Pierrick Gandolfo

Regulation of the expression of peripheral benzodiazepine receptors and their endogenous ligands during rat sciatic nerve degeneration and regeneration: a role for PBR in neurosteroidogenesis.

Peripheral benzodiazepine receptors (PBR) and their endogenous ligands, the diazepam-binding inhibitor derived-peptides, are present in Schwann cells in the peripheral nervous system. The aim of this study was to determine the influence of reversible (freeze-injury) and permanent (transection and ligature) nerve lesion on PBR density and on the levels of their endogenous ligands, by autoradiography (using [3H]PK11195) and radioimmunoassay (using antisera directed against the octadecaneuropeptide (ODN), a diazepam-binding inhibitor fragment). The potential role of PBR on peripheral nerve steroidogenesis, was studied by investigating the effect of specific PBR agonists and antagonists on pregnenolone levels in the sciatic nerve. Sixteen to 30 days after nerve lesion, PBR density and ODN-LI level were highly increased. Their expression returned to normal level when regeneration was completed 60 days after freeze-injury, but remained elevated when regeneration did not occur in transected distal stumps. Reverse-phase HPLC analysis of ODN-LI showed that in control nerve extracts, the major immunoreactive peak co-elutes with triakontatetraneuropeptide (TTN). After freeze-injury, intermediate molecular forms eluting between ODN and TTN were predominant and remained elevated at day 60. The greater accumulation of intermediate forms when regeneration is allowed to occur may indicate a particular role of these forms in axonal elongation and myelination. Ro5-4864, a high affinity PBR agonist increased pregnenolone concentration in the sciatic nerve. This effect was antagonised by PK11195, a high affinity PBR antagonist, which had no effect on pregnenolone basal level, indicating a specific action of PBR in neurosteroid production. These results suggest a role for PBR and their endogenous ligands in peripheral nerve regeneration. A trophic effect could be exerted via stimulation of steroid synthesis.

Role of PACAP and VIP in astroglial functions

Astrocytes represent at least 50% of the volume of the human brain. Besides their roles in various supportive functions, astrocytes are involved in the regulation of stem cell proliferation, synaptic plasticity and neuroprotection. Astrocytes also influence neuronal physiology by responding to neurotransmitters and neuropeptides and by releasing regulatory factors termed gliotransmitters. In particular, astrocytes express the PACAP-specific receptor PAC1-R and the PACAP/VIP mutual receptors VPAC1-R and VPAC2-R during development and/or in the adult. There is now clear evidence that PACAP and VIP modulate a number of astrocyte activities such as proliferation, plasticity, glycogen production, and biosynthesis of neurotrophic factors and gliotransmitters.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates endozepine release from cultured rat astrocytes via a PKA-dependent mechanism

Astroglial cells synthesize and release endozepines, neuropeptides that are related to the octadecaneuropeptide ODN. Glial cells also express PACAP/VIP receptors. We have investigated the possible effect of PACAP on the release of ODN‐like immunoreactivity (ODN‐LI) by cultured rat astrocytes. Administration of PACAP27 and PACAP38 induced a concentration‐dependent increase in secretion of ODN‐LI whereas VIP was ~1000‐fold less potent. The maximum effect of PACAP38 occurred after 5 min, then gradually declined during the next 10 min. The stimulatory effects of PACAP and VIP were abrogated by the PACAP antagonist PACAP6–38. PACAP38 stimulated cAMP formation, activated polyphosphoinositide turnover, and provoked calcium mobilization from IP3‐sensitive pools. The PKA inhibitor H89 suppressed PACAP‐induced secretion of ODN‐LI, whereas PLC inhibitor U73122 and the PKC inhibitor chelerythrine had no effect. In contrast, U73122 restored the stimulatory action of PACAP on ODN‐LI release and cAMP formation during prolonged (15 min) incubation with the peptide, and this effect was prevented by PMA. The present results demonstrate that PACAP stimulates endozepine release through activation of PAC1 receptors coupled to the AC/PKA pathway. Our data also show that activation of the PLC/PKC pathway down‐regulates the effect of PACAP on endozepine release.—Masmoudi, O., Gandolfo, P., Leprince, J., Vaudry, D., Fournier, A., Patte‐Mensah, C., Vaudry, H., Tonon, M.‐C. Pituitary adenylate cyclase‐activating polypeptide (PACAP) stimulates endozepine release from cultured rat astrocytes via a PKA‐dependent mechanism. FASEB J. 17, 17–27 (2003)

Biochemical and functional characterization of high-affinity urotensin II receptors in rat cortical astrocytes

The urotensin II (UII) gene is primarily expressed in the central nervous system, but the functions of UII in the brain remain elusive. Here, we show that cultured rat astrocytes constitutively express the UII receptor (UT). Saturation and competition experiments performed with iodinated rat UII ([125I]rUII) revealed the presence of high‐ and low‐affinity binding sites on astrocytes. Human UII (hUII) and the two highly active agonists hUII4‐11 and [3‐iodo‐Tyr9]hUII4‐11 were also very potent in displacing [125I]rUII from its binding sites, whereas the non‐cyclic analogue [Ser5,10]hUII4‐11 and somatostatin‐14 could only displace [125I]rUII binding at micromolar concentrations. Reciprocally, rUII failed to compete with [125I‐Tyr0,D‐Trp8]somatostatin‐14 binding on astrocytes. Exposure of cultured astrocytes to rUII stimulated [3H]inositol incorporation and increased intracellular Ca2+ concentration in a dose‐dependent manner. The stimulatory effect of rUII on polyphosphoinositide turnover was abolished by the phospholipase C inhibitor U73122, but only reduced by 56% by pertussis toxin. The GTP analogue Gpp(NH)p caused its own biphasic displacement of [125I]rUII binding and provoked an affinity shift of the competition curve of rUII. Pertussis toxin shifted the competition curve towards a single lower affinity state. Taken together, these data demonstrate that rat astrocytes express high‐ and low‐affinity UII binding sites coupled to G proteins, the high‐affinity receptor exhibiting the same pharmacological and functional characteristics as UT.

The endozepine ODN stimulates polyphosphoinositide metabolism in rat astrocytes

Astrocytes synthesize a series of peptides called endozepines which act as endogenous ligands of benzodiazepine receptors. The present study demonstrates that the endozepine ODN causes a dose‐dependent increase in inositol trisphosphate and a parallel decrease in phosphatidylinositol bisphosphate in cultured rat astrocytes. Pre‐incubation of astrocytes with the phospholipase C inhibitor U 73122 or with pertussis toxin totally blocked polyphosphoinositide metabolism. These data show that, in rat astrocytes, ODN stimulates a phospholipase C coupled to a pertussis toxin‐sensitive G protein.

The vasoactive peptides urotensin II and urotensin II-related peptide regulate astrocyte activity through common and distinct mechanisms: Involvement in cell proliferation

UII (urotensin II) and its paralogue URP (UII-related peptide) are two vasoactive neuropeptides whose respective central actions are currently unknown. In the present study, we have compared the mechanism of action of URP and UII on cultured astrocytes. Competition experiments performed with [125I]UII showed the presence of very-high- and high-affinity binding sites for UII, and a single high-affinity site for URP. Both UII and URP provoked a membrane depolarization accompanied by a decrease in input resistance, stimulated the release of endozepines, neuropeptides specifically produced by astroglial cells, and generated an increase in [Ca2+]c (cytosolic Ca2+ concentration). The UII/URP-induced [Ca2+]c elevation was PTX (pertussis toxin)-insensitive, and was blocked by the PLC (phospholipase C) inhibitor U73122 or the InsP3 channel blocker 2-APB (2-aminoethoxydiphenylborane). The addition of the Ca2+ chelator EGTA reduced the peak and abolished the plateau phase, whereas the T-type Ca2+ channel blocker mibefradil totally inhibited the Ca2+ response evoked by both peptides. However, URP and UII induced a mono- and bi-phasic dose-dependent increase in [Ca2+]c and provoked short- and long-lasting Ca2+ mobilization respectively. Similar mono- and bi-phasic dose-dependent increases in [3H]inositol incorporation into polyphosphoinositides in astrocytes was obtained, but the effect of UII was significantly reduced by PTX, although BRET (bioluminescence resonance energy transfer) experiments revealed that both UII and URP recruited Galphao-protein. Finally, UII, but not URP, exerted a dose-dependent mitogenic activity on astrocytes. Therefore we described that URP and UII exert not only similar, but also divergent actions on astrocyte activity, with UII exhibiting a broader range of activities at physiological peptide concentrations.

GABA inhibits endozepine release from cultured rat astrocytes.

In the mammalian brain, the endogenous ligands for benzodiazepine receptors (also called endozepines) are predominantly synthesized by glial cells. It has recently been reported that rat astrocytes in primary culture release substantial amounts of endozepines. The aim of the present study was to investigate the possible involvement of GABA in the control of endozepine release. Exposure of cultured rat astrocytes to GABA (10−7 to 10−5 M) induced a dose‐related inhibition of endozepine secretion. At higher doses (3 × 10−5 to 10−3 M), the effect of GABA gradually diminished. The inhibitory effect of GABA (10−5 M) was mimicked by the GABAB receptor agonist baclofen (10−5 M). In contrast, the GABAA receptor agonists 3APS and isoguvacine (10−5 M each) did not modify endozepine release. The inhibition of endozepine secretion evoked by GABA and baclofen (10−5 M each) was totally abrogated by the specific GABAB receptor antagonist phaclofen (10−4 M). GABA and baclofen caused a significant inhibition of forskolin‐evoked production of cAMP in astrocytes and this effect was abolished in the presence of phaclofen. In contrast, isoguvacine had no effect on cAMP production. Exposure of astrocytes to dbcAMP induced a time‐ and dose‐dependent stimulation of endozepine release. These data indicate that GABA, acting through GABAB receptors negatively coupled to adenylyl cyclase, inhibits endozepine release from cultured rat astrocytes. The secretion of endozepines thus appears to be a valuable marker to monitor astrocyte activity. GLIA 25:404–411, 1999.

Somatostatin down-regulates the expression and release of endozepines from cultured rat astrocytes via distinct receptor subtypes

Endozepines, a family of regulatory peptides related to diazepam‐binding inhibitor (DBI), are synthesized and released by astroglial cells. Because rat astrocytes express various subtypes of somatostatin receptors (sst), we have investigated the effect of somatostatin on DBI mRNA level and endozepine secretion in rat astrocytes in secondary culture. Somatostatin reduced in a concentration‐dependent manner the level of DBI mRNA in cultured astrocytes. This inhibitory effect was mimicked by the selective sst4 receptor agonist L803‐087 but not by the selective sst1, sst2 and sst3 receptor agonists L779‐591, L779‐976 and L797‐778, respectively. Somatostatin was unable to further reduce DBI mRNA level in the presence of the MEK inhibitor U0126. Somatostatin and the sst1, sst2 and sst4 receptor agonists induced a concentration‐dependent inhibition of endozepine release. Somatostatin and the sst1, sst2 and sst4 receptor agonists also inhibited cAMP formation dose‐dependently. In addition, somatostatin reduced forskolin‐induced endozepine release. H89 mimicked the inhibitory effect of somatostatin on endozepine secretion. In contrast the PLC inhibitor U73122, the PKC activator PMA and the PKC inhibitor calphostin C had no effect on somatostatin‐induced inhibition of endozepine release. The present data demonstrate that somatostatin reduces DBI mRNA level mainly through activation of sst4 receptors negatively coupled to the MAPK pathway, and inhibits endozepine release through activation of sst1, sst2 and sst4 receptors negatively coupled to the adenylyl cyclase/PKA pathway.

The endozepine ODN stimulates [3H]thymidine incorporation in cultured rat astrocytes

High concentrations of diazepam-binding inhibitor (DBI) mRNA have been detected in astrocytoma, suggesting that DBI-derived peptides may play a role in glial cell proliferation. In the present study, we have investigated the effect of a processing product of DBI, the octadecaneuropeptide ODN, on DNA synthesis in cultured rat astrocytes. At very low concentrations (10(-14) to 10(-11) M), ODN caused a dose-dependent increase of [3H]thymidine incorporation. At higher doses (10(-10) to 10(-5) M), the effect of ODN gradually declined. The central-type benzodiazepine receptor antagonist flumazenil (10(-6) M) completely suppressed the stimulatory action of ODN whereas the peripheral-type benzodiazepine receptor ligand, PK11195 (10(-6) M) had no effect. The ODN-induced stimulation of [3H]thymidine incorporation was mimicked by methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). The GABAA receptor antagonist bicuculline (10(-4) M) suppressed the effect of both ODN and DMCM on DNA synthesis. Exposure of cultured astrocytes to the specific GABAA agonist 3APS (10(-10) to 10(-4) M) also induced a dose-related increase of [3H]thymidine incorporation. The present study indicates that ODN, acting through central-type benzodiazepine receptors associated with the GABAA receptor complex, stimulates DNA synthesis in rat glial cells. These data provide evidence for an autocrine role of endozepines in the control of glial cell proliferation.

Protective effect of the octadecaneuropeptide on hydrogen peroxide‐induced oxidative stress and cell death in cultured rat astrocytes

J. Neurochem. (2011) 118, 416–428.