Membrane localization and guanine nucleotide sensitivity of medullary I-1-imidazoline binding sites

Imidazoline binding sites are labeled by [H-3]clonidine (I-1) or by [H-3]idazoxan (I-2). I-2-sites are mitochondrial. The subcellular localization of I-1-sites in brain is unknown. Crude membranes from bovine rostral ventrolateral medulla (RVLM) were further purified by discontinuous sucrose density gradient. Fractions were assayed for I-1-site density (B-max) with [I-125]p-iodoclonidine. Nonspecific binding was defined by 10 mu M BDF-6143, and alpha(2)-adrenergic binding was defined by 10 mu M epinephrine. The proportions of I-1 and alpha(2) in mitochondrial fractions were similar (28 +/- 3 and 24 +/- 4%, respectively), and both I-1 and alpha(2) showed the greatest enrichment within the membrane-enriched fraction (58 +/- 13 and 38 +/- 4%). The myelin fraction contained a higher proportion of alpha(2) than I-1 (38 +/- 4 and 15 +/- 2%), consistent with expression of alpha(2), but not I-1, by glia. The enrichment of I-1 and alpha(2) in cellular membranes and alpha(2) in myelin was confirmed by further purification of these fractions over a second discontinuous gradient. Following irreversible inactivation df alpha(2), the remaining I-1 sites in RVLM crude membranes were inhibited by Gpp(NH)p but not by ATP. We conclude that I-1-imidazoline sites are non-mitochondrial membrane proteins sensitive to guanine nucleotide and may be functional receptors. Copyright (C) 1996 Elsevier Science Ltd.