Vasopressin inhibits LTP in the CA2 mouse hippocampal area.

Growing evidence points to vasopressin (AVP) as a social behavior regulator modulating various memory processes and involved in pathologies such as mood disorders, anxiety and depression. Accordingly, AVP antagonists are actually envisaged as putative treatments. However, the underlying mechanisms are poorly characterized, in particular the influence of AVP on cellular or synaptic activities in limbic brain areas involved in social behavior. In the present study, we investigated AVP action on the synapse between the entorhinal cortex and CA2 hippocampal pyramidal neurons, by using both field potential and whole-cell recordings in mice brain acute slices. Short application (1 min) of AVP transiently reduced the synaptic response, only following induction of long-term potentiation (LTP) by high frequency stimulation (HFS) of afferent fibers. The basal synaptic response, measured in the absence of HFS, was not affected. The Schaffer collateral-CA1 synapse was not affected by AVP, even after LTP, while the Schaffer collateral-CA2 synapse was inhibited. Although investigated only recently, this CA2 hippocampal area appears to have a distinctive circuitry and a peculiar role in controlling episodic memory. Accordingly, AVP action on LTP-increased synaptic responses in this limbic structure may contribute to the role of this neuropeptide in controlling memory and social behavior

Vasopressin decreases EPSPs in CA2 after LTP induction.

<p>A: Top: typical recordings of the combined EPSP/IPSP evoked by stimulation of LIII entorhinal fibers (EC LIII) and recorded in CA2 pyramidal neurons (inset). Traces are averages of 12 successive responses recorded during 1 min, before high frequency stimulation (HFS) and during LTP before, during and after the response to AVP (10 nM ; 1 min). Bottom graph: typical example showing that the amplitude of the EPSP increased transiently (short-term facilitation) after HFS and then progressively to reach a plateau (long-term potentiation: LTP). Vasopressin (AVP 10 nM; 1 min) applied during LTP transiently decreased the EPSP amplitude. B) Average graph of EPSP amplitude <i>vs.</i> time (n = 6; * <i>p</i><0.05) demonstrating that AVP inhibited the CA2 EPSP evoked by EC LIII stimulation during LTP. Values are expressed as % of the mean of the responses recorded during 5 min before AVP application. C) Average graph (n = 6) showing that the IPSP component of the CA2 response to EC LIII stimulation was not affected by AVP. D) Typical graph (Left) and average graph (Right, n = 10) of EPSP amplitude <i>vs.</i> time showing that AVP did not affect the basal EC LIII-CA2 EPSP recorded before HFS stimulation. E) Left, Typical graph of the fEPSP evoked by Schaffer collaterals stimulation and recorded in a CA1 pyramidal neuron (SC-CA1); AVP did not affect the amplitude of the LTP-potentiated EPSP , as shown on the average graph (Right, n = 4).</p

Characterization of a Functional V1B Vasopressin Receptor in the Male Rat Kidney: Evidence for Cross Talk Between V1B and V2 Receptor Signaling Pathways

Although vasopressin V-1B receptor (V1BR) mRNA has been detected in the kidney, the precise renal localization as well as pharmacological and physiological properties of this receptor remain unknown. Using the selective V-1B agonist d[Leu(4), Lys(8)]VP, either fluorescent or radioactive, we showed that V1BR is mainly present in principal cells of the inner medullary collecting duct (IMCD) in the male rat kidney. Protein and mRNA expression of V1BR were very low compared with the V2 receptor (V2R). On the microdissected IMCD, d[Leu(4), Lys(8)]VP had no effect on cAMP production but induced a dose-dependent and saturable intracellular Ca2+ concentration increase mobilization with an EC50 value in the nanomolar range. This effect involved both intracellular Ca2+ mobilization and extracellular Ca2+ influx. The selective V1B antagonist SSR149415 strongly reduced the ability of vasopressin to increase intracellular Ca2+ concentration but also cAMP, suggesting a cooperation between V1BR and V2R in IMCD cells expressing both receptors. This cooperation arises from a cross talk between second messenger cascade involving PKC rather than receptor heterodimerization, as supported by potentiation of arginine vasopressin-stimulated cAMP production in human embryonic kidney-293 cells coexpressing the two receptor isoforms and negative results obtained by bioluminescence resonance energy transfer experiments. In vivo, only acute administration of high doses of V1B agonist triggered significant diuretic effects, in contrast with injection of selective V2 agonist. This study brings new data on the localization and signaling pathways of V1BR in the kidney, highlights a cross talk between V1BR and V2R in the IMCD, and suggests that V1BR may counterbalance in some pathophysiological conditions the antidiuretic effect triggered by V2R activation. New & Noteworthy: Although V1BR mRNA has been detected in the kidney, the precise renal localization as well as pharmacological and physiological properties of this receptor remain unknown. Using original pharmaceutical tools, this study brings new data on the localization and signaling pathways of V1BR, highlights a cross talk between V1BR and V-2 receptor (V2R) in the inner medullary collecting duct, and suggests that V1BR may counterbalance in some pathophysiological conditions the antidiuretic effect triggered by V2R activation

Recorded neurons are localized in the CA2 area and display a typical dendritic morphology.

<p>Upper left: Bright field image of the hippocampal slice as seen during the experiment. Note that the CA2 is well delimited between the large pyramidal layer and mossy fiber tract of the CA3 area on the right and the thin pyramidal layer of the CA1 area on the left. Upper right: Fluorescence image (×5) of a neuron filled with Alexa-594-cadaverine (Red) during whole-cell recording and typically located in the CA2 area. Blue: Hoechst labeling of cell nuclei. Lower left: Higher magnification (×10) confirms that the injected neuron in located in the characteristic dilatation of the pyramidal layer in the CA2 area and displays the typical morphological characteristics of a pyramidal CA2 neuron, with few dendritic branches in stratum oriens (SO) and a dense branching in stratum lacunosum molecular (SLM). Lower right: Merged high magnification (×40) fluorescence image showing in red the Alexa-594-cadaverine filling the injected neuron and in green the immunolabeling of α-actinin2, a protein enriched in CA2 neurons. Note the co-localization of cadaverin and α-actinin, demonstrating that the recorded neuron was located in the CA2 area. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; SLM, stratum lacunosum moleculare.</p

Vasopressin decreased the population fEPSP evoked by stimulation of LIII entorhinal fibers (EC LIII) and recorded in the CA2 dendritic field.

<p>A) Left : Typical recordings (left, 12 traces averaged over 1 min) of fEPSPs evoked by two successive stimulations before HFS (1) and during LTP before (2), during (3) and after (4) the response to AVP (100 nM ; 1 min). Note that the response to the second stimulation (S2) is larger than the first one (S1), indicative of a paired-pulse facilitation. Right, corresponding graph of the fEPSP amplitude <i>vs.</i> time. HFS induced a short- and then a long-term potentiation. Values correspond to S1. AVP (1 min) transiently decreased the LTP-potentiated fEPSP. B) Average graphs (n = 8; values expressed as % of the mean of the responses recorded during 5 min before AVP application) demonstrating that AVP (100 nM; 1 min) transiently and significantly decreased the amplitude of both S1 and S2 fEPSPs without affecting the paired-pulse facilitation (calculated as the ratio S2/S1). C) Left: fEPSP recorded in the presence of the GABA_A antagonist SR 95531. AVP was effective to decrease the fEPSP during LTP (n = 14) but failed to affect the fEPSP in slices in which no LTP was evoked (Middle; n = 7). Right, Absence of effect of AVP on the fiber volley (FV) amplitude (n = 5), demonstrating that AVP did not reduce the excitability of the EC LIII fibers. D) fEPSP recorded in the CA2 dendritic area during stimulation of SC fibers. Left, Typical recording in a slice in which HFS induced a LTP and AVP reduced the potentiated fEPSP. Right, Average graph (n = 7) demonstrating that AVP (100 nM; 1 min) transiently and significantly decreased the amplitude of the SC-CA2 fEPSPs.</p

Agonist-selective Dynamic Compartmentalization of Human Mu Opioid Receptor as Revealed by Resolutive FRAP Analysis*

Techniques for analyzing the membrane diffusion of molecules are the most promising methods for investigating the compartmentalization of G-protein-coupled receptors, particularly as relevant to receptor signaling processes. Here, we report fluorescence recovery after photobleaching (FRAP) measurements performed at variable spot radius for human mu opioid (hMOP) receptors on SH-SY5Y neuroblastoma cells in the presence of ligands. Although an antagonist did not affect the behavior of the receptors compared with the basal state, two different agonists, DAMGO and morphine, caused markedly different changes to receptor diffusion. Like receptors in the absence of ligand, receptors bound to morphine exhibited diffusion confined to joined semipermeable domains, but with smaller domain size and diffusion coefficient. This effect was inhibited by pertussis toxin, strongly suggesting that this dynamic behavior is associated with early steps of signaling. In the presence of DAMGO, half of the receptors displayed free long-range diffusion and the other half were confined to smaller isolated domains. Hypertonic sucrose buffer suppressed this effect, which we attribute to receptor entry into clathrin-coated pits. It is likely that the observation of distinct receptor dynamics in the presence of DAMGO and morphine involves the agonist-selective phosphorylation of the receptor

Terlipressin, a vasoactive prodrug recommended in hepatorenal syndrome, is an agonist of human V1, V2 and V1B receptors: Implications for its safety profile

International audienceTerlipressin is recommended as a gold standard to treat hepatorenal syndrome complicating liver cirrhosis. It is presented as a specific V1A receptor agonist, beyond its enzymatic conversion into lysine8-Vasopressin (LVP), able to counteract the splanchnic vasodilation. However, the complete pharmacological characterization of this drug with respect to the different vasopressin receptor subtypes is missing. We studied terlipressin intrinsic properties, focusing not only on V1A, but also on other vasopressin receptor subtypes. The experimental studies were conducted on rat and human cellular models. Binding experiments were performed on rat liver membranes and CHO cells transfected with the different human vasopressin receptor subtypes. Agonist status was assessed from inositol phosphate or cyclic AMP assays, and measurement of intracellular calcium variations, performed on cultured vascular smooth muscle cells from rat aorta and human uterine artery and CHO cells. Terlipressin binds to the rat and human V1A receptors with an affinity in the micromolar range, a value 120 fold lower than that of LVP. It induces a rapid and transient intracellular calcium increase, a robust stimulation of phospholipase C but with reduced maximal efficiencies as compared to LVP, indicating a partial V1A agonist property. In addition, terlipressin is also a full agonist of human V2 and V1B receptors, with also a micromomolar affinity

Bioactivity studies on atypical natural opioid hexapeptides processed from proenkephalin (PENK) precursor polypeptides

International audienceEndogenous opioids are derived from four related polypeptide precursors: proenkephalin (PENK), prodynorphin (PDYN), pronociceptin (PNOC) and proopiomelanocortin (POMC). In mammals PENK encodes for four copy of Met-enkephalin, one octapeptide Met-enkephalin-Arg-Gly-Leu, one heptapeptide Met-enkephalin-Arg-Phe and a single copy of Leu-enkephalin. Our detailed bioinformatic search on the existing PENK sequences revealed several atypical hexapeptide Met-enkephalins in different vertebrate animals. They are located either in the second enkephalin unit or in the seventh enkephalin core position at the C-terminus. Altogether four different hexapeptide sequences were obtained representing eleven animal species: Met-enkephalin-Arg(6) (YGGFMR) in the bird zebra finch, Met-enkephalin-Asp(6) (YGGFMD), Met-enkephalin-Ile(6) (YGGFMI) in zebrafish; and Met-enkephalin-Ser(6) (YGGFMS) in two pufferfish species. All novel peptides were chemically synthesized and studied in receptor binding and G-protein activation assays performed on rat brain membranes. The four novel enkephalins were equipotent in stimulating G-proteins. Affinities of the peptides determined by equilibrium competition assays in receptor binding experiments were statistically different. At the MOP receptors the highest affinity (Ki 4nM) was obtained with the zebra finch peptide Met-enkephalin-Arg(6). The pufferfish Met-enkephalin-Ser(6) exhibited the highest affinity (Ki 6.7nM) at the DOP receptor. Phylogenetic neuropeptide libraries, defined here as a collection of mutationally different species variants of orthologous and paralogous peptide sequences, represent the natural molecular diversity of the neuropeptides. Such libraries can provide a wide range of structural information establishing comparative functional analyses. Since DNA sequencing data are rapidly increasing, more development in the natural peptide library concept is expected

Long-Term Morphine Treatment Enhances Proteasome-Dependent Degradation of Gβ in Human Neuroblastoma SH-SY5Y Cells: Correlation with Onset of Adenylate Cyclase Sensitization

International audienceThe initial aim of this study was to identify protein changes associated with long-term morphine treatment in a recombinant human neuroblastoma SH-SY5Y clone (sc2) stably overexpressing the human mu-opioid (MOP) receptor. In MOP receptor-overexpressing sc2 cells, short-term morphine exposure was found to be much more potent and efficacious in inhibiting forskolin-elicited production of cAMP, and long-term morphine exposure was shown to induce a substantially higher degree of opiate dependence, as reflected by adenylate cyclase sensitization, than it did in wild-type neuroblastoma cells. Differential proteomic analysis of detergent-resistant membrane rafts isolated from untreated and chronically morphine-treated sc2 cells revealed long-term morphine exposure to have reliably induced a 30 to 40% decrease in the abundance of five proteins, subsequently identified by mass spectrometry as G protein subunits alphai(2), alphai(3), beta(1), and beta(2), and prohibitin. Quantitative Western blot analyses of whole-cell extracts showed that long-term morphine treatment-induced down-regulation of Gbeta but not of the other proteins is highly correlated (r(2) = 0.96) with sensitization of adenylate cyclase. Down-regulation of Gbeta and adenylate cyclase sensitization elicited by long-term morphine treatment were suppressed in the presence of carbobenzoxy-l-leucyl-l-leucyl-l-norvalinal (MG-115) or lactacystin. Thus, sustained activation of the MOP receptor by morphine in sc2 cells seems to promote proteasomal degradation of Gbeta to sensitize adenylate cyclase. Together, our data suggest that the long-term administration of opiates may elicit dependence by altering the neuronal balance of heterotrimeric G proteins and adenylate cyclases, with the ubiquitin-proteasome pathway playing a pivotal role