Maria Angela Sortino

Relative Contribution of Different Receptor Subtypes in the Response of Neuroblastoma Cells to Tumor Necrosis Factor-α

Abstract: The effect of tumor necrosis factor‐α (TNF‐α) on neuronal viability has been investigated in the SK‐N‐BE neuroblastoma cell line. These cells undergo differentiation upon chronic treatment with retinoic acid. Exposure of SK‐N‐BE cells to TNF‐α produced a proliferative response in undifferentiated cells, whereas a reduced cell number was observed in retinoic acid (RA)‐differentiated cultures. This biphasic response may be related to the different expression of TNF‐α receptors (TNFRs); a significant increase in the density of TNFR1 was in fact observed following RA‐induced differentiation. Under these conditions, a pronounced increase in the formation of ceramide‐1‐phosphate (which was prevented by the selective inhibitor of phosphatidylcholine‐specific phospholipase C, D609) and an activation of caspase‐3 upon TNF‐α challenge were evident. Selective blockade of each TNFR subtype allowed a more detailed analysis of the effect observed. Preincubation with an anti‐TNFR1 antibody prevented the cytotoxic effect of TNF‐α in RA‐differentiated SK‐N‐BE cells, whereas the anti‐TNFR2 antibody blocked the proliferative activity of the cytokine in undifferentiated cultures.

Changing hypothalamopituitary function: Its role in aging of the female reproductive system

Changes in female reproductive function occur relatively early during the life span in many mammalian species. Therefore, this physiological system is an excellent model system in which to study the effects of age on specific endocrine relationships since changes occur prior to the occurrence of multiple pathologies associated with later stages of aging. Data from several laboratories suggest that changes in hypothalamic, pituitary and ovarian function may contribute to age-related deterioration of fertility in females. We will focus our attention on the role of hypothalamic changes in the cascade of events that eventually lead to acyclicity and infertility. Data suggest that changes in the diurnal rhythmicity of catecholaminergic neurotransmitters and their receptors occur during middle age. These changes may regulate the pattern of release of GnRH since alterations in the pulsatile pattern of LH secretion also become detectable at this age. Some age-related changes in hypothalamic and pituitary function are not irreversible or absolutely determined. Instead it appears that the ovarian steroidal milieu modulates the rate of aging of several aspects of hypothalamohypophysial function. In summary, changes in hypothalamic and pituitary function appear to contribute to the aging of the female reproductive system.

Antiproliferative effects induced by guanine-based purines require hypoxanthine-guanine phosphoribosyltransferase activity.

Abstract Guanine (GUA), guanosine and GMP exert a marked growth inhibition on the U87 glioma cell line that is not seen with other tested nucleotides, nucleosides and nucleobases. This effect could be replicated in several different human tumoral cell lines. Guanine shows a higher potency than guanosine or GMP, and co-treatments with adenosine or adenine are able to antagonize or revert the antiproliferative effect of guanine. The loss of the guanine effect in a cell line bearing a mutated inactive hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and the decreased potency of GUA in U87 cells silenced for HGPRT transcripts, demonstrates the central role of the intracellular metabolism of GUA for growth-inhibitory effects. Considering the potential application of growth-inhibitory substances in anticancer therapy, knowledge of the molecular mechanism underlying GUA-induced effects encourages studies aimed at defining possible tumoral targets for experimental therapies.

Involvement of norepinephrine activity in the regulation of α1 adrenergic receptors in the medial preoptic nucleus of estradiol-treated rats

To establish whether the diurnal decrease in the density of alpha 1 receptors observed in the medial preoptic nucleus (MPN) of estrogen (E2)-treated rats is related to the concomitant diurnal increase in norepinephrine (NE) turnover rates, we quantitated the density of [3H]-Prazosin binding to alpha 1 receptors after blockade of NE turnover with alpha-methyl-paratyrosine (alpha MPT). A series of preliminary studies was performed to rule out an interference of this drug with [3H]-Prazosin binding to alpha 1 adrenergic receptors in vitro and in vivo. Incubation of brain slices with alpha MPT produced a dose-dependent inhibition of [3H]-Prazosin binding to alpha 1 adrenergic receptors with an IC50 of approximately 6 mM. Scatchard analysis demonstrated that alpha MPT exhibited a simple competitive interaction with [3H]-Prazosin binding sites as shown by an increase in the apparent dissociation constant (Kd) of the ligand and no change in the number of alpha 1 receptors (Bmax). In contrast, preincubation of brain slices with alpha MPT and prior in vivo administration of alpha MPT did not affect [3H]-Prazosin binding to alpha 1 adrenergic receptors. Once we established that alpha MPT could be used to suppress NE turnover without interfering with the measurement of alpha 1 receptor densities, we repeatedly injected this drug to ovariectomized (OVX) and E2-implanted rats. The density of alpha 1 adrenergic receptors in MPN was quantitated autoradiographically. Blockade of NE turnover with alpha MPT only partially prevented the reduction in alpha 1 receptor density observed in the E2-treated rats, suggesting that the decrease in the level of [3H]-Prazosin binding sites cannot be completely ascribed to increased NE turnover rates.