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Increases in both Ca2+and nitric oxide levels are vital for a variety of cellular processes; however, the interaction between these two crucial messengers is not fully understood. Here, we demonstrate that expression of inducible nitric-oxide synthase in hepatocytes, in response to inflammatory mediators, dramatically attenuates Ca2+signaling by the inositol 1,4,5-trisphosphate-forming hormone, vasopressin. The inhibitory effects of induction were reversed by nitric oxide inhibitors and mimicked by prolonged cyclic GMP elevation. Induction was without effect on Ca2+signals in response to AlF4−or inositol 1,4,5-trisphosphate, indicating that phospholipase C activation and release of Ca2+from inositol 1,4,5-trisphosphate-sensitive Ca2+stores were not targets for nitric oxide inhibition. Vasopressin receptor levels, however, were dramatically reduced in induced cultures. Our data provide a possible mechanism for hepatocyte dysfunction during chronic inflammation.

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 vitroPI3K assays performed without the addition of any exogenous substrates revealed that guanosine 5′-(γ-thio)triphosphate (GTPγS) specifically stimulated the nuclear synthesis of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), whereas guanosine 5′-(β-thio)diphosphate was ineffective. PI3K inhibitors wortmannin and LY294002 prevented GTPγS-induced PtdIns(3,4,5)P3synthesis. Moreover, pertussis toxin inhibited partially PtdIns(3,4,5)P3accumulation, suggesting that nuclear Gi/G0proteins are involved in the activation of PI3K. Immunoblot experiments showed the presence of Gα0proteins in VSMC nuclei. In contrast with previous reports, immunoblots and indirect immunofluorescence failed to detect the p85α subunit of the heterodimeric PI3K within VSMC nuclei. By contrast, we have detected the presence of a 117-kDa protein immunologically related to the PI3Kγ. These results indicate the existence of a G protein-activated PI3K inside VSMC nucleus that might be involved in the contol of VSMC proliferation and in the pathogenesis of vascular proliferative disorders.

The muscarinic agonist, carbachol (CCh), was shown to stimulate the production of inositol phosphates (IP) in isolated cells from rabbit fundic mucosa. This stimulatory effect was time- and dose-dependent: EC 50values for IP 1, IP 2and IP 3accumulation were not statistically different. The mean value was 30 ± 8 μM ( n= 6). The corresponding maximal stimulation (% of basal value) observed after 20 min incubation in the presence of 100 μM CCh was 160 ± 15%. CCh-induced IP accumulation was abolished by atropine ( Ki= 0.32 ± 0.18 nM ( n=3)). The CCh concentrations leading to half-maximal inhibition of N-[ 3H]methylscopolamine binding and half-maximal IP accumulation were similar. The half-maximal value for CCh-induced aminopyrine accumulation was 8-times lower. These results indicate that IP 3-mediated mobilization of intracellular Ca 2+might be involved in CCh-induced acid secretion by parietal cells.

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.

Nordidemnin (NorD) is a cyclic depsipeptide isolated from a Caribbean tunicate. This drug is thought to affect cell proliferation by acting on protein, RNA, and DNA syntheses. We studied the ability of NorD to arrest sea urchin embryos at the prophase stage. We tested whether NorD could alter the synthesis and phosphorylation of proteins as well as polyphosphoinositide (PPI) metabolism, as the activation of these processes is necessary for progression through the cell cycle. The dose-response effect on protein synthesis and cell cleavage suggests that NorD acts in the same way as emetine, a well-known protein synthesis inhibitor. We observed that treatment of eggs with emetine or NorD, even at concentrations that inhibited DNA and protein synthesis as well as phosphorylation of proteins, led to modifications in the incorporation of 32P into phosphatidylinositol phosphate and phosphatidylinositol bisphosphate without any alteration in the chemical amounts of these lipids. However, fluctuations in the PPI messenger system that occur during the cell cycle were maintained in the presence of either drug. We suggest that proteins and PPI are linked in an intricate network to control mitosis and cellular proliferation.

Partially purified plasma membranes prepared from myo-[ 3H]inositol-prelabeled WRK1 cells exhibit a phosphatidylinositol 4,5-bisphosphate (PIP 2) phospholipase C activity sensitive to NaF. NaF increased the production of IP 2and IP 3in a time- and concentration-dependent manner. The maximal increase in IP 2and IP 3production 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 IP 3and IP 2, accumulation in a concentration-dependent manner. The effect of fluoride on the PIP 2phospholipase C from WRK1 cell membranes appears to be similar to the well-documented effect of F −on the well-characterized N s. N iand 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 PIP 2phospholipase C.

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 (EC 50= 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.

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 IP2and IP3in a time‐ and concentration‐dependent manner. The maximal increase in IP2and IP3production 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 IP3and IP2, accumulation in a concentration‐dependent manner. The effect of fluoride on the PIP2phospholipase C from WRK1 cell membranes appears to be similar to the well‐documented effect of F−on the well‐characterized Ns. Niand 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 PIP2phospholipase C.

The muscarinic agonist, carbachol (CCh), was shown to stimulate the production of inositol phosphates (IP) in isolated cells from rabbit fundic mucosa. This stimulatory effect was time‐ and dose‐dependent: EC50values for IP1, IP2and IP3accumulation were not statistically different. The mean value was 30 ± 8 μM (n= 6). The corresponding maximal stimulation (% of basal value) observed after 20 min incubation in the presence of 100 μM CCh was 160 ± 15%. CCh‐induced IP accumulation was abolished by atropine (Ki= 0.32 ± 0.18 nM (n=3)). The CCh concentrations leading to half‐maximal inhibition of N‐[3H]methylscopolamine binding and half‐maximal IP accumulation were similar. The half‐maximal value for CCh‐induced aminopyrine accumulation was 8‐times lower. These results indicate that IP3‐mediated mobilization of intracellular Ca2+might be involved in CCh‐induced acid secretion by parietal cells.

To study the vasopressin receptor domains involved in the hormonal binding, we synthesized natural and modified fragments of V1avasopressin receptor and tested their abilities to affect hormone-receptor interactions. Natural fragments mimicking the external loops one, two, and three were able to inhibit specific vasopressin binding to V1areceptor. In contrast, the natural N-terminal part of the V1avasopressin receptor was found inactive. One fragment, derived from the external second loop and containing an additional C-terminal cysteine amide, was able to fully inhibit the specific binding of both labeled vasopressin agonist and antagonist to rat liver V1avasopressin receptor and the vasopressin-sensitive phospholipase C of WRK1 cells. The peptide-mediated inhibition involved specific interactions between the V1areceptor and synthetic V1avasopressin receptor fragment since 1) it was dependent upon the vasopressin receptor subtype tested (Ki(app)for the peptide: 3.7, 14.6, and 64.5 μmfor displacing [3H]vasopressin from rat V1a, V1b, and V2receptors, respectively; 2) it was specific and did not affect sarcosin 1-angiotensin II binding to rat liver membranes; 3) it was not mimicked by vasopressin receptor unrelated peptides exhibiting putative detergent properties; and 4) no direct interaction between [3H]vasopressin and synthetic peptide linked to an affinity chromatography column could be observed. Such an inhibition affected both the maximal binding capacity of the V1avasopressin receptor and its affinity for the labeled hormone, depending upon the dose of synthetic peptide used and was partially irreversible. Structure-activity studies using a serie of synthetic fragments revealed the importance of their size and cysteinyl composition. These data indicate that some peptides mimicking extracellular loops of the V1avasopressin receptor may interact with the vasopressin receptor itself and modify its coupling with phospholipase C.

Although vasopressin V1Breceptor (V1B-R) mRNA was detected in the kidney, the precise renal localization, pharmacological and physiological properties of this receptor remain unknown. Using the selective V1Bagonist d[Leu4, Lys8]VP, either fluorescent or radioactive, we showed that V1B-R is mainly present in principal cells of the inner medullary collecting duct (IMCD) in the male rat kidney. Protein and mRNA expression of V1B-R were very low compared to V2 receptor (V2-R). On microdissected IMCD, d[Leu4, Lys8]VP had no effect on cAMP production but induced a dose-dependent and saturable [Ca2+]iincrease mobilization with an EC50in the nanomolar range. This effect involved both intracellular calcium mobilization and extracellular calciuminflux. The selective V1Bantagonist SSR149415 strongly reduced the ability of vasopressin to increase [Ca2+]ibut also cAMP suggesting a cooperation between V1B-R and V2-R in IMCD cells expressing both receptors. This cooperation arises from a crosstalk between second messenger cascade involving protein kinase C rather than receptor heterodimerization as supported by potentiation of the AVP-stimulated cAMP production in HEK293 cells co-expressing the two receptor isoforms and negative results obtained by bioluminescence resonance energy transfer experiments. In vivo, only acute administration of high doses of V1Bagonist triggered significant diuretic effects by contrast with injection of selective V2agonist. This study brings new data on the localization and signaling pathways of V1B-R in the kidney, highlights a crosstalk between V1B-R and V2-R in IMCD and suggests that V1B-R may counterbalance in some pathophysiological conditions the antidiuretic effect triggered by V2-R activation.

Arginine vasopressin elicits elaborate Ca2+ signals in the liver (intercellular Ca2+ waves), the functional implications of which are not understood. Waves propagate across hepatocyte plates following a lobular gradient in Via vasopressin receptor density. Here, we report that changes in this receptor distribution control Ca2+ wave propagation and bile flow. Although basal circulating vasopressin levels do not play a major role in the regulation of V1a receptor expression, increases in vasopressin concentration within physiological limits for 24 h can abolish the lobular gradient in V1a receptor, as assessed by spectrofluorimetry, videomicroscopy, binding studies, and RNase protection assays. In animals in which the V1a receptor gradient was abolished, intercellular Ca2+ waves were impaired due to the equalization of Ca2+ responses in the various zones of the lobule. In the isolated perfused liver, the early increase in vasopressin‐induced bile flow observed in control rats was much smaller if the V1a receptor density gradient was abolished. These findings suggest that V1a vasopressin receptor distribution controls intercellular Ca2+ wave propagation and bile flow. The control of hormone receptor distribution in a tissue by an agonist may turn the signaling and function of this agonist on or off.

In rat portal vein myocytes, a2A-adrenoceptors activate voltage-dependent Ca2+ channels via a transduction pathway requiring protein kinase C activation mediated by a pertussis-toxin-sensitive G-protein. As revealed by the use of antibodies directed against the different a-subunits expressed in portal vein myocytes, we show that the clonidine-induced stimulation of voltage-dependent Ca2+ channels is mainly mediated by a Gi1–2-protein.

We have investigated whole-cell Ba2+ currents through Ca2+ channels (IBa) in single myocytes freshly isolated from the aortic media of neonatal (1-day-old) and adult (12-week-old) rats. In neonatal myocytes, IBa was undetectable even in presence of the dihydropyridine (DHP) agonist Bay K 8644. Binding of [3H]Nitrendipine on crude plasma membrane preparation of media confirmed the absence of DHP-receptors. By contrast, a robust DHP-sensitive ‘L-type’ IBa was recorded in adults which was consistent with the presence of specific [3H]Nitrendipine binding sites. In conclusion, neonatal aortic myocytes do not express any Ca2+ channels. The acquisition of L-type Ca2+ channels may be related to cell differentiation and acquisition of contractility during postnatal development.