Peptide analogs of neuropeptide Y (NPY) with a Tyr-32 and Leu-34 replacement resulted in the decapeptide TyrIleAsnLeuIleTyrArgLeuArgTyr-NH2 (9; Table 1) and a 3700-fold improvement in affinity at Y2 (rat brain; IC50 = 8.2 +/- 3 nM) receptors when compared to the native NPY(27-36) C-terminal fragment. In addition, compound 9 was an agonist at Y1 (human erythroleukemia (HEL) cell; ED50 = 8.8 +/- 0.5 nM) receptors with potency comparable to that of NPY(1-36) (ED50 = 5 nM). Molecular dynamics and 1H-NMR were used to propose a solution structure of decapeptide 9 and for subsequent analog design. The replacement of Leu with Pro at position 4 of decapeptide 9 afforded an antagonist of NPY in HEL cells (18, TyrIleAsnProIleTyrArgLeuArgTyr-NH2; IC50 = 100 +/- 5 nM). Deletion of the N-terminal tyrosine of 18 resulted in a 10-fold improvement in antagonistic activity with a parallel 4-fold decrease in Y2 affinity. This potent antagonist may provide further insight into the physiological role(s) for NPY in the mammalian and peripheral nervous system.

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Cultures of bovine adrenomeduilary chromaffIn cells accumulated 1-methyl4nphenylpyridinium (MPP+) in a time-and concentration-dependent manner by-a process that was prevented by desmethylimipramine. The (14).Uptake studies were performed 1 day after changing to serum-free medium at 370C and were initiated by adding to the DMEM/F12 medium 10 ttl of [3H]MPP+ (1 uCi; 5.9-5000 pmol in 0.5 ml of medium). Uptake was terminated by rapid removal of the medium, followed by two washes of the cells with Earle's medium. The cellular contents were released by adding 0.5 ml of 0.1 M HClO4 per 106 cells, followed by freezing and thawing.Subcellular fractionation of chromaffin cells was performed as described by Diliberto et al. (15). Briefly, the cells were homogenized and then centrifuged at 800 x g for 10 min to yield supernatant S1' and pellet P1' fractions. S1' was then centrifuged at 26,000 x g for 10 min, which resulted in supernatant S2 and pellet P2 fractions. P2 was then resusAbbreviations: TyrOHase, tyrosine hydroxylase; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MPP', 1-methyl-4-phenylpyridinium.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.8160

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