Membrane localization and guanine nucleotide sensitivity of medullary I1-imidazoline binding sites.
Imidazoline binding sites are labeled by [3H]clonidine (I1) or by [3H]idazoxan (I2). I2-sites are mitochondrial. The subcellular localization of I1-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 I1-site density (Bmax) with [125I]p-iodoclonidine. Nonspecific binding was defined by 10 microM BDF-6143, and alpha 2-adrenergic binding was defined by 10 microM epinephrine. The proportions of I1 and alpha 2 in mitochondrial fractions were similar (28 +/- 3 and 24 +/- 4%, respectively), and both I1 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 I1 (38 +/- 4 and 15 +/- 2%), consistent with expression of alpha 2, but not I1, by glia. The enrichment of I1 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 of alpha 2, the remaining I1 sites in RVLM crude membranes were inhibited by Gpp(NH)p but not by ATP. We conclude that I1-imidazoline sites are non-mitochondrial membrane proteins sensitive to guanine nucleotide and may be functional receptors.
Ernsberger
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Department of Medicine, Case Western Reserve School of Medicine, Cleveland, OH 44106-4982, USA.
Shen
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IH
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry*; D003000:Clonidine / Q000031:analogs & derivatives / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D019329:Idazoxan / Q000378:metabolism; D054755:Imidazoline Receptors; D008526:Medulla Oblongata / Q000648:ultrastructure*; D011955:Receptors, Drug / Q000032:analysis* / Q000187:drug effects* / Q000378:metabolism; D014316:Tritium
D000345:Affinity Labels; D006150:Guanine Nucleotides; D054755:Imidazoline Receptors; D011955:Receptors, Drug; D014316:Tritium; C053113:4-iodoclonidine; D006165:Guanylyl Imidodiphosphate; D003000:Clonidine; D019329:Idazoxan