title: Catecholamine binding to the alpha-adrenergic receptors of hamster adipocytes. Evidence that guanine nucleotides regulate this binding to the alpha 2-receptor subtype.
abstract: The binding characteristics of the alpha-component of (-)-[3H]norepinephrine to hamster adipocyte membranes were studied. Binding was rapid, equilibrium in 20 min at 25 degrees C. Dissociation of specific binding by 10 microM phentolamine suggested dissociation from two different sites. The time course of dissociation induced by a 50-fold dilution was unchanged by the addition of norepinephrine, suggesting the absence of cooperative binding sites. [3H]norepinephrine binding was saturable, yielding curvilinear Scatchard plots. Computer modeling of these data further supported the existence of two classes of binding sites, one with high affinity (KD = 23 nM) but low binding capacity (96 fmol/mg protein) and one with low affinity (KD = 400 nM) but high binding capacity (1000 fmol/mg protein). Adrenergic ligands competed with [3H]norepinephrine binding in the following order of potency: (-)-norepinephrine greater than (-)-epinephrine much greater than (+)-norepinephrine greater than (-)-isoproterenol. Displacement by the selective alpha-adrenergic drugs prazosin, clonidine, and yohimbine yielded biphasic curves consistent with binding of [3H]norepinephrine to both alpha 1-(14-22%) and alpha 2-(78-86%) receptor subtypes. Although Gpp(NH)p failed to alter the binding of [3H] dihydroergocryptine, it severely reduced the binding affinity of (-)-epinephrine, (-)-norepinephrine and the selective alpha 2-agonist, clonidine. The inhibitory effects of clonidine and of the alpha-component of (-)-epinephrine on the adrenocorticotropin-stimulated cyclic AMP production in the intact adipocyte were closely correlated with their effects on the binding of both [3H]norepinephrine and [3H]dihydroergocryptine. Conversely, yohimbine but not prazosin markedly antagonised the alpha-inhibitory effect of norepinephrine on cyclic AMP production. These data led to conclude that [3H]norepinephrine can be successfully used to study the entire alpha-adrenergic receptor population of hamster fat cells and that the predominant alpha 2-receptor subtype exists in two different affinity states for agonists, the proportions of which are modulated by guanine nucleotides.
mesh_terms: D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D003000:Clonidine / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004088:Dihydroergotoxine / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D008647:Mesocricetus; D009638:Norepinephrine / Q000378:metabolism*; D011941:Receptors, Adrenergic / Q000378:metabolism*; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects / Q000378:metabolism*
chemical_list: D011941:Receptors, Adrenergic; D011942:Receptors, Adrenergic, alpha; D004088:Dihydroergotoxine; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D000324:Adrenocorticotropic Hormone; D000242:Cyclic AMP; D003000:Clonidine; D009638:Norepinephrine