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G Guillon

Publications and source records attributed to G Guillon.

At least 19 recordsLinked to original sources
PubMed2004

Key amino acids located within the transmembrane domains 5 and 7 account for the pharmacological specificity of the human V1b vasopressin receptor.

In mammals, the vasopressin V(1b) receptor (V(1b)-R) is known to regulate ACTH secretion and, more recently, stress and anxiety. The characterization of the molecular determinant responsible for its pharmacological selectivity was made possible by the recent discovery of the first V(1b) antagonist, SSR149415. Based upon the structure of the crystallized bovine rhodopsin, we established a three-dimensional molecular model of interaction between the human V(1b)-R (hV(1b)-R) and SSR149415. Four amino acids located in distinct transmembrane helices (fourth, fifth, and seventh) were found potentially responsible for the hV(1b)-R selectivity. To validate these assumptions, we selectively replaced the leucine 181, methionine 220, alanine 334, and serine 338 residues of hV(1a)-R by their corresponding amino acids present in the hV(1b)-R (phenylalanine 164, threonine 203, methionine 324, and asparagine 328, respectively). Four mutants, which all exhibited nanomolar affinities for vasopressin and good coupling to phospholipase C pathway, were generated. hV(1a) receptors mutated at position 220 and 334 exhibited striking increase in affinity for SSR149415 both in binding and phospholipase C assays at variance with the hV(1a)-R modified at position 181 or 338. In conclusion, this study provides the first structural features concerning the hV(1b)-R and highlights the role of few specific residues in its pharmacological selectivity.

Amino Acid Sequence↗
PubMed2004

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 V(1b) vasopressin receptor. By contrast, many selective vasopressin-related compounds are available for characterization of the vasopressor (V(1a)) or antidiuretic (V(2)) vasopressin receptor subtypes. Recently, SSR149415, a selective nonpeptide molecule, was discovered with nanomolar affinity for mammalian V(1b) 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 [deaminocysteine(1),arginine(8)]vasopressin (dAVP), modified in position 4 by various amino acid residues. Some of these, d[cyclohexylalanine(4)]AVP or d[lysine(4)]AVP, have a high affinity and an excellent selectivity for the human V(1b) receptor subtype. However, they exhibit a mixed V(1b)/V(2) 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[cyclohexylalanine(4)]AVP was tritiated and represents the first selective radiolabelled ligand available for studying the human V(1b) receptors. The discovery of these new selective V(1b) agonists and V(1b) 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.

Animals↗
PubMed2002

Haemodynamic effects of an angiotensin-converting enzyme inhibitor and angiotensin receptor antagonist during hypovolaemia in the anaesthetized pig.

BACKGROUND: Renin-angiotensin system antagonists, either angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor (AT(1)) antagonists, may interfere with regulation of arterial pressure during anaesthesia. This study aimed to compare the haemodynamic profile of anaesthetized pigs, which were subjected to haemorrhage in the presence of the ACE inhibitor enalaprilat or the AT(1) antagonist valsartan. METHODS: Thirty-six pigs were assigned randomly to placebo, enalaprilat or valsartan groups. After a 30-min period of stabilization following anaesthesia and injection of the study drug, the animals were bled in two equal steps of 20% of their estimated blood volume (20% BV and 40% BV). RESULTS: After bleeding of 20% BV, the mean arterial pressure (MAP) decreased significantly but similarly in each group (20-25%) but the placebo and the enalaprilat groups had a significant decrease in cardiac index (CI, 22% and 16%, respectively) without significant change in systemic vascular resistance (SVR). Conversely, in the valsartan group, SVR decreased significantly (23%, P<0.02 vs other groups) without significant change in CI (-4%). After bleeding of 40% BV, the CI decreased significantly compared with 20% BV in the three groups (19% in the placebo and enalaprilat groups, 14% in the valsartan group) but the MAP decreased significantly in the enalaprilat group only (23%). The SVR increased significantly in the placebo group (P<0.01 vs each of the other groups), but there were no differences in the change in SVR between the other groups. CONCLUSION: Blockade of the renin-angiotensin system by either enalaprilat or valsartan leads to a similar decrease in arterial pressure during anaesthesia and haemorrhage but the haemodynamic profiles are quite different.

Angiotensin Receptor Antagonists↗
PubMed2002

[1-deamino-4-cyclohexylalanine] arginine vasopressin: a potent and specific agonist for vasopressin V1b receptors.

To date, there are no vasopressin (VP) agonists that exhibit a high affinity and selectivity for the VP V1b receptor with respect to the V1a, V2, and oxytocin receptors. In this study, we describe the synthesis and pharmacological properties of [1-deamino-4-cyclohexylalanine] arginine vasopressin (d[Cha4]AVP). Binding experiments performed on various membrane preparations revealed that d[Cha(4)]AVP exhibits a nanomolar affinity for V1b receptors from various mammalian species (rat, bovine, human). It exhibits high V1b/V1a and V1b/oxytocin selectivity for rat, human, and bovine receptors. Furthermore, it exhibits high V1b/V2 specificity for both bovine and human vasopressin receptors. Functional studies performed on biological models that naturally express V1b receptors indicate that d[Cha4]AVP is an agonist. Like VP, it stimulated basal and corticotropin-releasing factor-stimulated ACTH secretion and basal catecholamine release from rat anterior pituitary and bovine chromaffin cells, respectively. In vivo experiments performed in rat revealed that d[Cha4]AVP was able to stimulate both ACTH and corticosterone secretion and exhibits negligible vasopressor activity. It retains about 30% of the antidiuretic activity of VP. This long-sought selective VP V1b receptor ligand with nanomolar affinity will allow a better understanding of V1b-mediated VP physiological effects and is a promising new tool for V1b receptor structure-function studies.

Adrenocorticotropic Hormone↗
PubMed2002

Evidence for a direct negative coupling between dopamine-D2 receptors and PLC by heterotrimeric Gi1/2 proteins in rat anterior pituitary cell membranes.

Dopamine (DA) is known to inhibit basal and hormone TRH- or angiotensin II (AngII)-stimulated PRL secretion and inositol phosphate accumulation in rat pituitary cells in primary culture. This inhibition persists when cells are incubated in a calcium-free medium (a condition in which DA could not inhibit PLC activities by blocking calcium influx) and is abolished by a Pertussis toxin treatment. These data suggest that DA receptor could be negatively coupled to PLC by a direct mechanism involving a Pertussis toxin-sensitive G protein. To demonstrate this hypothesis, we measured PLC activities on crude plasma membranes obtained from rat pituitary cells in primary culture grown in the presence of tritiated myo-inositol. We showed that 1) DA and quinpirole or RU24926 (specific D2 agonists) inhibited both basal and TRH- or AngII-stimulated membrane PLC activities. 2) Such inhibitions were completely prevented by sulpiride (specific D2 antagonist). 3) Heterotrimeric Gi1/2 proteins coupled the DA receptors to PLC because DA inhibitions were completely reversed by preincubation either with Pertussis toxin or with a specific G(alpha)i1/(alpha)i2 antibody. Such data are in favor of the existence of a direct negative coupling between DA-D2 receptor and PLC on a native physiological plasma membrane model.

Angiotensin II↗
PubMed2001

Characterization of a G protein-activated phosphoinositide 3-kinase in vascular smooth muscle cell nuclei.

Recent studies highlight the existence of an autonomous nuclear polyphosphoinositide metabolism related to cellular proliferation and differentiation. However, only few data document the nuclear production of the putative second messengers, the 3-phosphorylated phosphoinositides, by the phosphoinositide 3-kinase (PI3K). In the present paper, we examine whether GTP-binding proteins can directly modulate 3-phosphorylated phosphoinositide metabolism in membrane-free nuclei isolated from pig aorta smooth muscle cells (VSMCs). In vitro PI3K assays performed without the addition of any exogenous substrates revealed that guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) specifically stimulated the nuclear synthesis of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), whereas guanosine 5'-(beta-thio)diphosphate was ineffective. PI3K inhibitors wortmannin and LY294002 prevented GTPgammaS-induced PtdIns(3,4,5)P(3) synthesis. Moreover, pertussis toxin inhibited partially PtdIns(3,4,5)P(3) accumulation, suggesting that nuclear G(i)/G(0) proteins are involved in the activation of PI3K. Immunoblot experiments showed the presence of Galpha(0) proteins in VSMC nuclei. In contrast with previous reports, immunoblots and indirect immunofluorescence failed to detect the p85alpha subunit of the heterodimeric PI3K within VSMC nuclei. By contrast, we have detected the presence of a 117-kDa protein immunologically related to the PI3Kgamma. These results indicate the existence of a G protein-activated PI3K inside VSMC nucleus that might be involved in the control of VSMC proliferation and in the pathogenesis of vascular proliferative disorders.

Cell Nucleus↗
PubMed2001

ATP induces intracellular calcium increases and actin cytoskeleton disaggregation via P2x receptors.

The consequences of purinoceptor activation on calcium signalling, inositol phosphate metabolism, protein secretion and the actin cytoskeleton were demonstrated in the WRK-1 cell line. Extracellular ATP was used as a secretagogue to induce a rise in intracellular Ca(2+) concentration ([Ca(2+)](i)), acting via P2x purinergic receptors, which causes actin skeleton disaggregation and protein secretion. ATP bound specifically to purinergic receptors, with Ki of 0.8 microM. The magnitude order for binding of different nucleotides was alpha beta-Met-ATP >or= dATPalphaS > ATP >or= ADP > UTP > AMP > suramin. No increase in inositol phosphates (IPs) was observed after ATP application suggesting that the purinergic sites in WRK-1 cells are not of a P2y type. ATP (1-100 microM) caused a concentration-dependent increase in [Ca(2+)](i)(EC(50)= 30 microM). The responses were reproducible without any desensitization over several applications. The response to ATP was abolished when extracellular calcium ([Ca(2+)](e)) was reduced to 100 nM. A non-specific purinergic antagonist, suramin, reversibly inhibited the ATP-response suggesting that ATP is able to bind to P2x purinergic sites to trigger Ca(2+) entry and increase of [Ca(2+)](i). ATP induced a concentration-dependent disaggregation of actin and exocytotic release of proteins both, which were dependent upon [Ca(2+)](e). Similarly, alpha,beta-Met-ATP, a potent P2x agonist also stimulated Ca(2+) mobilization, actin network destructuration, and protein release. In the isolated rat neurohypophysial nerve terminals, ATP was shown to act as a physiological stimulus for vasopressin release via Ca(2+) entry through a P2x receptor [6]. Here, we show that in these nerve terminals, ATP is also able to induce actin disaggregation by a Ca(2+) dependent mechanism. Thus, actin cytoskeleton alterations induced by ATP through activation of P2x receptors could be a prelude to exocytosis.

Actins↗
PubMed2001

Haemodynamic and renal effects of intravenous enalaprilat during coronary artery bypass graft surgery in patients with ischaemic heart dysfunction.

Renal dysfunction occurring after open heart surgery is multifactorial in origin but activation of the renin-angiotensin system may have a prominent role. Fourteen patients with ischaemic heart dysfunction scheduled for elective coronary artery bypass graft (CABG) surgery were allocated to a treatment group [enalaprilat for 2 days; ACEI (angiotensin-converting enzyme inhibitor) group, n=7] or a control group (n=7). The cardiac index was significantly higher in ACEI-treated patients than in the controls before and after cardiopulmonary bypass (CPB) (P<0.05) and on postoperative day 2 (P<0.05). The systemic vascular resistance was significantly lower in the ACEI-treated patients than in the controls before and after CPB (P<0.05). Renal plasma flow, measured as [131I]orthoiodohippuran clearance (ClH), was higher in the ACEI group than in the control group before CPB, as was endogenous creatinine clearance after CPB (P<0.05). On post-operative day 7, ClH was significantly higher in the ACEI group than in the control group (P<0.05). Plasma renin activity and vasopressin concentration increased in both groups during CPB (P<0.05). The study demonstrates that administration of an i.v. ACEI, enalaprilat, improves cardiac output during CABG surgery in patients with ischaemic heart dysfunction. Moreover, renal perfusion was better maintained during surgery, and this effect was sustained up to post-operative day 7.

Aged↗
PubMed2001

Expression and regulation of adenylyl cyclase isoforms in the human adrenal gland.

The aim of the present study was to identify which adenylyl cyclase isoforms were expressed in the human adrenal gland and to determine which isoform(s) may be coupled to ACTH action. Our results indicate that, in both glomerulosa and fasciculata zones, adenylyl cyclase 1 was detected in cells at the membrane level, adenylyl cyclases 3 and 2 in both the cytoplasm and the plasma membrane, whereas adenylyl cyclase 5/6 and adenylyl cyclase 4 were found mainly in cytoplasm. The levels of expression of each isoform were similar between the two adrenocortical zones, except for adenylyl cyclase 5/6, which had a lower level of expression in the zona fasciculata. We next evaluated the role of the various adenylyl cyclase isoforms during ACTH-stimulated cAMP production in both glomerulosa and fasciculata cell preparations. Corroborating with previous observations, we found that calcium had a biphasic effect on cAMP production. Interestingly, pertussis toxin treatment increased cAMP production, indicating that, in addition to Gs, ACTH is coupled to a Gi protein. Incubation with the betagamma-subunit sequestrant peptide QEHA decreased cAMP production, as did incubation with inhibitory antibodies against either adenylyl cyclase 2 or adenylyl cyclase 5/6. Inhibitory adenylyl cyclase 3 antibodies interfered with ACTH action only in the zona fasciculata. Altogether these data indicate that adrenocortical cells express one or two isoforms of each class of adenylyl cyclases and, thus, have the ability to produce cAMP in response to various regulatory, intracellular mediators. Importantly, our results indicate that in the human adrenal gland, ACTH acts mainly through adenylyl cyclase 5/6 and adenylyl cyclase 2/4, whereas the effect of ACTH on adenylyl cyclase 3 activity may be a consequence of calcium influx.

Adenylate Cyclase Toxin↗
PubMed2000

Binding properties of a selective tritiated vasopressin V2 receptor antagonist, [H]-SR 121463.

BACKGROUND: [3H]-SR 121463 is the first radiolabeled selective nonpeptide vasopressin V2 receptor antagonist ligand that has been reported to date. In the present work, we studied the binding properties of [3H]-SR 121463 for renal V2 receptors from animal and human origins. METHODS: Binding studies were performed with [3H]-SR 121463 in Chinese hamster ovary (CHO) cells transfected with the human V2 receptor and in various kidney preparations expressing the native V2 receptors (rat, rabbit, dog, pig, monkey, and human). Autoradiographies were performed in rat and human kidney sections. RESULTS: [3H]-SR 121463 binding to CHO cells stably transfected with the cloned human renal V2 receptor was specific, highly stable, time dependent, saturable, and reversible. A single population of high-affinity binding sites was identified (Kd = 0.94 +/- 0.34 nmol/L, Bmax = 9876 +/- 317 fmol/mg protein). Of note, [3H]-SR 121463 revealed a higher number (about 40%) of V2 sites than [3H]-AVP in the same preparation. Displacement of [3H]-SR 121463 binding by reference peptide and nonpeptide vasopressin/oxytocin compounds exhibited a typical AVP V2 profile. [3H]-SR 121463 also displayed a high affinity for native V2 receptors in several kidney preparations from rat, pig, dog, rabbit, bovine, monkey, and human. The autoradiographic experiments using rat and human kidney sections showed intense labeling in the medullopapillary region and lower intensity in the cortex, consistent with a main localization of V2 receptors on collecting tubules. CONCLUSION: [3H]-SR 121463 is a useful ligand for the specific labeling of animal and human V2 receptors and could be a suitable probe for the search and in situ localization of V2 sites.

Animals↗
PubMed1999

The relationship among carbon dioxide pneumoperitoneum, vasopressin release, and hemodynamic changes.

UNLABELLED: We assessed the role of vasopressin (VP) for the hemodynamic response to pneumoperitoneum in pigs. Four groups of anesthetized pigs were investigated. Nine pigs were intraabdominally insufflated with CO2 and eight were intraabdominally insufflated with argon; eight pigs received an i.v. injection of 1 mg/kg SR 49059, a VP antagonist, before CO2 insufflation; and six pigs received SR 49059 alone. Hemodynamics, plasma concentrations of VP and vasoactive hormones, and Paco2 were measured. Data were analyzed by using analysis of variance, Student's t-test, and Mann-Whitney U-test. Five minutes after insufflation, changes in systemic vascular resistance (SVR) were significantly correlated with changes in VP (r = 0.72; P = 0.005) but not with changes in epinephrine, norepinephrine, renin activity, or Paco2. SVR increased during CO2 insufflation but not during argon insufflation or CO2 insufflation with a preceding infusion of SR 49059. The SR 49059 injection itself resulted in increases in heart rate and cardiac output and decreases in blood pressure and SVR. We conclude that, during CO2 pneumoperitoneum in pigs, absorbed CO2 initiates a pathophysiological process that stimulates VP release. Hence, VP most likely plays a key role in the hemodynamic response to a CO2-induced pneumoperitoneum. IMPLICATIONS: Intraabdominal insufflation of CO2 is associated with hemodynamic and hormonal changes. Investigating CO2 and argon-insufflated pigs and using a vasopressin antagonist, we found that CO2 insufflation released vasopressin, which, in turn, induced hemodynamic perturbances.

Animals↗
PubMed1999

Distribution of signaling molecules involved in vasopressin-induced Ca2+ mobilization in rat hepatocyte multiplets.

In freshly isolated rat hepatocyte multiplets, Ca2+ signals in response to vasopressin are highly organized. In this study we used specific probes to visualize, by fluorescence and confocal microscopy, the main signaling molecules involved in vasopressin-mediated Ca2+ responses. V1a receptors were detected with a novel fluorescent antagonist, Rhm8-PVA. The Galphaq/Galpha11, PLCbeta3, PIP2, and InsP3 receptors were detected with specific antibodies. V1a vasopressin receptors and PIP2 were associated with the basolateral membrane and were not detected in the bile canalicular domain. Galphaq/Galpha11, PLCbeta3, and InsP3 receptors were associated with the basolateral membrane and also with other intracellular structures. We used double labeling, Western blotting, and drugs (cytochalasin D, colchicine) known to disorganize the cytoskeleton to demonstrate the partial co-localization of Galphaq/Galpha11 with F-actin.

Actins↗
PubMed1999

Comparative involvement of cyclic nucleotide phosphodiesterases and adenylyl cyclase on adrenocorticotropin-induced increase of cyclic adenosine monophosphate in rat and human glomerulosa cells.

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.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Vasopressin receptors in human adrenal medulla and pheochromocytoma.

The nature of vasopressin (VP) receptors present in normal and tumoral human adrenal was investigated using various experimental approaches. Specific VP-binding sites were detected by autoradiography using [3H]arginine VP as a radioligand in adrenal cortex and medulla. The V1a receptor subtype was expressed in the two parts of the gland, as shown by pharmacological studies and RT-PCR experiments. By contrast, the V1b receptor subtype was only expressed in medullary chromaffin cells. This was confirmed by the characterization of V1b transcripts detected in adrenal medulla tissues. In pheochromocytoma, we also detected functional V1b receptors. These receptors triggered intracellular calcium mobilization from intracellular pools and were involved in catecholamine secretion. Binding experiments performed on pheochromocytoma plasma membrane preparations also revealed V1a vasopressin-binding sites, whose roles and cellular localization have not yet been determined. RT-PCR experiments confirmed these data; 100% and 80% of the five tumors tested exhibited V1a and V1b transcripts, respectively. Perifusion experiments also demonstrated that some pheochromocytomas may secrete large amounts of VP. Our findings imply that VP locally secreted by human adrenal medulla may regulate adrenal function by acting on V1a or V1b receptors. More interestingly, we demonstrate that one pheochromocytoma oversecretes VP. In this particular case, this may contribute to the increase in blood pressure observed.

Adrenal Gland Neoplasms↗
PubMed1998

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.

Animals↗