Giacomo Pozzoli

Erythropoietin exerts anti‐apoptotic effects on rat microglial cells in vitro

Erythropoietin (EPO), a renal cytokine regulating haematopoiesis, is also produced by different cell types within the central nervous system, where it acts via the activation of specific receptors. Current evidence shows that EPO exerts neurotrophic and neuroprotective activities in different in vivo and in vitro models of brain damage. In the present study we investigated the effects of EPO on primary cultures of rat cortical microglia and astrocytes. We found that: (i) EPO exerted a marked stimulatory effect on microglial cell viability, assessed through the MTS assay, whereas astrocytes were almost unaffected; (ii) the cytokine increased microglial cell population size in a concentration‐dependent manner; however, as microglia cultures undergo spontaneous apoptosis after separation from astrocytes, the apparent effect on cell proliferation could be attributed to EPO antagonism of normal apoptosis; (iii) subsequent flow cytometry analysis on microglial cells demonstrated both the trophic role of factor(s) released by astrocytes in mixed cultures, and the putative anti‐apoptotic action of EPO; (iv) the latter was further confirmed through the assessment of gene expression of anti‐ and pro‐apoptotic factors, which showed that EPO is able to shift the Bcl : Bax ratio towards a net anti‐apoptotic effect; (v) EPO did not affect the pro‐inflammatory function of microglial cells.

Interleukin-1β and Interleukin-6 Specifically Increase the Release of Prostaglandin E2 from Rat Hypothalamic Explants in vitro

It has previously been shown that the cytokines interleukin-1 beta and interleukin-6 (IL-1 beta and IL-6) stimulate directly the release of corticotrophin-releasing-hormone-41 from the rat hypothalamus in vitro, while IL-1 beta can also stimulate the release of somatostatin. These effects can be antagonized by drugs which block prostaglandin (PG) synthesis. PGs are also involved in the control of hypothalamic neuropeptides by other neurotransmitters. In the present study, we have characterized the production of PGs from the rat hypothalamus in vitro, and investigated the effects of IL-1 beta and IL-6, as well as the neurotransmitters norepinephrine, acetylcholine and 5-hydroxytryptamine, on the acute release of PGs, using a well-validated acute hypothalamic incubation system. The rate of release of PGs [PGE2, PGF2 alpha, 6-keto-PGF1 alpha (6KPGF1 alpha) and thromboxane B2 (TXB2) in the medium was found to stabilize after 60 min of preincubation and thereafter remain constant, with TXB2 being the predominant species. Twenty-minute incubation in the presence of human recombinant IL-1 beta or IL-6, in the dose range 1-100 U/ml, had no effect on the release of PGF2 alpha, 6KPGF1 alpha or TXB2; however, the release of PGE2 was significantly increased by both IL-1 beta and IL-6. The effect of IL-1 beta was antagonized by both indomethacin and dexamethasone. None of the other neurotransmitters tested had any effect on the release of any of the PGs.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that corticotropin-releasing hormone inhibits cell growth of human breast cancer cells via the activation of CRH-R1 receptor subtype

It has been previously shown that corticotropin-releasing hormone (CRH) exerts antiproliferative activity on an estrogen-dependent tumor cell line, i.e. human endometrial adenocarcinoma Ishikawa (IK) cells. Here we have investigated the effects of CRH on another estrogen-dependent tumor cell line, human breast cancer MCF7 cells. In this paradigm, CRH given at a fixed concentration of 100 nM significantly inhibited cell growth induced by 100 nM estradiol (E2) after 48 and 72 h of incubation. This effect was not associated with the induction of apoptosis. CRH inhibition of cell proliferation was counteracted in a concentration-dependent manner by the non-selective CRH receptor antagonist, astressin, as well as by a CRH-R1 selective receptor antagonist, antalarmin. RNase protection assays carried out on MCF7 under basal conditions showed that these cells express in a constitutive manner the CRH-R1 receptor subtype. We have also investigated the putative source of CRH acting on breast cancer cells; we found that MCF7 cells express CRH mRNA under basal conditions and secrete sizable amounts of immunoreactive CRH, which leads to postulate the existence of paracrine-autocrine inhibitory mechanism operated by CRH in breast cancer cells.

Vasoactive Intestinal Peptide and Forskolin Stimulate Interleukin 6 Production by Rat Cortical Astrocytes in Culture via a Cyclic AMP‐Dependent, Prostaglandin‐Independent Mechanism

Abstract: In this study we analyzed the involvement of the cyclic AMP (cAMP)‐protein kinase A system in the regulation of interleukin 6 production by cultured cortical astrocytes. Vasoactive intestinal peptide strongly increased, in a dose‐dependent manner, interleukin 6 production. This effect was reduced when protein kinase A was blocked by KT‐5720; it was not affected by calphostin C, a protein kinase C inhibitor. Forskolin caused a concentration‐dependent increase in interleukin 6 release that was also inhibited by KT‐5720. Because prostaglandins are believed to play a role in interleukin 6 production, we tried to determine whether the stimulatory effects of vasoactive intestinal peptide and forskolin on cytokine release might be mediated by stimulation of prostaglandin production in cortical astrocytes. Vasoactive intestinal peptide did not increase the production of either prostaglandin E2 or F2α. Conversely, forskolin concentration‐dependently stimulated the production of both prostaglandins, an effect that was blocked by indomethacin. Indomethacin did not affect either vasoactive intestinal peptide‐ or forskolin‐stimulated interleukin 6 production. To exclude the possibility that prostaglandins participate in interleukin 6 production induced by forskolin, we tested prostaglandins E2 and F2α. The former was completely ineffective in eliciting the cytokine production, whereas prostaglandin F2α slightly increased interleukin 6 production only at the highest concentrations. 8‐Bromo‐cAMP and dibutyryl‐cAMP stimulated interleukin 6 production to a lesser extent than vasoactive intestinal peptide and forskolin. In conclusion, we provide evidence that vasoactive intestinal peptide increases interleukin 6 production by astrocytes through the stimulation of the cAMP‐protein kinase A pathway, an effect that is reproduced by cAMP analogues. In addition, we point out that prostaglandins are not involved in vasoactive intestinal peptide‐ and forskolin‐mediated induction of interleukin 6 production in cultured astrocytes.

Evidence for the neuronal origin of immunoreactive interleukin-1β released by rat hypothalamic explants

In this study, we have investigated the release of immunoreactive interleukin-1 beta (irIL-1 beta) from the rat hypothalamus in vitro. It was found that (1) tissue explants release sizable amounts of irIL-1 beta (ranging from 0.43 to 0.52 pg/mg of wet tissue) in 20 min incubations; (2) basal release in significantly increased by depolarization induced with 56 mM KCl; (3) K(+)-induced irIL-1 beta release is inhibited by the specific blocker of N-type calcium channels, omega-conotoxin, and by verapamil, but not by nifedipine; (4) K(+)-induced release is also inhibited by the Na+ channel blockers tetrodotoxin and lidocaine; (5) irIL-1 beta release is significantly increased by noradrenalin; such increase is antagonized by verapamil and the beta-blocker propranolol, but not by the alpha-blocker phentolamine. The present evidence suggests that irIL-1 beta released by rat hypothalamic explants following KCl depolarization is neuronal in origin.

Interleukin-18 displays effects opposite to those of interleukin-1 in the regulation of neuroendocrine stress axis

The effects of interleukin-18 (IL-18), a putative member of the IL-1 family, were investigated on basal and stimulated release of corticotropin-releasing hormone (CRH) and prostanoids from rat hypothalamic explants and glial cells in vitro. We found that IL-18 decreases basal and KCl-stimulated CRH release from the hypothalamus. IL-18 also reduced CRH gene expression after 1- and 3-h incubation. The cytokine did not modify basal PGE2 production by hypothalamic explants but abolished production stimulated by IL-1beta. Similar effects were also observed on cultured glial cells. The present findings show that IL-18 possesses a profile of in vitro neuroendocrine activities opposing to, and even antagonizing, those of IL-1beta.

Nociceptin (1–13)NH2 Inhibits Stimulated Calcitonin-Gene-Related-Peptide Release From Primary Cultures of Rat Trigeminal Ganglia Neurones

In this work we have developed and characterized primary cultures of neonatal rat trigeminal ganglia neurones; calcitonin-gene-related-peptide (CGRP) released from cells was taken as a marker of neuronal function. A significant and consistent increase in CGRP secretion was elicited by non-specific (56 mM KCl or veratridine) or specific (capsaicin) depolarizing stimuli. This paradigm was subsequently used to investigate the effects of nociceptin, an opioid-like peptide involved in central and peripheral control of nociception. We found that the nociceptin analogue nociceptin (1–13)NH2 (NOC) did not affect baseline CGRP release, but it reduced in a concentration-dependent manner CGRP release induced by all tested stimuli. NOC-induced reduction was statistically significant from 0.01 nM onward and achieved maximal effects at 10 nM. Such effects of NOC were seemingly mediated by the activation of specific ORL1 receptors, as a well-known nociceptin antagonist, N(Phe1)nociceptin (1–13)NH2, was able to completely revert NOC inhibition of capsaicin-stimulated CGRP release.

HIV-1 Gp120 protein modulates corticotropin releasing factor synthesis and release via the stimulation of its mRNA from the rat hypothalamus in vitro: involvement of inducible nitric oxide synthase ☆

In the present study, we examined whether the human immunodeficiency virus type I (HIV-I) gp120 coat protein can modulate corticotropin releasing factor (CRF) secretion by using the incubation of rat hypothalamic explants as an in vitro model. Treatment of the hypothalamic fragments with recombinant gp120 resulted in a time- and concentration-dependent increase in CRF release. The maximal dose of 10 nM gp120 increased CRF release by 56.4% after 1 h, and 78.4% after 3 h, as compared with their respective controls. The intra-hypothalamic amount of CRF was also increased by 54.7% and 77.3% vs. controls after 1 and 3 h, respectively. Moreover, the action of gp120 was blocked by pretreatment with cycloheximide, suggesting that the viral protein modulates CRF secretion via an increase in its synthesis. We also investigated the effects of gp120 on CRF gene expression. RNase protection analyses of total RNA isolated from the explants indicated that 10 nM gp120 significantly increases CRF mRNA in a time-dependent manner. Furthermore, gp120 did not modify CRF mRNA stability, suggesting that the viral protein modulates CRF gene expression at the transcriptional level. Analysis of the mechanisms that mediate gp120-induced CRF synthesis was conducted. The incubation of the explants with recombinant interleukin-1 (IL-1) type I receptor antagonist (hrIL-1 ra) did not antagonize the actions of gp120 at 1 and 3 h, indicating that the effect of the latter is independent of IL-1 mediated mechanisms. The involvement of some second messenger pathways was also investigated. Specific inhibitors of cAMP-PKA, cyclo-oxygenase or heme oxygenase pathways failed to antagonize the gp120-induced increase in CRF production. By contrast, incubation with nonselective inhibitors of nitric oxide synthase (NOS), L-NAME and L-NNA, or aminoguanidine (AG), a selective inhibitor of inducible NOS (iNOS), blocked CRF release and, AG, its mRNA accumulation, stimulated by gp120, whereas selective inhibitors of endothelial and neuronal NOS had no effect. In addition, only L-NAME, L-NNA and AG were able to inhibit the gp120-stimulated production of nitrites. These results indicate that gp120 directly stimulates CRF gene expression and peptide synthesis from the rat hypothalamus in vitro via the activation of iNOS. Therefore, the actions of this viral protein on the HPA axis may, in part, reflect its ability to modulate CRF synthesis.

The effects of 5-HT3 receptor antagonists on cisplatin-induced emesis in the pigeon

In the present study, the emetic effect of the anticancer drug cisplatin, and the protective effects of 5-HT3 receptor antagonists against cisplatin emesis were investigated in the pigeon. The experimental set-up involved the i.v. administration of drugs and subsequent observation of the percentage of vomiting animals and the number of emetic episodes per vomiting animal. It was observed that cisplatin induced dose-dependent emesis in the pigeon. 5-HT3 receptor antagonists afforded partial protection against cisplatin emesis, although some of them, i.e. indole, indole-like derivatives and zacopride, displayed intrinsic emetic activity. A serotonergic mechanism appears to be involved in both cisplatin- and 5-HT3 receptor antagonist-induced emesis, since pretreatment with an inhibitor of 5-HT synthesis, para-chlorophenylalanine (pCPA), prevented vomiting induced by either cisplatin or 5-HT3 receptor antagonists. It is concluded that the intrinsic emetic effects of 5-HT3 receptor antagonists provide pharmacological evidence of species differences in the properties of 5-HT3 receptors.

Prostaglandin E2 and Bacterial Lipopolysaccharide Stimulate Bioactive Interleukin-1 Release from Rat Hypothalamic Explants

While interleukin-1 (IL-1) is intimately involved in locally modulating the acute inflammatory response, it is also able to influence processes at remote sites, i.e., in an endocrine manner. While there is as yet little evidence that IL-1 can cross the blood-brain barrier, many effects such as fever, increased slow-wave sleep, anorexia and the modulation of neuroendocrine function suggest an action of circulating IL-1 at regulatory sites within the hypothalamus. However, there is accumulating evidence for IL-1 originating within the central nervous system (CNS), and it is currently unclear as to whether the neurally mediated manifestations of the acute inflammatory response are due to activation of central or peripheral (circulating) IL-1. In this study we have characterized the release of IL-1 from rat hypothalamic explants, and we have investigated the effects of putative modulators of IL-1 release, lipopolysaccharide (LPS) and the prostaglandins E2 (PGE2) and F2 alpha (PGF2 alpha). After 1 h of incubation, IL-1-like activity in hypothalamic supernatants ranged between 175 and 2,304 munits/mg of protein; this was substantially inhibited by the addition to the bioassay system of antibodies (1:200) against IL-1 alpha, but not against IL-1 beta. LPS and PGE2 significantly stimulated IL-1 release at 100 and 1 ng/ml respectively, whereas PGF2 alpha had no effect in the range of doses tested. It is therefore concluded that the control of hypothalamic IL-1 release may be investigated by means of acute rat hypothalamic explants.(ABSTRACT TRUNCATED AT 250 WORDS)