title: Hydrophobic calcium channel ligands: methodical problems and their solution.
abstract: Niguldipine is a 1,4-dihydropyridine derivative that combines L-type Ca2+ channel-blocking effects and alpha 1-adrenolytic activity within a single molecule, exemplifying a novel approach in the treatment of hypertension. As niguldipine is a very hydrophobic compound, it (1) readily adsorbs to surfaces of the plastic-ware often used in radioligand binding assays and (2) partitions into the hydrophobic membrane compartments. Both phenomena decrease the actual free drug concentration in radioligand-binding assays and lead to gross underestimation of the affinity of niguldipine (and other hydrophobic ligands) for the 1,4-dihydropyridine binding domain of the L-type Ca2+ channel or for alpha 1A adrenoceptors, respectively. Partitioning of the hydrophobic molecules into the membrane phase leads to a dependence of the Ki value on "total receptor" concentration despite mathematic corrections of the experimentally determined IC50 values. The Ki dependence was mimicked by adding denatured membranes (devoid of high-affinity receptor-binding activity) to native membrane preparations. Loss to pipet tips and tubes was avoided by a special dilution protocol. Partitioning into the hydrophobic membrane compartments needed more elaborate correction procedures.
authors.affiliation: Institute of Biochemical Pharmacology, University of Innsbruck, Austria.
authors.forename: I
authors.initials: I
authors.lastname: Graziadei
authors.forename: G
authors.initials: G
authors.lastname: Zernig
authors.forename: A
authors.initials: A
authors.lastname: Grassegger
authors.forename: R
authors.initials: R
authors.lastname: Boer
authors.forename: C
authors.initials: C
authors.lastname: Schudt
authors.forename: H
authors.initials: H
authors.lastname: Glossmann
mesh_terms: D000327:Adsorption; D000818:Animals; D002021:Buffers; D002121:Calcium Channel Blockers; D015220:Calcium Channels / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D004095:Dihydropyridines / Q000493:pharmacokinetics; D006168:Guinea Pigs; D008024:Ligands; D009994:Osmolar Concentration; D051381:Rats; D011919:Rats, Inbred Strains; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism; D012107:Research Design; D014867:Water
chemical_list: D002021:Buffers; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004095:Dihydropyridines; D008024:Ligands; D011942:Receptors, Adrenergic, alpha; D014867:Water; C054074:niguldipine