Marie Christine Tonon

Assessment of trh as a potential msh release stimulating factor in xenopus-laevis

This study considers the possible involvement of the tripeptide TRH (thyrotropin releasing hormone) in the physiological regulation of melanophore stimulating hormone (MSH) secretion from the pars intermedia of the toad, Xenopus laevis. TRH was shown to stimulate release of MSH from superfused neurointermediate lobes obtained from white-background adapted animals, but had no effect on secretion from lobes of black-background adapted animals. Immunohistochemical analysis revealed a rich TRH-containing neuronal network terminating in the neural lobe of the Xenopus pituitary. Plasma levels of TRH, determined with a specific radioimmunoassay, proved to be extremely high and no significant difference in this level could be found between white- and black-adapted animals. Plasma TRH probably originates from the skin, and our results show that its concentration is within the effective concentration range established for this peptide in stimulating MSH release from the pars intermedia. Therefore, while both our superfusion and immunohistochemical results argue favourably for a function of TRH in the regulation of MSH secretion, we conclude that, in any regulatory role, it would likely have to function within the pars intermedia at concentrations exceeding the high plasma values. While TRH could be involved in short-term activation of the secretory process in white-background adapted animals or in animals undergoing the initial stages of black background adaptation, our results indicate that this peptide may have no function in the maintenance of secretion from the pars intermedia of animals fully adapted to black background.

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.

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.

Localization and characterization of diazepam-binding inhibitor (DBI)-like peptides in the brain and pituitary of the trout (Salmo gairdneri)

The distribution of diazepam-binding inhibitor (DBI)-like peptide(s) in the brain and pituitary of the trout was determined by the indirect immunofluorescence technique using an antiserum raised against synthetic rat octadecaneuropeptide (ODN). Numerous immunoreactive perikarya and processes were observed in the basal hypothalamus, within the pars lateralis of the nucleus lateralis tuberis. In the pituitary, ODN-immunoreactive processes were visualized in the neurohypophysial tract, projecting into the pars intermedia, and the pars distalis. Reverse-phase high-performance liquid chromatography combined to radioimmunoassay quantification was used to characterize the DBI-related material in tissue extracts. In both pituitary and hypothalamic extracts, the major immunoreactive form eluted with a retention time higher than that of rat ODN. In the hypothalamus, a minor peak co-eluting with the synthetic ODN standard was also resolved. The existence of peptides related to mammalian DBI in the hypothalamo-hypophysial complex of the trout suggests these neuropeptides may participate in the control of pituitary hormone release.

Distribution and characterization of endozepine-like immunoreactivity in the central nervous system of the frog Rana ridibunda

The localization of endozepine-like immunoreactivity in the brain of the frog Rana ridibunda was investigated by indirect immunofluorescence, using an antiserum against synthetic rat octadecaneuropeptide (ODN). A specific immunoreaction was detected in ependymal cells lining the ventricular system of the brain and in circumventricular organs. Numerous immunoreactive cells were found covering the walls of the lateral ventricles in the telencephalon, as well as in the diencephalic and mesencephalic ventricles. In the hypothalamus, both the preoptic nucleus and the infundibular region showed numerous immunopositive cells. Ependymal cells lining the rhomboencephalic fourth ventricle and the central canal of the spinal cord were also immunoreactive. The concentration of endozepine-like immunoreactivity was measured in various regions of the brain using a sensitive and specific radioimmunoassay for rat ODN. The highest levels of ODN-like immunoreactivity were found in the infundibulum, cerebellum and preoptic area. Reverse phase high performance liquid chromatography and radioimmunoassay quantification were used to characterize endozepines in the frog brain. The elution profiles of the different brain regions revealed four major immunoreactive peaks. The present results demonstrate the presence of peptides immunologically related to the endozepine family in the central nervous system of the frog. The localization of immunoreactive endozepines in ependymal cells suggests that these peptides play important neuromodulatory functions in the amphibian brain.

VIP and PACAP stimulate tsh release from the bullfrog pituitary

We have recently shown that corticotropin-releasing hormone (CRH) is a major thyrotropin (TSH)-releasing factor in amphibians, but we have also found that, besides CRH, other hypothalamic substances stimulate TSH secretion in frog. In order to characterize novel TSH secretagogues, we have investigated the effect of frog (Rana ridibunda) vasoactive intestinal polypeptide (VIP) (fVIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) (fPACAP38 and PACAP27) on TSH release from bullfrog (Rana catesbeiana) pituitary cells in primary culture. Incubation of pituitary cells for 24h with graded concentrations of fVIP, fPACAP38 and PACAP27 (10(-9) to 10(-6)M) induced a dose-dependent stimulation of TSH release with minimum effective doses of 10(-9)M for fVIP and 10(-8)M for fPACAP38 and PACAP27. The PAC1-R/VPAC2-R antagonist PACAP(6-38) (10(-7) and 10(-6)M) dose-dependently suppressed the stimulatory effects of fVIP and fPACAP38 (10(-7)M each). Likewise, this antagonist (10(-6) and 10(-5)M) dose-dependently attenuated the stimulatory effect of PACAP27 (10(-7)M). On the other hand, the VPAC1-R/VPAC2-R antagonist [d-pCl-Phe(6), Leu(17)]VIP (10(-6) and 10(-5)M) dose-dependently inhibited the stimulatory effect of fVIP (10(-9)M) and PACAP27 (10(-8)M), but did not affect the response to fPACAP38 (10(-8)M). These data indicate that, in amphibians, the activity of thyrotrophs can be regulated by VIP and PACAP acting likely through VPAC2-R and PAC1-R.

Beta-amyloid peptides stimulate endozepine biosynthesis in cultured rat astrocytes

Accumulation of β‐amyloid peptide (Aβ), which is a landmark of Alzheimers disease, may alter astrocyte functions before any visible symptoms of the disease occur. Here, we examined the effects of Aβ on biosynthesis and release of diazepam‐binding inhibitor (DBI), a polypeptide primarily expressed by astroglial cells in the CNS. Quantitative RT–PCR and specific radioimmunoassay demonstrated that aggregated Aβ25−35, at concentrations up to 10−4 m, induced a dose‐dependent increase in DBI mRNA expression and DBI‐related peptide release from cultured rat astrocytes. These effects were totally suppressed when aggregation of Aβ25−35 was prevented by Congo red. Measurement of the number of living cells revealed that Aβ25−35 induced a trophic rather than a toxic effect on astrocytes. Administration of cycloheximide blocked Aβ25−35‐induced increase of DBI gene expression and endozepine accumulation in astrocytes, indicating that protein synthesis is required for DBI gene expression. Altogether, the present data suggest that Aβ‐induced activation of endozepine biosynthesis and release may contribute to astrocyte proliferation associated with Alzheimers disease.

D2 Dopamine receptor subtype mediates the inhibitory effect of dopamine on TRH-induced prolactin release from the bullfrog pituitary.

Dopamine receptors in mammals are known to consist of two D1-like receptors (D1 and D5) and three D2-like receptors (D2, D3 and D4). The aim of this study was to determine the dopamine receptor subtype that mediates the inhibitory action of dopamine on the release of prolactin (PRL) from the amphibian pituitary. Distal lobes of the bullfrog (Rana catesbeiana) were perifused and the amount of PRL released in the effluent medium was measured by means of a homologous enzyme-immunoassay. TRH stimulated the release of PRL from perifused pituitaries. Dopamine suppressed TRH-induced elevation of PRL release. Quinpirole (a D2 receptor agonist) also suppressed the stimulatory effect of TRH on the release of PRL, whereas SKF-38393 (a D1 receptor agonist) exhibited no such an effect. The inhibitory action of dopamine on TRH-induced PRL release from the pituitary was nullified by the addition of L-741,626 (a selective D2 receptor antagonist) to the medium, but not by the addition of SCH-23390 (a selective D1 receptor antagonist). These data indicate that the inhibitory effect of dopamine on TRH-evoked PRL release from the bullfrog pituitary gland is mediated through D2 dopamine receptors.

Beta-amyloid peptide stimulates endozepine release in cultured rat astrocytes through activation of N-formyl peptide receptors

Astroglial cells synthesize and release endozepines, a family of neuropeptides derived from diazepam‐binding inhibitor (DBI). The authors have recently shown that β‐amyloid peptide (Aβ) stimulates DBI gene expression and endozepine release. The purpose of this study was to determine the mechanism of action of Aβ in cultured rat astrocytes. Aβ25–35 and the N‐formyl peptide receptor (FPR) agonist N‐formyl‐Met‐Leu‐Phe (fMLF) increased the secretion of endozepines in a dose‐dependent manner with EC50 value of ≈2 μM. The stimulatory effects of Aβ25–35 and the FPR agonists fMLF and N‐formyl‐Met‐Met‐Met (fMMM) on endozepine release were abrogated by the FPR antagonist N‐t‐Boc‐Phe‐Leu‐Phe‐Leu‐Phe. In contrast, Aβ25–35 increased DBI mRNA expression through a FPR‐independent mechanism. Aβ25–35 induced a transient stimulation of cAMP formation and a sustained activation of polyphosphoinositide turnover. The stimulatory effect of Aβ25–35 on endozepine release was blocked by the adenylyl cyclase inhibitor somatostatin, the protein kinase A (PKA) inhibitor H89, the phospholipase C inhibitor U73122, the protein kinase C (PKC) inhibitor chelerythrine and the ATP binding cassette transporter blocker glyburide. Taken together, these data demonstrate for the first time that Aβ25–35 stimulates endozepine release from rat astrocytes through a FPR receptor positively coupled to PKA and PKC.

Amphibian Melanotrope Subpopulations Respond Differentially to Hypothalamic Secreto-Inhibitors

The melanotrope population of the frog intermediate lobe consists of two subtypes of cells, referred to as high-(HD) and low-density (LD) melanotrope cells, which differ markedly in their basal morphofunctional features as well as their in vitro response to hypothalamic factors, such as the stimulator thyrotropin-releasing hormone (TRH) and the inhibitor dopamine. In this study, we have investigated whether other major hypothalamic regulators of the release of α-melanocyte-stimulating hormone (α-MSH), such as γ-aminobutyric acid (GABA) and neuropeptide Y (NPY), also differentially regulate frog melanotrope subpopulations. Our results show that in LD cells, both factors markedly inhibited proopiomelanocortin (POMC) mRNA accumulation and α-MSH secretion. In contrast, the secretory and biosynthetic activity of HD cells was not modified by GABA. NPY inhibited POMC transcript accumulation and tended to reduce α-MSH secretion in HD cells, yet these effects were less pronounced than those evoked in LD cells. In addition, GABA and NPY inhibited the KCl-induced rise in cytosolic free calcium levels in both subpopulations. Taken together, these results further indicate that frog melanotrope subpopulations are differentially regulated by the hypothalamus and strongly suggest that the intensity of such regulation is directly related to the activity of the cell subset. Thus, the LD subpopulation represents a highly responsive cell subset which is regulated by multiple neuroendocrine factors (TRH, dopamine, GABA and NPY), whereas the hormone storage HD subpopulation shows a moderate response to single stimulatory (TRH) and inhibitory (NPY) inputs.