering of pH from 7.4 to 6.5 was found to enhance specific glibenclamide binding. Interestingly, the magnitude of this effect was much larger in cardiac membranes. The specific nature of the regulation of glibenclamide binding by guanine nucleotides, divalent cations and pH remains to be explored.

Identification of high and low (GTP-sensitive) affinity [3H]glibenclamide binding sites in cardiac ventricular membranes.

Glibenclamide is an antagonist of the ATP-modulated K+ channel in cardiac tissue. This study showed glibenclamide to bind to high (0.2 nM) and low (40 nM) affinity binding sites in canine ventricular membranes. Gpp [NH]p significantly altered the binding characteristics of the low affinity site, while those of the high affinity site were unchanged. This indicates independence of the two sites and suggests the low affinity site may be coupled to a G-binding protein. Although we have identified two [3H]glibenclamide binding sites, the importance of these sites to the cardiac effects of glibenclamide remains to be determined.

[3H]BAY K 8644, a 1,4-dihydropyridine Ca++ channel activator: characteristics of binding to high and low affinity sites in cardiac membranes.

The binding of [3H]BAY K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)- pyridine-5-carboxylate] to high and low affinity sites in rabbit ventricular membranes was characterized. Binding affinities were 0.66 and 138 nM at 15 degrees C and 9.1 and 72 nM at 37 degrees C, for the high and low affinity sites, respectively, and binding site densities were 0.3 and 14 pmol/mg at 15 degrees C and 0.41 and 1.4 pmol/mg at 37 degrees C, for the respective sites. The modification of high affinity [3H]BAY K 8644 binding by verapamil, diltiazem, tiapamil, Ca++ and EDTA appeared to be the same as that for nitrendipine binding, consistent with the hypothesis that the high affinity binding site for [3H]BAY K 8644 on isolated membranes is the same as the 1,4-dihydropyridine antagonist binding site. The binding of [3H]BAY K 8644 to a low affinity binding site was modified by temperature, Ca++ and diltiazem, but the lack of stereoselectivity, lack of denaturation by heat and the large number of sites indicated that most of the low affinity binding sites were not associated with Ca++ channels. It is concluded that the high affinity binding site for BAY K 8644 is associated with Ca++ channels, and is modified by at least some of the factors that modify the binding site for Ca++ channel antagonists, whereas many or all of the low affinity binding sites detected are not related to Ca++ channels.

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.

Kinetics of binding of membrane-active drugs to receptor sites. Diffusion-limited rates for a membrane bilayer approach of 1,4-dihydropyridine calcium channel antagonists to their active site.

Using the model of 1,4-dihydropyridine calcium channel-blocking drug binding to receptors in the cardiac sarcolemmal membrane, diffusion-limited rates of association were calculated for two distinct approaches. In the "aqueous approach," the drug reaches the receptor by diffusion through the bulk solvent, whereas in the "membrane approach," the drug partitions into the membrane bilayer and then diffuses laterally to a specific receptor site. Calculated rates for the membrane approach were approximately 3 orders of magnitude greater than those for the aqueous approach. The membrane approach diffusion-limited rate depends weakly on the sizes of the binding site, the drug molecule, and the vesicle, but depends strongly on ligand asymmetry. Although the measured binding rates for several 1,4-dihydropyridines were all slower than the calculated diffusion-limited rates for either model, other experimental data (such as very high partition coefficients and specific positions of these drugs in the membrane bilayer) suggest that the membrane approach is the most likely. These results have important implications for specifying critical characteristics of active 1,4-dihydropyridines.

Characteristics of the binding of [3H]nitrendipine to rabbit ventricular membranes: modification by other Ca++ channel antagonists and by the Ca++ channel agonist Bay K 8644.

This study was carried out to characterize [3H]nitrendipine binding to cardiac membranes and to test the hypothesis that high affinity binding of Ca++ channel antagonists and agonists is to Ca++ channels. Binding was specific, rapid, reversible and stereoselective. The relative order of potency of nifedipine analogs for inhibition of binding was the same as that for inhibition of smooth and cardiac muscle contraction. Results with diltiazem, verapamil and lidoflazine were consistent with the hypothesis that nondihydropyridine Ca++ channel antagonists act at one or more sites allosterically linked to the 1,4-dihydropyridine site in cardiac cells. The Ca++ channel agonist Bay K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyr idine- 5-carboxylate] displaced specifically bound [3H]nitrendipine in an apparently competitive manner with an IC50 value of 5 nM. The results suggest that organic antagonists do not act by physically blocking the Ca++ channel. The data also support the hypothesis that the high affinity binding sites for [3H]nitrendipine in isolated cardiac membranes are associated with Ca++ channels that are inactivated or are otherwise unavailable for opening.

Comparison of high affinity binding of calcium channel blocking drugs to vascular smooth muscle and cardiac sarcolemmal membranes.

The binding of the 1,4-dihydropyridine calcium channel blocker [3H]nitrendipine to canine cardiac sarcolemmal and bovine aortic membranes was found to be rapid, specific, saturable, and reversible. Dissociation constants (Kd) determined by Scatchard analysis were 0.14 and 0.16 nM and the maximal numbers of binding sites (Bmax) were 0.96 +/- 0.2 and 0.08 +/- 0.01 pmole/mg protein for cardiac and aortic membranes respectively. Values of Kd calculated from kinetic data were approximately 0.10 nM for both membrane preparations. Competition assays with the enantiomers of a nisoldipine derivative indicated that [3H]nitrendipine binds stereoselectively. The order of potency of several nifedipine analogs for inhibition of binding of [3H]nitrendipine to cardiac and aortic membranes paralleled their relative potencies for inhibition of contraction in smooth muscle. It is concluded that the high affinity binding sites for nitrendipine in bovine aortic smooth muscle membranes are similar to those of canine ventricular sarcolemma.

Effects of metal cations and calmodulin antagonists on [3H] nitrendipine binding in smooth and cardiac muscle.

It was previously reported that [3H]nitrendipine binding to a microsomal fraction from intestinal smooth muscle was dependent upon the presence of divalent metal cations (Bolger et al., J. Pharmacol. Exp. Ther. 225: 291-309, 1983). The effects of cations and calmodulin antagonists on [3H]nitrendipine binding in smooth and cardiac muscle have been studied further. Treatment of ileal and aortic smooth muscle and cardiac muscle with EDTA reduced specific [3H]nitrendipine binding by 70 to 95%. Microsomes from rabbit ventricle were more resistant to EDTA treatment than were those from ileal smooth muscle, but low concentrations of Ca++ (less than 10(-5) M) produced half-maximal restoration of binding in both tissues. The ability of cations at a concentration of 10(-3) M to restore binding to membranes from guinea-pig ileum was in the sequence, Ca++ = Sr++ greater than Mg++ = Mn++ = Co++ greater than Ba++ = Ni++ greater than Zn++ = Cd++ greater than La = Sm = Tm . In contrast to the activation of calmodulin-dependent processes, the ability of these cations to restore [3H]nitrendipine binding did not correlate linearly with ionic radius. However, calmodulin antagonists were found to inhibit [3H]nitrendipine binding with the order of potency: pimozide greater than less than calmidazolium (R 24571) greater than trifluoperazine greater than chlorpromazine greater than promethazine greater than chlorpromazine sulfoxide, that correlates quite well with the potency of these drugs as inhibitors of calmodulin-dependent processes. The results suggest that calmodulin antagonists bind to a protein associated with the [3H]nitrendipine binding site that has a hydrophobic domain similar to that exposed on calmodulin by Ca++, but that this protein is not calmodulin itself.

Specific binding of a calcium channel activator, [3H]BAY k 8644, to membranes from cardiac muscle and brain.

BAY k 8644 is a member of a new class of drugs that directly activates Ca2+ channels. This 1,4-dihydropyridine was found to bind to both high and low affinity sites on rabbit ventricular microsomes and guinea pig brain synaptosomes. The dissociation constant obtained from Scatchard analysis with [3H]BAY k 8644 was 2 to 3 nM for the high affinity binding site, and the estimated maximal number of binding sites was 0.8 and 0.4 pmol/mg protein for heart and brain membranes, respectively, at 15 degrees C. Competition between nitrendipine and [3H]BAY k 8644 indicated a common high affinity binding site for Ca2+ channel activators and antagonists. The results suggest that the 1,4-dihydropyridine Ca2+ channel antagonists do not act as simple channel plugs.

Binding of the calcium channel blocker nitrendipine to its receptor in purified sarcolemma from canine cardiac ventricle.

High affinity binding of the 1,4-dihydropyridine calcium channel blocker [3H]nitrendipine was found in cardiac sarcolemma but not cardiac sarcoplasmic reticulum or mitochondria. Sarcolemmal binding of [3H]nitrendipine was saturable and reversible, with a maximum (Bmax) of approximately 1 pmol/mg protein and a Kd of approximately 0.14 nM. Displacement of sarcolemma-bound [3H]nitrendipine by other nifedipine analogs was stereospecific. The Kd for nitrendipine binding was approximately three orders of magnitude lower than the IC50 for the negative inotropic effect of this drug on isolated cat myocardium.

Binding of [3H]nimodipine to cardiac and smooth muscle membranes.

Specific binding of [3H]nimodipine to membranes from rat ventricle and guinea pig ileal longitudinal smooth muscle was studied. Dissociation constants were 0.24 and 0.12 nM, and the maximal number of binding sites were 0.4 and 0.75 pmol/mg protein for cardiac and smooth muscle, respectively. The values obtained for both types of muscle were similar to those obtained for [3H]nitrendipine binding, as were the potencies of a series of dihydropyridines for competing with [3H]nimodipine. These results support the hypothesis that the binding site characterized is that mediating the pharmacological effects of these compounds.