Cycling of polyamines (spermine and spermidine) in the brain was examined by measuring polyamine transport in synaptic vesicles, synaptosomes and glial cells, and the release of spermine from hippocampal slices. It was found that membrane potential-dependent polyamine transport systems exist in synaptosomes and glial cells, and a proton gradientdependent polyamine transport system exists in synaptic vesicles. The glial cell transporter had high affinities for both spermine and spermidine, whereas the transporters in synaptosomes and synaptic vesicles had a much higher affinity for spermine than for spermidine. Polyamine transport by synaptosomes was inhibited by putrescine, agmatine, histidine, and histamine. Transport by glial cells was also inhibited by these four compounds and additionally by norepinephrine. On the other hand, polyamine transport by synaptic vesicles was inhibited only by putrescine and histamine. These results suggest that the polyamine transporters present in glial cells, neurons, and synaptic vesicles each have different properties and are, presumably, different molecular entities. Spermine was found to be accumulated in synaptic vesicles and was released from rat hippocampal slices by depolarization using a high concentration of KCl. Polyamines, in particular spermine, may function as neuromodulators in the brain.

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Prostaglandin E2 (PGE2) was shown to induce neuronal death in the CNS. To characterize the neurotoxicity of PGE2 and E-prostanoid receptors (EP) in motor neurons, we investigated PGE2-induced cell death and the type(s) of EP responsible for mediating it in NSC-34, a motor neuron-like cell line. Immunoblotting studies showed that EP2 and EP3 were dominantly expressed in NSC-34 cells and motor neurons in mice. Exposure to PGE2 and butaprost, an EP2 agonist, but not sulprostone, an EP1/3 agonist, resulted in decreased viability of these cells. These results suggest that PGE2 induces cell death by activation of EP2 in NSC-34 cells.

The antiproliferating effect of nine kinds of bis(ethy1)polyamine analogues [three kinds each of bis(ethyl)triamine, bis(ethy1)tetraamine and bis(ethy1)pentaaminel was compared using FM3 A cells.The inhibitory effect was in the order BE4444 > BE3443 > BE4334ZBE444 > BE343 > BE333 > BE44 > BE34 > BE33. Our results indicate that not only polyamine deficiency but also the accumulation of polyamine analogues is involved in the inhibition of cell growth. Accumulation of bis(ethy1)polyamine analogues caused the inhibition of protein synthesis and the decrease in the ATP content. The protein synthetic system in mitochondria was more strongly inhibited by bis(ethy1)polyamine analogues than that in the cytoplasm. Under conditions such that cytoplasmic protein synthesis was inhibited by 50% by bis(ethy1)polyamine analogues, mitochondrial protein synthesis was almost completely inhibited. Mitochondria1 Ile-tRNA formation was inhibited by bis(ethy1)polyamine analogues at the concentrations that cytoplasmic Ile-tRNA formation was stimulated. This may be one of the reasons for the selective inhibition of mitochondrial protein synthesis. This inhibition was followed by the decrease in ATP content, swelling of mitochondria and depletion of mitochondrial DNA. These results suggest that the early event of metabolic change caused by bis(ethy1)polyamine analogues in cells is the inhibition of protein synthesis, especially of mitochondrial protein synthesis.Since the polyamines putrescine, spermidine and spermine are essential for the maintenance of eukaryotic cell proliferation [ 11, inhibitors of polyamine biosynthesis to deplete cellular polyamines have been developed as antiproliferative reagents [2, 31. Bis(ethy1)polyamine analogues have also been developed as antiproliferative reagents [4, 51. These reagents can not only negatively regulate the synthesis of omithine decarboxylase (OmDC) and S-adenosylmethionine decarboxylase (AdoMetDC) [6], but can also induce spermidinekpermine A"-acetyltransferase (SSAT) activity [7, 81. Thus, the analogues can deplete intracellular polyamines almost completely, and they are thought to inhibit cell growth through this process. We found that A", W'-bis(ethyl)spermine (BE343) can substitute for the functions of spermine in various aspects and accumulate in cells at a concentration fivefold that of spermine in control cells [9, lo], and suggested that not only polyamine deficiency but also the accumulation of BE343 may be involved in the inhibition of cell growth [lo]. Recently, it has been reported that the inhibition of cell growth by BE343 correlated with the intracellular acCorrespondence to K. Igarashi, Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba, Japan 263Fax: +81 43 290 2900. Abbreviations. OmDC, ornithine decarboxylase; AdoMetDC, S-adenosylmethionine decarboxylase; SSAT, spermidinehpermine W-acetyltransferase.Enzymes. Ornithine decarboxylase (EC 4.1.1.17) ; S-adenosylmethionine decarboxylase (EC 4.1.1.50) ; spermidinehpermine W-acetyltransferase...

The mechanism of the antiproliferation effect of N1, N"-bis(ethyl)spemine (BESPM) was studied in detail using mouse FM3A cells, since this polyamine analogue mimics the functions of spermine in severalcaspects [lgarashi. K., Kashiwagi, K., Fukuchi, J., Isobe, Y., Otomo, S. & Shirahata, A.(1 990) Biochem. Biophys. Res. Commun. 172,7201. Our results indicate that not only the decrease in spcrimine and spermine caused by BESPM but also its accumulation play important roles on the inhibition of cell growth by BESPM, since BESPM accumulated in cells at a concentration fivefold that of spermidine in control cells. In comparison with the polaymine-deficient cells caused by ol-difluoromethylornithine, an inhibitor of ornithine decarboxylase, and ethylglyoxal bis-(guanylhydrazone), an inhbitor of S-adenosylmethionine decarboxylase, the behavior of polyaminedeficient cells caused by BESPM was different as follows: the inhibition of cell growth by BESPM was not abrogated by spermine or spermidine; polyamine uptake, which is stimulated during polyamine deficiency, was greatly inhibited, while spermidine/spermine N'-acetyltransferase activity, which is inhibited during polyamine deficiency, was enhanced in BESPM-treated cells ; thymidine kinase activity did not decrease in BESPM-treated cells; inhibition of cell growth and macromolecule synthesis by BESPM correlated with the swelling of mitochondria and the decrease in ATP content; BESPM caused cell death when incubated together for several days. The role of BESPM accumulation on inhibition of cell growth is discussed.