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.

Association of the G protein alpha(q)/alpha11-subunit with cytoskeleton in adrenal glomerulosa cells: role in receptor-effector coupling.

In 3-day primary cultures of rat glomerulosa cells, a 30-min pre-incubation with either 10 microM colchicine (a microtubule-disrupting agent) or 10 microM 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 alpha(q)/alpha11-subunit of the G(q/11) protein was associated with both structures. Ang II stimulation induced a rapid translocation of alpha(q)/alpha11, microfilaments, and microtubules to the membrane and induced a time-dependent increase in the level of alpha(q)/alpha11 associated with both microfilaments and microtubules. Moreover, double immunofluorescence staining clearly showed a colocalization of the alpha(q)/alpha11-subunit of the G(q/11) coupling protein and microfilament distribution. These associations and plasma membrane redistribution under Ang II stimulation indicate that microfilaments and microtubules are both involved in phospholipase C activation and inositol phosphate production. Moreover, our results indicate that the alpha(q)/alpha11 protein is closely associated with cytoskeletal elements and is found both at the plasma membrane level as well as on intracellular stress fibers.

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.

Characterization of a monoclonal antibody produced in an attempt to mimic the active site of HIV aspartyl protease using haptens based on inhibitor models.

The high binding affinity and specificity of antibodies for a great variety of ligands has been widely exploited in structure-activity relationship studies of biomolecules and more recently in the development of new catalysts for several chemical reactions. It is assumed that antibodies generated against haptenic protease inhibitors would recognize both these haptens and the substrate of the model proteolytic reaction. We have produced antibodies against HIV PRp12 aspartyl protease substrate analogues, chemically modified at the scissile bond, Phe-Pro. Identical chemical modifications have been reported for related HIV protease inhibitors. We finally selected an anti-hapten monoclonal antibody that specifically recognized the substrate and those haptens with both the phenylalanyl side chain and the prolyl pyrrolidine ring. This selectivity of recognition suggests that such an antibody might mimic the catalytic site of the model protease.

Antineoplastic activity of didemnin congeners: nordidemnin and modified chain analogues.

Nordidemnin (2), a natural analogue of the marine cyclodepsipeptide didemnin B (1b), showed cytotoxic activity against L1210 leukemia and antineoplastic activity against P388 leukemia as well as B16 melanoma; nordidemnin (2) was as active as didemnin B (1b). The influence of synthetic modifications in the linear peptidic chain on in vitro and in vivo activity was also studied. Replacement of the terminal lactyl residue by mandelyl and 3-(p-hydroxyphenyl)propionyl residues in compounds 3 and 4, respectively, did not affect the cytotoxic activity against L1210 leukemia (ID50 of 1.1 nM and 1.2 nM, respectively) or the in vivo activity against P388 leukemia. Unlike these aromatic substituants, the lipophilic palmityl residue induced a dramatic loss in cytotoxic activity. The inverted chirality of the MeLeu joining residue in compound 6 caused a marked reduction in the in vitro activity.

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.

G proteins in aortic endothelial cells and bradykinin-induced formation of nitric oxide.

In bovine aortic endothelial cells (BAEC), pertussis toxin (PTx) ADP-ribosylated two major substrates with apparent molecular masses of 40 and 41 kDa, whereas cholera toxin (CTx) ADP-ribosylated two other substrates of 44 and 50 kDa. [alpha-32P]GTP bound to three bands in the 22-27 kDa range. Immunoblot analysis revealed the simultaneous presence of G alpha i1, G alpha i2, G alpha i3, G alpha q or G alpha 11 and of different forms of G alpha s but did not detect significant levels of G alpha 0. Bradykinin caused a 9-fold increase in intracellular cyclic GMP level in BAEC (measured as an index of NO production). Preincubation of BAEC with CTx, but not with PTx, inhibited bradykinin-dependent production of cyclic GMP. These results show that G alpha s, G alpha q or alpha 11, Gi and small GTP-binding proteins are present in BAEC and suggest that a CTx-sensitive G-protein (possibly either small G-protein, G alpha q or G alpha 11) could be associated with the bradykinin-mediated NO formation.

Up-regulation in late pregnancy of both Go1 alpha and Go2 alpha isoforms in human myometrium.

The nature of the 39 kDa pertussis toxin substrate previously detected in human pregnant myometrium was investigated. Comparison of membranes from non-pregnant and from 39-40 week pregnant myometrium revealed a higher level at 39 kDa of pertussis toxin ADP-ribosylation and Go alpha immunoreactivity in late pregnancy. Furthermore, quantification of both Go alpha isoforms with specific anti-alpha o1- and alpha o2-antibodies revealed an increase in their expression in late pregnancy. At the same time, only limited changes in the levels of Gi1,2, Gi3 and Gq were observed. In rat myometrial membranes, neither pertussis toxin substrate nor Go alpha immunoreactivity could be detected at the 39 kDa level. These results demonstrate that, in the human myometrium in late pregnancy, there is an increase in the expression of both isoforms of the Go alpha subunits, suggesting a role for these proteins throughout gestation and/or near term for parturition.

Effect of nordidemnin on the cell cycle of sea urchin embryos. Role in synthesis and phosphorylation of proteins and in polyphosphoinositide turnover in mitosis progression.

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.

Mechanism of the antibiotic action of alpha-dehydrobiotin.

alpha-Dehydrobiotin, a naturally occurring biotin analogue, exhibits antibiotic properties [Hanka, L. J., Reineke, L. M., & Martin, D. G.(1969) J. Bacteriol. 100, 42--46]. It is shown in this paper that in addition to its activity as corepressor of the transcription of the biotin locus [Guha, A., Saturen, Y., & Szybalski, W. (1971) J. Mol. Biol. 56, 53--62] alpha-dehydrobiotin acts at the enzyme level. The synthesis of specifically tritiated alpha-dehydrobiotin has been achieved. By use of this labeled compound and a biotin-department strain of Escherichia coli, it has been demonstrated that alpha-dehydrobiotin can be linked covalently to proteins without further transformation. The fixation of biotin to apocarboxylases is inhibited irreversibly after preincubation with alpha-dehydrobiotin. This strongly supports the hypothesis that alpha-dehydrobiotin can be specifically linked to apocarboxylases in place of biotin and leads to carboxylases that are inactive. Thus, the antibiotic properties of alpha-dehydrobiotin would be partly due to the fact that it compete with biotin for the fixation on the apocarboxylases, producing irreversibly inactive enzymes.

Close association of the alpha subunits of Gq and G11 G proteins with actin filaments in WRK1 cells: relation to G protein-mediated phospholipase C activation.

A selective polyclonal antibody directed toward the C-terminal decapeptide common to the alpha subunits of Gq and G11 G proteins (G alpha q/G alpha 11) was prepared and used to investigate the subcellular distribution fo these proteins in WRK1 cells, a rat mammary tumor cell line. In immunoblots, the antibody recognized purified G alpha q and G alpha 11 proteins and labeled only two bands corresponding to these alpha subunits. Functional studies indicated that this antibody inhibited vasopressin- and guanosine 5'-[alpha-thio]triphosphate-sensitive phospholipase C activities. Immunofluorescence experiments done with this antibody revealed a filamentous labeling corresponding to intracytoplasmic and perimembranous actin-like filament structures. Colocalization of G alpha q/G alpha 11 and F-actin filaments (F-actin) was demonstrated by double-labeling experiments with anti-G alpha q/G alpha 11 and anti-actin antibodies. Immunoblot analysis of membrane, cytoskeletal, and F-actin-rich fractions confirmed the close association of G alpha q/G alpha 11 with actin. Large amounts of G alpha q/G alpha 11 were recovered in the desmin- and tubulin-free F-actin-rich fraction obtained by a double depolymerization-repolymerization cycle. Disorganization of F-actin filaments with cytochalasin D preserved G alpha q/G alpha 11 and F-actin colocalization but partially inhibited vasopressin- and fluoroaluminate-sensitive phospholipase C activity, suggesting that actin-associated G alpha q/G alpha 11 proteins play a role in signal transduction.

Dual effects of nordidemnin on WRK1 cells: inhibition of phosphoinositide metabolism and cell proliferation.

Nordidemnin (NorD), a cyclodepsipeptide isolated from marine invertebrates, exhibits antiproliferative and antitumoral properties identical to didemnin B on many cell lines. On WRK1 cells, a rat mammary tumor cell line, NorD considerably reduced the vasopressin-stimulated accumulation of inositol phosphates. This effect was more pronounced on dividing cells and of weak amplitude on quiescent ones. It was observed with nanomolar concentrations of NorD and became significative after 3 hr of incubation at 37 degrees C. The maximal effect was observed after a 14-hr incubation period. In contrast, the inactive analog epinordidemnin, as well as the structurally related immunosuppressive cyclosporin A, had no significant effect on phosphoinositide metabolism. More detailed analysis demonstrated that NorD reduced the amounts of all intracellular inositol phosphate isomers, including inositol pentakisphosphate and inositol hexakisphosphate. Vasopressin-stimulated inositol (1,4,5)-trisphosphate accumulation was reduced by 80% and, as a consequence, the intracellular calcium mobilization was strongly affected. Similarly, NorD reduced both the level of inositol (1,4,5)-trisphosphate and the intracellular free calcium concentration of unstimulated cells. NorD blocked phosphoinositide metabolism by reducing the myoinositol transporter and, by a consequence, the pool of inositol lipids. NorD also strongly inhibited WRK1 cell proliferation with the same EC50 as that observed for the effect on phosphoinositide metabolism. Epinordidemnin, which was unable to inhibit inositol phosphate accumulation, had no effect on cell growth. Cyclosporin A, which slightly inhibited WRK1 cell growth, did not significantly affect the calcium-phosphatidylinositol cascade. Taken together, these results suggest that NorD might interfere with WRK1 cell growth by inhibiting phosphoinositide turnover.

Synthetic rat V1a vasopressin receptor fragments interfere with vasopressin binding via specific interaction with the receptor.

To study the vasopressin receptor domains involved in the hormonal binding, we synthesized natural and modified fragments of V1a vasopressin 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 V1a receptor. In contrast, the natural N-terminal part of the V1a vasopressin 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 V1a vasopressin receptor and the vasopressin-sensitive phospholipase C of WRK1 cells. The peptide-mediated inhibition involved specific interactions between the V1a receptor and synthetic V1a vasopressin 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 microM for displacing [3H]vasopressin from rat V1a, V1b, and V2 receptors, 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 V1a vasopressin 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 V1a vasopressin receptor may interact with the vasopressin receptor itself and modify its coupling with phospholipase C.

Mutation of Asn-391 within the conserved NPXXY motif of the cholecystokinin B receptor abolishes Gq protein activation without affecting its association with the receptor.

Among the most conserved regions in the G-protein-coupled receptors is the (N/D)PX(2-3)Y motif of the seventh transmembrane domain (X represents any amino acid). The mutation of the Asn/Asp residue of this motif in different G-protein-coupled receptors was shown to affect the activation of either adenylyl cyclase or phospholipase C. We have mutated the Asn residue (Asn-391) of the NPXXY motif in the CCKBR to Ala and determined the effects of the mutation on binding, signaling, and G-proteins coupling after expression of the mutated receptor in COS cells. The mutated receptor displayed similar expression levels and high affinity CCK binding compared with the wild type CCKBR. However, unlike the wild type CCKBR, the mutated receptor was completely unable to mediate activation of either phospholipase C and protein kinase C-dependent and -independent mitogen-activated protein kinase pathways, indicating an essential role of Asn-391 in CCKBR signaling. Coimmunoprecipitation experiments allowed us to show that the inactive mutant retains an intact capacity to form stable complexes with G(q)alpha subunits in response to CCK. These results indicate that the formation of high affinity CCK-receptor-G(q) protein complexes is not sufficient to activate G(q) and suggest that Asn-391 is specifically involved in G(q) proteins activation.