Gilles Guillon

Inhibition of oxytocin receptor function by direct binding of progesterone

The steroid hormone progesterone (P4) is essential for establishing and maintaining pregnancy in mammals. One of its functions includes maintenance of uterine quiescence by decreasing uterine sensitivity to the uterotonic peptide hormone oxytocin. Although it is generally held that steroid hormones such as P4 act at a genomic level by binding to nuclear receptors and modulating the expression of specific target genes, we show here that the effect of P4 on uterine sensitivity to oxytocin involves direct, non-genomic action of P4 on the uterine oxytocin receptor (OTR), a member of the G-protein-coupled receptor family. P4 inhibits oxytocin binding to OTR-containing membranes in vitro, binds with high affinity to recombinant rat OTR expressed in CHO cells, and suppresses oxytocin-induced inositol phosphate production and calcium mobilization. These effects are highly steroid- and receptor-specific, because binding and signalling functions of the closely related human OTR are not affected by P4 itself but by the P4 metabolite 5β-dihydroprogesterone. Our findings provide the first evidence for a direct interaction between a steroid hormone and a G-protein-coupled receptor and define a new level of crosstalk between the peptide- and steroid-hormone signalling pathways.

Oxytocin and Vasopressin Agonists and Antagonists as Research Tools and Potential Therapeutics

We recently reviewed the status of peptide and nonpeptide agonists and antagonists for the V1a, V1b and V2 receptors for arginine vasopressin (AVP) and the oxytocin receptor for oxytocin (OT). In the present review, we update the status of peptides and nonpeptides as: (i) research tools and (ii) therapeutic agents. We also present our recent findings on the design of fluorescent ligands for V1b receptor localisation and for OT receptor dimerisation. We note the exciting discoveries regarding two novel naturally occurring analogues of OT. Recent reports of a selective VP V1a agonist and a selective OT agonist point to the continued therapeutic potential of peptides in this field. To date, only two nonpeptides, the V2/V1a antagonist, conivaptan and the V2 antagonist tolvaptan have received Food and Drug Administration approval for clinical use. The development of nonpeptide AVP V1a, V1b and V2 antagonists and OT agonists and antagonists has recently been abandoned by Merck, Sanofi and Pfizer. A promising OT antagonist, Retosiban, developed at Glaxo SmithKline is currently in a Phase II clinical trial for the prevention of premature labour. A number of the nonpeptide ligands that were not successful in clinical trials are proving to be valuable as research tools. Peptide agonists and antagonists continue to be very widely used as research tools in this field. In this regard, we present receptor data on some of the most widely used peptide and nonpeptide ligands, as a guide for their use, especially with regard to receptor selectivity and species differences.

Receptor‐oriented intercellular calcium waves evoked by vasopressin in rat hepatocytes

Agonist‐induced intracellular calcium signals may propagate as intercellular Ca2+ waves in multicellular systems as well as in intact organs. The mechanisms initiating intercellular Ca2+ waves in one cell and determining their direction are unknown. We investigated these mechanisms directly on fura2‐loaded multicellular systems of rat hepatocytes and on cell populations issued from peripheral (periportal) and central (perivenous) parts of the hepatic lobule. There was a gradient in vasopressin sensitivity along connected cells as demonstrated by low vasopressin concentration challenge. Interestingly, the intercellular sensitivity gradient was abolished either when D‐myo‐inositol 1,4,5‐trisphosphate (InsP3) receptor was directly stimulated after flash photolysis of caged InsP3 or when G proteins were directly stimulated with AlF4−. The gradient in vasopressin sensitivity in multiplets was correlated with a heterogeneity of vasopressin sensitivity in the hepatic lobule. There were more vasopressin‐binding sites, vasopressin‐induced InsP3 production and V1a vasopressin receptor mRNAs in perivenous than in periportal cells. Therefore, we propose that hormone receptor density determines the cellular sensitivity gradient from the peripheral to the central zones of the liver cell plate, thus the starting cell and the direction of intercellular Ca2+ waves, leading to directional activation of Ca2+‐dependent processes.

Activation of membrane phospholipase C by vasopressin: A requirement for guanyl nucleotides

Vasopressin stimulates the liberation of labelled inositol phosphate in partially purified plasma membranes prepared from myo‐[3H]inositol prelabelled WRK1 cells. This stimulatory effect was very rapid (165% stimulation of inositol trisphosphate accumulation after a 10 s incubation period in the presence of 1 μM vasopressin), concentration dependent (EC50= 12 nM) and was abolished by an antagonist of the vasopressor response to vasopressin. GTP, even at high concentrations (0.1 mM), did not increase inositol phosphate release: it was found to be absolutely necessary for hormonal stimulation of phospholipase C activity. Non‐hydrolysable analogues of GTP may also stimulate this enzyme activity.

Association of the G Proteinα q/α11-Subunit with Cytoskeleton in Adrenal Glomerulosa Cells: Role in Receptor-Effector Coupling1

In 3-day primary cultures of rat glomerulosa cells, a 30-min preincubation with either 10 μm colchicine (a microtubule-disrupting agent) or 10 μm cytochalasin B (a microfilament-disrupting agent) decreased angiotensin II (Ang II)-induced inositol phosphate accumulation by 50%. Moreover, both drugs decreased inositol phosphate production induced by fluoroaluminate (a nonspecific activator of all G proteins), indicating that both microtubules and microfilaments are essential for phospholipase C activation. Analysis of microfilament- and microtubule-enriched fractions and immunoprecipitation of actin and tubulin revealed that the αq/α11-subunit of the Gq/11 protein was associated with both structures. Ang II stimulation induced a rapid translocation ofα q/α11, microfilaments, and microtubules to the membrane and induced a time-dependent increase in the level ofα q/α11 associated with both microfilaments and microtubules. Moreover, double immunofluorescence staining clearly showed a colocalization of theα q/α...

Cyclic AMP-independent effects of ACTH on glomerulosa cells of the rat adrenal cortex.

The aim of the present paper is to point out the complexity of ACTH action in glomerulosa cells of the adrenal cortex. We demonstrate that the increase in cAMP production induced by ACTH is the result of a balance between activation of adenylyl cyclase and direct modulation of a PDE2 phosphodiestease activity, an effect mediated by inhibition of cGMP content. Moreover, Ca2+ is essential for cAMP production and aldosterone secretion, but its exact primary action is not clearly determined. We recently described that ACTH activated a chloride channel, via the Ras protein, which can be involved in steroidogenesis. ACTH also increases tyrosine phosphorylation of several proteins. These data, together with those of phospholipase C activation, indicate that ACTH action in the adrenal is complex, and most certainly not limited to cAMP production, in particular for the low concentrations of the hormone. Some years ago, cAMP was considered to be the unique second messenger of ACTH action; now it becomes more and more evident that ACTH triggers complex signaling pathways using several second messengers in a closely interacting way. The most predominant point is that these signals are observed for low concentrations of ACTH.

Activation of polyphosphoinositide phospholipase C by fluoride in WRK1 cell membranes

Partially purified plasma membranes prepared from myo‐[3H]inositol‐prelabeled WRK1 cells exhibit a phosphatidylinositol 4,5‐bisphosphate (PIP2) phospholipase C activity sensitive to NaF. NaF increased the production of IP2 and IP3 in a time‐ and concentration‐dependent manner. The maximal increase in IP2 and IP3 production rates represented 400 ± 18 and 360 ± 40% of the basal production rate, respectively. Half‐maximum stimulation was reached with 2–4 mM NaF. The observed effect was specific for F−. Aluminium potentiated fluoride‐induced IP3 and IP2, accumulation in a concentration‐dependent manner. The effect of fluoride on the PIP2 phospholipase C from WRK1 cell membranes appears to be similar to the well‐documented effect of F− on the well‐characterized Ns. Ni and transducin GTP‐binding proteins. This observation constitutes an additional argument to suggest that a GTP‐binding protein is involved in the process of receptor‐mediated activation of PIP2 phospholipase C.

Genomic and non-genomic mechanisms of oxytocin receptor regulation.

Our recent studies have shown that regulation of uterine oxytocin (OT) binding involves at least two different mechanism: Estradiol (E2)-induced upregulation is accompanied by an increase in OT receptor (OTR) mRNA accumulation, implying that the E2 effect is mediated via increased OTR gene transcription and/or OTR mRNA stabilization. In contrast, P (P)-induced OTR down-regulation occurs via a novel non-genomic mechanism, involving a direct interaction of P with the OTR at the level of the cell membrane. We found that P specifically binds to the OTR and inhibits its ligand binding and signalling functions. Physiological levels of P repress in vitro the ligand binding capacity (Bmax) of the OTR by > 50%. When expressed in CHO cells, the OTR provides a high affinity (Kd: 20nM) membrane binding site for P. OT-induced inositol phosphate production and intracellular calcium mobilization is inhibited 85% and 90%, respectively, by P. These effects are specific as signalling and binding functions of the closely related V1a vasopressin receptor remain unaffected by P, and as other, related steroids are devoid of any effect on OTR binding or signalling functions. The present observation of a specific interaction of a steroid with a G-protein-linked receptor defines a new mechanism of non-genomic steroid action and uncovers a novel level of crosstalk between steroid and peptide hormone action.

Comparative Involvement of Cyclic Nucleotide Phosphodiesterases and Adenylyl Cyclase on Adrenocorticotropin-Induced Increase of Cyclic Adenosine Monophosphate in Rat and Human Glomerulosa Cells1

The present study investigated the role and identity of cyclic nucleotide phosphodiesterases (PDEs) in the regulation of basal and ACTH-stimulated levels of intracellular cAMP in human and rat adrenal glomerulosa cells. Comparative dose-response curves indicated that maximal hormone-stimulated cAMP accumulation was 11- and 24-fold higher in human and rat cells, compared with cAMP production obtained in corresponding membranes, respectively. Similarly to 3-isobutyl-1-methyl-xanthine, 25 microM erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA, a specific PDE2 inhibitor), caused a large increase in ACTH-stimulated cAMP accumulation; by contrast, it did not change cAMP production in membranes. Moreover, in membrane fractions, addition of 10 microM cGMP inhibited ACTH-induced cAMP production, an effect completely reversed by addition of 25 microM EHNA. These results indicate that PDE2 activity is involved in the regulation of cAMP accumulation induced by ACTH, and suggest that ACTH inhibits this activity. Indeed, time-course studies indicated that ACTH induced a rapid decrease in cGMP production, resulting in PDE2 inhibition, which in turn, contributed [with adenylyl cyclase (AC) activation] to an accumulation in cAMP for 15 min. Thereafter, cAMP content decreased, because of cAMP-stimulated PDE2, as confirmed by measurement of PDE activity that was activated by ACTH, but only after a 10-min incubation. Hence, we demonstrate that the ACTH-induced increase in intracellular cAMP is the result of a balance between activation of AC and direct modulation of PDE2 activity, an effect mediated by cGMP content. Although similar results were observed in both models, PDE2 involvement is more important in rat than in human adrenal glomerulosa cells, whereas AC is more stimulated in human than in rat glomerulosa cells.

The Discovery of Novel Vasopressin V1b Receptor Ligands for Pharmacological, Functional and Structural Investigations

Until recently, pharmacological studies dealing with vasopressin receptor isoforms were severely hampered by the lack of selective agonists or antagonists that recognize the pituitary V1b vasopressin receptor. By contrast, many selective vasopressin‐related compounds are available for characterization of the vasopressor (V1a) or antidiuretic (V2) vasopressin receptor subtypes. Recently, SSR149415, a selective nonpeptide molecule, was discovered with nanomolar affinity for mammalian V1b receptors and good selectivity for the other vasopressin and oxytocin receptor isoforms. This molecule exhibits potent antagonist properties both in vitro and in vivo. We also designed synthetic peptides derived from [deaminocysteine1,arginine8]vasopressin (dAVP), modified in position 4 by various amino acid residues. Some of these, d[cyclohexylalanine4]AVP or d[lysine4]AVP, have a high affinity and an excellent selectivity for the human V1b receptor subtype. However, they exhibit a mixed V1b/V2 pharmacological profile for the rat vasopressin receptor isoforms. Whatever the species considered, these peptides behave as agonists both in bioassays performed in vitro and in vivo. The d[cyclohexylalanine4]AVP was tritiated and represents the first selective radiolabelled ligand available for studying the human V1b receptors. The discovery of these new selective V1b agonists and V1b antagonist allows an accurate pharmacological characterization of all the vasopressin receptor isoforms. As emphasized in this review, attention to the vasopressin and oxytocin receptor species differences is of critical importance in studies with all vasopressin and oxytocin ligands.