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When normal human peripheral lymphocytes are treated with mitogen and grown in the presence of [3H]putrescine or [terminal methylenes-3H]spermidine, label is incorporated predominantly into one cellular protein. The radioactive constituent of this protein was identified as the unusual amino acid hypusine [Nt-4-amino-2-hydi-oxybutyl)lysine]. This was accomplished by isolation of the component from proteolytic digests or acid hydrolysates and comparison with authentic hypusine by chromatography, conversion to the 2,4-dintitrophenyl derivative, and oxidative degradation. The observed relationships among intracellular levels of labeled putrescine, polyamines, and protein bound hypusine after growth of cells with the various labeled amines and with or without an inhibitor of polyamine biosynthesis supply evidence that spermidine is the immediate amine precursor of hypusine and that the 4-amino-2-hydroxybutyl portion ofhypusine derives from the butylamine moiety of spermidine.While studying the role of polyamines as physiological substrates for transglutaminases, we observed an unidentified radiolabeled material in proteolytic digests ofthe protein fraction from normal human peripheral lymphocytes that had been treated with mitogen in the presence of [3H]putrescine. This labeled basic component of lymphocyte protein was initially thought to be N'-(y-glutamyl.)spermidine because it chromatographed in a position close to that ofthis y-glutamylpolyamine in the ion exchange system used. It was subsequently recognized and reported (1) as an unknown componentof lymphocyte protein. This conclusion was based on its observed stability to acid hydrolysis. We now report that this compound, which is the major labeled component formed in the lymphocyte protein fraction during growth ofthe cells with eitherlabeled putrescine or labeled spermidine, is the unusual amino acid hypusine. The present report provides evidence for the identity of this amino acid and for the direct precursor role of spermidine in its biosynthesis. In addition, it is shown that hypusine occurs predominantly in one protein of lymphocytes.Hypusine was discovered by Nakajima and coworkers (2), who isolated the free amino acid from homogenates of bovine brain and determined its structure as N8-(4-amino-2-hydroxybutyl)lysine. In addition to its detection in free form in a number of organs of several mammals (3), hypusine was found to be a constituent of animal proteins (4). Attempts to find any specific protein abundant in this amino acid were, however, without success (4). EXPERIMENTAL PROCEDURE Materials. [2,3-3H(N)]Putrescine-2HC1, [terminal, methylenes-3H(N)]spermidine-3HCl, and [3-aminopropyl-3-3H(N)]-spermine-4HCI were purchased from New England Nuclear. Their specific radioactivities were >20 Ci/mmol (1 Ci = 3.7 X 10'°becquerels) and each was used without dilution of specific radioactivity. Methylglyoxalbis(guanylhydrazone)-2HCl'H20 (MGBG) was from Aldrich. Hypusine, isolated by T. Nakajima and his colleagues and provided by them to John W. Daly o...

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The present study examined the effect of opioid receptor agonists in the rat forced swim assay. The ␦ -opioid receptor agonists SNC80 (( ϩ )-4-[( ␣ R)-␣ -((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,Ndiethylbenzamide) and ( 7 ␣ ,5]classes of compounds have also been investigated and have potential antidepressant activity. One such class of agents is the opioids. Studies have been performed using a variety of different opioid system-enhancing agents that suggest an antidepressant role for opioid agonists. Such studies have demonstrated antidepressant activity with the opioid ligand cyclazocine (Fink et al. 1970; open, placebo-controlled study) and with the opioid peptide ␤ -endorphin (Kline et al. 1977; open study). Interestingly, the enkephalinase inhibitors RB101 and BL-2401 exert an antidepressant-like action in a learned helplessness assay in rats, presumably by inhibiting the degradation of endogenous opioids (Baamonde et al. 1992;Kita et al. 1997). The effect of RB101 was reversed by the ␦ -opioid receptor antagonist nal-

Agonists at ␦, , and opioid receptors produce interacting effects in rodents and nonhuman primates. To further evaluate the determinants of these interactions, this study examined the effects of mixtures of ␦ ϩ and ␦ ϩ agonists in rhesus monkeys (n ϭ 4 -5) using two behavioral procedures, an assay of schedulecontrolled responding for food reinforcement and an assay of thermal nociception. Results were analyzed using dose-addition analysis. In the assay of schedule-controlled responding, the; the agonists methadone, fentanyl, morphine, and nalbuphine; and the agonists (5␣,7␣,8␤)-(Ϫ)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl) benzeneacetamide (U69,593) and bremazocine all dose dependently decreased rates of food-maintained responding when administered alone. Fixed ratio mixtures of SNC80 ϩ agonists produced additive or subadditive effects, whereas SNC80 ϩ agonist mixtures produced only additive effects. In the assay of thermal nociception, SNC80 produced no measurable effects when administered alone, whereas and agonists produced dose-dependent antinociception. SNC80 ϩ agonist mixtures produced superadditive effects manifested as leftward shifts in agonist dose-effect curves. This synergism was antagonized by the ␦-selective antagonist naltrindole, suggesting that SNC80-induced enhancement of agonist antinociception was ␦ receptor-mediated. SNC80 did not enhance the antinociceptive effects of the highly selective agonist U69,593, and it produced only a marginal enhancement of antinociception produced by the less selective agonist bremazocine. These results suggest that ␦ agonists may selectively enhance the antinociceptive effects of agonists in rhesus monkeys. These results also confirm that opioid agonist interactions may depend on the receptor selectivity and relative doses of the agonists and on the experimental endpoint.Biochemical and behavioral evidence indicates the existence of three opioid receptor types, the ␦, , and receptors (Martin et al., 1976;Lord et al., 1977;Evans et al., 1992;Thompson et al., 1993;Yasuda et al., 1993). Agonists selective for ␦, , and receptors produce distinct profiles of physiological and behavioral effects (Gutstein, 2001). In addition, selective ␦, , and agonists may also produce interacting effects. Studies of opioid receptor interactions have been conducted primarily with ␦ and agonists in rodents using assays of antinociception, and results from these studies suggest that the nature of the interaction depends on such variables as the particular agonists used, the relative doses tested, and the behavioral endpoint (Heyman et al., 1989;Jiang et al., 1990;Adams et al., 1993). For example, the peptidic ␦ agonists [D-Pen 2 ,D-Pen 5 ]-enkephalin (DPDPE) and deltorphin II enhanced the effects of some intermediate-efficacy agonists (morphine, normorphine, and codeine), but not of higher efficacy -agonists (PL017, fentanyl, or sufentanil), in an assay of thermal nociception in mice (Heyman et al., 1989;Jiang et al., 1990). In a subsequent study, DPDPE enhanced ...