Spermine oxidation leads to necrosis with plasma membrane phosphatidylserine redistribution in mouse leukemia cells.

Oxidation by copper/quinone-containing serum amine oxidases (SAO) is a well-known cause of polyamine cytotoxicity. Spermine oxidation exerts potent immunosuppressive effects in animal cells, but the cell death mechanism involved remains unclear. We compared biochemical and morphological parameters of SAO-mediated cell death in L1210 mouse leukemia cells with normal or amplified ornithine decarboxylase gene expression with those observed during apoptosis induced by deregulated polyamine uptake or by okadaic acid. None of the characteristic features of apoptotic cell death (e.g., chromatin condensation, nuclear fragmentation, internucleosomal DNA cleavage, poly(ADP-ribose) polymerase cleavage) were observed during spermine oxidation-mediated cell death, which was clearly necrotic by morphological criteria. Inhibition of a wide spectrum of caspases did not prevent SAO-dependent cell death, whereas N-acetylcysteine completely abolished the cytotoxic effects of spermine oxidation. Catalase only delayed spermine oxidation-induced cell death without affecting its modality or preventing depletion of intracellular glutathione, suggesting that both H(2)O(2) and aminoaldehydes generated by SAO-mediated spermine oxidation contribute to SAO-induced necrosis. Interestingly, redistribution of phosphatidylserine to the outer leaflet of the plasma membrane, usually a diagnostic feature of apoptosis, preceded necrotic cytolysis triggered by spermine oxidation. Thus, L1210 cell death caused by SAO-mediated spermine oxidation has all the attributes of primary necrosis, but is also accompanied by loss of phospholipid asymmetry, indicating that the latter phenomenon may not be unique to apoptosis. Phosphatidylserine exposure, a potent engulfment signal for phagocytes, might contribute to the immunosuppressive effects of plasma polyamines through a controlled and rapid necrotic process involving SAO.

Synthesis of spermidine and norspermidine dimers as high affinity polyamine transport inhibitors.

A series of novel spermidine and sym-norspermidine dimers was synthesized by crosslinking the polyamine backbones via alkylation of their secondary amino groups to butyl, trans-2-butenyl, 2-butynyl or p-xylyl bridges. The resulting hexamines behaved as high-affinity antagonists of polyamine uptake, with a relative potency that was dependent on the geometry of the linker structure.

Influence of the estrous cycle on c-fos and CRH gene transcription in the brain of endotoxin-challenged female rats.

The purpose of this study was to investigate whether the ovulatory cycle interferes with the effect of the acute-phase response of a systemic immune activation on the transcription of the immediate early gene c-fos and the stress-related neuropeptide corticotropin-releasing hormone (CRH) in the brains of female rats. Throughout the day of proestrus and diestrus-2 (09.00, 12.00, 15.00 h), adult rats received either a single intraperitoneal injection of the endotoxin lipopolysaccharide (LPS, 200 micrograms/100 g body weight) or the vehicle solution and were killed 3 h later (12.00, 15.00, 18.00 h). Frozen brains were mounted on a microtome, cut in 30-micron slices and then processed for the detection of c-fos mRNA and CRH primary transcript (heteronuclear [hnRNA]) by means of in situ hybridization histochemistry using 35S-labeled exonic and intronic probes, respectively. LPS injection induced a profound expression of c-fos mRNA in the several nuclei and areas of the brain, such as the organum vasculosum of the lamina terminalis/medial preoptic area, supraoptic nucleus, parvo- and magnocellular divisions of the hypothalamic paraventricular nucleus (PVN), arcuate nucleus/median eminence, central nucleus of the amygdala, locus coeruleus, nucleus of the solitary tract, area postrema and ventrolateral medulla. Interestingly, the intensity of expression of c-fos mRNA depended on the phase of the estrous cycle and/or the time of the day. Indeed, in several of the structures described above, LPS induced a more pronounced c-fos signal in the morning of proestrus than the afternoon and diestrus-2. CRH primary transcript was significantly increased by LPS treatment selectively in the parvocellular division of the PVN and the highest hybridization signal was observed in the morning of proestrus, a period where a large number of c-fos-positive cells were colocalized in CRH-immunoreactive neurons. A significant increase in the levels of AVP hnRNA was also observed in the parvocellular PVN of animals sacrificed at noon and early afternoon of both pro- and diestrus days. These results provide evidence that the neuroendocrine events regulating the reproductive cyclicity influence the endotoxin-induced activation of the early gene c-fos in selective structures of the brain and the stimulation of neurons directly involved in the regulation of the HPA axis. It is possible that the gonadal status of female mammals plays a crucial role in the integration of the organism in the presence of foreign material in preventing an exaggerated immune response during particular phases of the ovulatory cycle. The capacity of female animals to modulate the intensity through which the neuronal circuitry activated during immunogenic processes is likely to be an elegant sexual dimorphism participating in the adjustment of the responses in line with the physiological demand.

Differential effects of a steroidal antiestrogen, EM-139, on prostaglandin and cyclic adenosine 3',5'-monophosphate production in the circular and longitudinal layers of bovine myometrium.

The regulation of prostaglandin (PG) production and cAMP generation was studied in vitro in cultured smooth muscle cells isolated specifically from the circular or longitudinal layers of the bovine myometrium. We found that prostacyclin (PGI2) was the principal PG produced by the myometrium, especially in the longitudinal layer, followed closely by PGE2 and marginally by PGF2 alpha. The PG production (fg/ml, mean +/- SD) in the circular and longitudinal layers was, respectively, PGE2 (424.4 +/- 162.0) > PGI2 (189.5 +/- 19.0) > PGF2 alpha (9.5 +/- 3.0) versus PGI2 (751 +/- 36) > PGE2 (515.7 +/- 94.0) > PGF2 alpha (16.3 +/- 3.0); production was stimulated up to 15-fold 24 h after addition of phorbol 12-myristate (PMA; 100 nM). Hormonal control of PG production was assessed by use of a steroidal antiestrogen, EM-139. PG production was inhibited in a concentration-dependent manner by EM-139 in both circular and longitudinal layers, with maximal inhibition at 1 microM. In parallel studies, chronic treatment with EM-139 resulted in significant increases in isoproterenol-induced cAMP production in both muscle layers, but more especially in the circular layer. This antiestrogenic effect was reversed by addition of 17 beta-estradiol. These results indicate that the two smooth muscle layers of the bovine myometrium have distinct patterns of PG production and that the adenylate cyclase/cAMP response of the circular layer is more sensitive to estrogen modulation. Our findings with a cell culture model of separated myometrial layers provide strategic information for a better understanding of the regulation of uterine contractility during pregnancy.