Federica Pimpinelli

Presence of a reduced opioid response in interleukin-6 knock out mice

Cytokines are known to influence neuronal functions. The purpose of this study was to investigate the putative role of the cytokine interleukin‐6 (IL‐6) in the pathways involved in opioid‐mediated responses, by using IL‐6‐deficient mice. We reported that with a thermal stimulus IL‐6‐knock‐out (IL‐6KO) mice presented nociceptive thresholds similar to those measured in their controls. However, they showed a reduced analgesic response both to the restraint stress and to the administration of low doses of morphine. Hypothalamic levels of the opioid peptide β‐endorphin were significantly higher in IL‐6KO mice than they were in their controls. The development of tolerance to the analgesic effect of morphine was more rapid in IL‐6‐deficient mice than in wild‐type controls. Binding experiments showed that the number of opioid receptors in the midbrain, but not in the hypothalamus, decreased in IL‐6KO mice. Autoradiographic binding analysis revealed that the density of μ receptors diminished while the δ‐opioid receptors did not. These results suggest that IL‐6 is necessary for a correct development of neuronal mechanisms involved in the response to both endogenous and exogenous opiates.

Depolarization differentially affects the secretory and migratory properties of two cell lines of immortalized luteinizing hormone-releasing hormone (LHRH) neurons.

In this report we studied and compared the biochemical and the electrophysiological characteristics of two cell lines (GT1‐7 and GN11) of immortalized mouse LHRH‐expressing neurons and the correlation with their maturational stage and migratory activity. In fact, previous results indicated that GN11, but not GT1‐7, cells exhibit an elevated motility in vitro. The results show that the two cell lines differ in terms of immunoreactivity for tyrosine hydroxylase and nestin as well as of production and release of 3,4‐dihydroxyphenylalanine (DOPA) and of intracellular distribution and release of the LHRH. Patch‐clamp recordings in GN11 cells, reveal the presence of a single inward rectifier K+ current indicative of an immature neuronal phenotype (neither firing nor electrical activity). In contrast, as known from previous studies, GT1‐7 cells show the characteristics of mature LHRH neurons with a high electrical activity characterized by spontaneous firing and excitatory postsynaptic potentials. K+‐induced depolarization induces in GT1‐7 cells, but not in GN11 cells, a strong increase in the release of LHRH in the culture medium. However, depolarization of GN11 cells significantly decreases their chemomigratory response. In conclusion, these results indicate that GT1‐7 and GN11 cells show different biochemical and electrophysiological characteristics and are representative of mature and immature LHRH neurons, respectively. The early stage of maturation of GN11 cells, as well as the low electrical activity detected in these cells, appears to correlate with their migratory activity in vitro.

Cholinergic muscarinic mechanisms regulate neuropeptide Y gene expression via protein kinase C in human neuroblastoma cells

Neuropeptide Y (NPY) participates in the control of several functions in the nervous system. NPYergic neurons present in brain areas involved in cognitive processes are linked to ascending projections of the cholinergic system, a finding that suggests a role for acetylcholine in the control of these cells. In the present study, the effect of the activation of cholinergic muscarinic receptors on the expression of the human NPY gene was assessed. The SH-SY5Y neuroblastoma cell line was used as an in vitro model of human neurons; NPY mRNA levels were evaluated by Northern blot analysis. The results indicate that: (a) the expression of the human NPY gene in SH-SY5Y cells is stimulated by the cholinergic muscarinic agonist, carbachol; (b) this effect is mediated by the M3 muscarinic receptor subtype, as indicated by the inhibitory effect of the M3 antagonist 4-DAMP; (c) protein kinase C (PKC) activation plays an important role in the induction of NPY gene expression in this system, as suggested by experiments with the PKC activator, TPA, and the PKC inhibitor, GF 109203X; (d) the stimulation of NPY mRNA levels by TPA and by carbachol in SH-SY5Y cells requires de novo synthesis of RNA and protein. In conclusion, the present study shows that the activation of PKC-coupled muscarinic receptors as the M3 subtype positively modulates the expression of the human NPY gene in SH-SY5Y neuroblastoma cells, via PKC-related mechanisms.

Polarization of caveolins and caveolae during migration of immortalized neurons.

During CNS development neurons undergo directional migration to achieve their adult localizations. To study neuronal migration, we used a model cell line of immortalized murine neurons (gonadotropin‐releasing hormone expressing neurons; GN11), enriched with caveolins and caveolae invaginations that show in vitro chemotaxis upon serum exposure. Cholesterol depletion with methyl‐β‐cyclodextrin induced the loss of caveolae and the inhibition of chemotaxis, thus suggesting that GN11 migration depends upon the structural integrity of caveolae. Polarization of proteins and organelles is a hallmark of cell migration. Accordingly, GN11 cells transmigrating through filter pores exhibited a polarized distribution of caveolin‐1 isoform (cav‐1) in the leading processes. In contrast, during two‐dimensional migration cav‐1 and caveolae polarized at the trailing edge. As caveolae are enriched with signaling molecules, we suggest that asymmetrical localization of caveolae may spatially orient GN11 neurons to incoming migratory signals thereby transducing them into directional migration.

Presence of delta opioid receptors on a subset of hypothalamic gonadotropin releasing hormone (GnRH) neurons

Opioid peptides exert an inhibitory effect on hypothalamic gonadotropin releasing hormone (GnRH) secretion mainly by interacting with mu-opioid receptors. Although a direct role for opioids via delta-opioid receptors (DORs) has been suggested, the presence of these receptors on GnRH neurons has never been demonstrated. In the present study, we determined the distribution of DORs in the basal hypothalamus of rat with special focus on their relation to GnRH neurons. Double-labelling immunofluorescence and confocal microscopy revealed that DORs are exclusively present in a subpopulation of GnRH nerve terminals, with the highest density in the external layer of the median eminence. We then studied the functional characteristics of DORs in an immortalized GnRH-secreting neuronal cell line (GT1-1) known to endogenously express this receptor. Here, pertussis toxin pretreatment abolished the delta-agonist (DPDPE) inhibitory effect on cAMP accumulation. We also analyzed the type of G proteins involved in the signal transduced by the DOR and showed that GT1-1 cells express the inhibitory Go and Gi2 alpha-subunits. However, only Go was down-regulated under chronic DPDPE exposure. Finally, since DOR is expressed postnatally in brain, we compared GnRH neuronal cells immortalized at different developmental stages (the more mature GT1-1 and GT1-7 cells, versus the more immature GN11 cells), evidencing that only mature neurons express DOR. In conclusion, our study indicates that a direct control of opioids via delta-receptors occurs on GnRH neurons and validates the use of GT1 cells to further investigate the nature of the DOR present on GnRH neurons.

Decrease of the Number of Opioid Receptors and of the Responsiveness to Morphineduring Neuronal Differentiation Induced by 17β-Estradiol in Estrogen Receptor-Transfected Neuroblastoma Cells (SK-ER3)

Estrogens modulate the density of opioid receptors in selected brain areas; however, it is not clear whether they exert such an effect directly on the cells which express the opioid receptors. Therefore, we analyzed the binding of [3H]-diprenorphine in human neuroblastoma cells stably transfected with the estrogen receptor cDNA (SK-ER3 cell line). A 16-hour exposure of these cells with 1 nM 17β-estradiol induces a progressive morphological differentiation which appears clearly established 6 days after the suspension of the treatment. The binding of [3H]-diprenorphine was then measured immediately after the exposure to 17β-estradiol (16 h) as well as 6 days later. The results shows that the number of opioid receptors in SK-ER3 cells is unaffected at 16 h but appears significantly reduced at 6 days. This effect is blocked by the estrogen antagonist ICI-182780, and is coincident to a decrease of the inhibitory effect of morphine on cyclic AMP accumulation. Binding experiments performed using selective ligands suggest that the µ subclass of opioid receptors is down-regulated by estradiol in SK-ER3 cells.

Antiprogestins inhibit the binding of opioids to μ-opioid receptors in nervous membrane preparations

The present study showed that the glucocorticoid/progesterone antagonists, 17 beta-hydroxy-1 1 beta-(4-dimethylamino-phenyl-1)-17-(prop-1-ynyl)estra-4,9-dien+ ++-3-one (RU486) and 17 beta-hydroxy-11 beta-(4-dimethylamino-phenyl-1)-17-(propan-3-ol)estra-4,9-dien-3-o ne (ZK 98299), inhibit the binding of labeled dihydromorphine to mu-opioid receptors present on membrane preparations derived from rat and mouse brain, as well as from human neuroblastoma cells. The inhibitory effect of RU486 was dose-dependent and linked to a decrease of the affinity of labeled dihydromorphine to the mu-opioid receptors. Kinetic experiments have shown that RU486 induces a decrease of the association rate constant (k + 1) of dihydromorphine. RU486 also proved able to dissociate the dihydromorphine-mu-opioid receptor complex, although at a rate slower than that exhibited by unlabeled dihydromorphine. Finally, the addition of NaCl (100 mM) to the incubation buffer induced a 50% decrease of the inhibitory effect of RU486. A 6-day treatment of neuroblastoma cells with RU486 eliminated the inhibitory effect morphine exerts on the intracellular accumulation of cyclic AMP induced by prostaglandin E1. These results indicate that RU-486 may interact with brain mu-opioid receptors in vitro, by decreasing the affinity of opioid ligands.

Effects and Metabolism of Steroid Hormones in Human Neuroblastoma Cells

The development of the central nervous system is influenced by sex steroids and by their metabolites. However, little information on the possible effects of steroid hormones on neuroblastoma cells is available. Human neuroblastoma cell lines have been used as a model of human neuroblasts in vitro to study the metabolism of steroid hormones; in addition, the effects of steroids and steroid antagonists on neuroblastoma cell growth have also been investigated. The results obtained show that SH-SY5Y human neuroblastoma cells may actively metabolize testosterone and progesterone to their respective 5 alpha-reduced metabolites and that differentiation of neuroblastoma cells is paralleled by a significant increase in expression of the type-1 5 alpha-reductase and of the formation of steroid metabolites. All these data are suggestive of a potential role of steroid 5 alpha-reduced metabolites in the biology of neuroblastoma cells. Studies performed to analyze the role of steroid hormones on neuroblastoma cell proliferation show that progesterone at low doses may induce minor stimulation, and at higher doses, a toxic effect on the neuroblastoma cell line SK-N-SH is seen. Moreover, the antiprogestin 17 beta-hydroxy-11 beta-(4-dimethylamino-phenyl-1)-17-(prop-1-ynyl)estra-4,9-dien+ ++-3-one (RU486) decreases the proliferation of these cells in a dose-dependent manner. The effect of RU486 is not antagonized by either progesterone or dexamethasone, a result that seems to exclude the action of RU486 via classic intracellular steroid hormone receptors.

Expression of prostacyclin receptors in luteinizing hormone-releasing hormone immortalized neurons: role in the control of hormone secretion.

PGs of the E series are involved in the control of LHRH secretion. The present experiments were conducted to clarify whether PGI2 (prostacyclin) might be also involved in such a control, using multiple methodological approaches on immortalized LHRH-secreting neurons. A RT-PCR procedure to detect mouse PGI2 receptor (IP) messenger RNA was first applied, and the results obtained showed the presence of a specific transcript in two cell lines of immortalized LHRH neurons (GT1-1 and GN11 cell lines). Receptor binding assays on membrane preparations from GT1-1 cells showed the presence of a single specific and saturable class of binding sites (Kd = 4.6 nM; 10,000 sites/cell) for [3H]iloprost, a stable analog of PGI2. Competition experiments showed that the binding sites labeled by [3H]iloprost possess the pharmacological characteristics of IP receptors. In functional studies, PGI2 and its analogs, iloprost and cicaprost, were able to stimulate LHRH release from the GT1-1 cells with elevated potencies (EC50 = 0.6-4.3 nM); PGE1 was only slightly less active (EC50 = 28.5 nM), whereas PGE2, considered the major PG involved in LHRH secretion, was poorly effective (EC50 = 921 nM). The relative potencies (EC50) of these compounds in stimulating the intracellular accumulation of cAMP were in line with their LHRH-releasing activities. In conclusion, these results indicate that immortalized LHRH-secreting neurons express IP receptors through which PGI2 may exert relevant effects on LHRH release.