VISUALIZATION OF MULTIPLE IMIDAZOLINE GUANIDINIUM-RECEPTIVE SITES

Compounds with an imidazoline or guanidinium moiety elicit a variety o f stimulatory and inhibitory cell responses in both central and periph eral tissues. Many of these effects are mediated by interaction with a lpha-adrenergic receptors, but these molecules also selectively recogn ize other membrane-bound proteins with high affinity. We used a functi onalized derivative of the imidazoline molecule cirazoline to visualiz e the imidazoline/guanidinium-receptive site (IGRS). 2-[3-Aminophenoxy ]methyl imidazoline was radioiodinated and subsequently converted to t he arylazide to generate the photoaffinity adduct 2-[3-azido-4-[I-125] iodophenoxy]methyl imidazoline ([I-125]AZIPI). Both 2-[3-amino-4-[I-12 5]iodophenoxy]methyl imidazoline and [I-125]AZIPI exhibited saturable high affinity binding in rat liver membrane preparations (K(i) = 2-5 n M). In rat liver mitochondrial membranes, [I-125]AZIPI photoincorporat es into two peptides with apparent molecular weights of approximately 55,000 and approximately 61,000 as determined by SDS-polyacrylamide ge l electrophoresis. The labeling of these two species is blocked by var ious competing ligands (10 muM) with a potency order expected for an I GRS. The photolabeling of both peptides is blocked by the imidazolines cirazoline and idazoxan or by the guanidinium guanabenz, but it is no t altered the alpha2-adrenergic receptor antagonist rauwolscine or by the adrenergic receptor agonist epinephrine. Photoincorporation of [I- 125]AZIPI is minimally inhibited by the imidazoline clonidine or by th e alpha1-adrenergic receptor antagonist prazosin. However, the guanidi nium ligand amiloride exhibits higher affinity for the M(r) = 61,000 p eptide as compared with the M(r) = 55,000 peptide, suggesting that the two labeled species differ in their ligand recognition properties. An additional IGRS was identified by photolabeling in membranes prepared from PC-12 pheochromocytoma cells. In PC-12 membranes, [I-125]AZIPI p hotolabels a major M(r) = approximately 61,000 peptide; the photoincor poration is blocked by cirazoline, guanabenz, and amiloride but not by idazoxan (competing ligands = 10 muM). These data indicate the existe nce of at least three subtypes of IGRS that differ in their ligand rec ognition properties, their apparent molecular weight, and their tissue distribution.