Comparative ACTH-stimulated responses in rat and human glomerulosa cells.
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Adolescent↗
Biomedical subjects
G Guillon
Publications and source records attributed to G 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 5beta-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.
Animals↗
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.
Animals↗
Vasopressin regulates adrenal functions by acting through different vasopressin receptor subtypes.
In mammals, vasopressin is known to be synthesized in the hypothalamus and released in the blood stream at the pituitary level. This neuropeptide is also synthesized and secreted by the adrenal medulla in many species including human. Moreover, agents like acetylcholine and corticotropin releasing factor stimulates its basal secretion. V1a vasopressin receptors are present in the adrenal cortex and are involved in steroids secretion (aldosterone in the zona glomerulosa and glucocorticoids in the zona fasciculata of some species). These receptors are coupled to phospholipase C beta and to dihydropyridine-sensitive calcium channels via heterotrimeric G proteins differing by their sensitivities to pertussis toxin. The adrenal medulla, from many species, exhibits V1a vasopressin receptors. In rat adrenal medulla, functional V1b vasopressin receptors could also be characterized. These receptors stimulate catecholamines secretion via activation of phospholipase C beta and subsequent mobilization of intracellular calcium. The adrenal medulla secretes AVP and exhibits functional vasopressin receptors. The adrenal cortex also possesses functional vasopressin receptors and is in contact with adrenal medulla via "medullary rays". We may thus reasonably conclude that AVP physiologically regulates adrenal gland functions via autocrine/paracrine mechanisms.
Adrenal Cortex↗
Signaling mechanisms induced by endothelin agonists in human adrenal glomerulosa cells.
Endothelin-1 (ET-1) exhibits secretagogue and trophic actions on the adrenal zona glomerulosa (ZG). Little information is available on the intracellular signaling events that follow stimulation of ET receptors on ZG cells. This study examined the expression of ET receptor subtypes and their involvement in transduction mechanisms induced by ET agonists on human ZG cells in primary culture. RT-PCR allowed the detection of both ETA and ETB receptor mRNAs in these cells. ET-1 induced a concentration-dependent increase in inositol phosphate (IP) accumulation in the presence of LiCl, whereas ETB agonists were inactive. The ET-1-induced increase in IP accumulation was prevented by BQ-123. ET-1 evoked an increase in [Ca2+]i, which was partially prevented by BQ-788. IRL 1620 also delayed the rise in [Ca2+]i. These results show that in human adrenal ZG cells, ET-1 induces an increase in IP accumulation through ETA receptor activation and evokes a rise in [Ca2+]i via stimulation of both ETA and ETB receptors.
Adrenal Glands↗
[Intracellular calcium channels, hormone receptors and intercellular calcium waves].
The hormone-mediated intercellular Ca2+ waves were analyzed in multiplets of rat hepatocytes by video imaging of fura2 fluorescence. These multicellular systems are composed of groups of several cells (doublets to quintuplets) issued from the liver cell plate, a one cell-thick cord of about 20 hepatocytes long between portal and centrolobular veins. When the multiplets were homogeneously bathed with the glycogenolytic agonists vasopressin, noradrenaline, angiotensin II and ATP, they showed highly organized Ca2+ signals. Surprisingly, for a given agonist, the primary rises in intracellular Ca2+ concentration ([Ca2+]i) originated invariably in the same hepatocyte, then was propagated in a sequential manner to the nearest connected cells (cell 2, then 3, cell 4 in a quadruplet, for example). The sequential activation of the cells appeared to be an intrinsic property of multiplets of rat hepatocytes. The same sequence was observed at each train of oscillations occurring between cells. The order of [Ca2+]i responses was modified neither by repeated additions of hormones nor by the hormonal dose. The mechanical disruption of an intermediate cell did not prevent the activation of the next cell. These results suggest that each hepatocyte in the multiplet displays its own sensitivity to the hormone and that a gradient of sensitivity between each cell could be responsible for directing the intercellular Ca2+ wave. To test this hypothesis, we selectively isolated rat hepatocytes from periportal (PP) and perivenous (PV) areas of the liver cell plate. Periportal (PP) and perivenous (PV) rat hepatocyte suspensions were loaded with quin2/AM and hormonal responses were studied in a spectrofluorimeter. Noradrenaline, angiotensin II, and vasopressin-induced [Ca2+]i rises were greater in PV than in PP hepatocytes. In contrast, PP cells were more responsive than PV cells to ATP. The function of the InsP3 receptor (InsP3R) was also studied by measuring the InsP3-mediated 45Ca2+ release from permeabilized PP and PV hepatocytes. In permeabilized PP and PV hepatocytes, internal Ca2+ stores displayed the same loading-kinetics, the responses to InsP3 were similar, and the sizes of InsP3-sensitive compartment were not different. In a further study, we investigated by video microscopy in fura2-loaded multicellular systems of rat hepatocytes, the mechanisms controlling intercellular propagation of the Ca2+ wave and coordination of Ca2+ signals induced by the different hormones. Using focal microperfusion which allows local perfusion of any cell of the multiplet, rapid agonist removal during the Ca2+ response and microinjection, we found that second messengers and [Ca2+]i rises in one hepatocyte cannot trigger Ca2+ responses in connected adjacent cells, suggesting that diffusion across gap junctions, while required for coordination, is not sufficient by itself for the propagation of the intercellular Ca2+ wave. In addition, focal microperfusion and intermediate cell disruption experiments revealed very fine functional differences (hormonal delay, frequency of [Ca2+]i oscillations) between hormone-induced Ca2+ signals, even between two adjacent connected hepatocytes. Recent unpublished results performed in suspensions of PP and PV rat hepatocytes supported the view of a major role played by vasopressin receptors (V1a) in genesis and orientation of the Ca2+ wave. Vasopressin binding sites, V1a mRNAs detected by RNAse Protection Assay, and vasopressin-induced InsP3 production, were more abundant in PV than in PP cells. A gradient of hormone receptors could orientate the propagation of the Ca2+ wave in multicellular systems and in liver cell plate. These results suggest that the intercellular Ca2+ wave in multicellular systems of rat hepatocytes is propagated through mechanisms involving at least three factors. (ABSTRACT TRUNCATED)
Animals↗
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.
1-Sarcosine-8-Isoleucine Angiotensin II↗
Efficient photoaffinity labeling of the rat V1a vasopressin receptor using a linear azidopeptidic antagonist.
We have synthesized and fully characterized by fast-atom-bombardment-mass, NMR and ultraviolet spectroscopies the vasopressin antagonist 3-azidophenylpropionyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr(3I )-NH2. Easily radioiodinatable just before use, it has a high affinity for the natural rat liver V1a receptor [dissociation constant (Kd) = 54 +/- 20 pM; Carnazzi, E., Aumelas, A., Barberis, C., Guillon, G. & Seyer, R. (1994) J. Med. Chem. 37, 1841-1849] and for both the rat vasopressin V1a receptor expressed in Spodoptera frugiperda 9 cells (Sf9 cells, Kd = 688 +/- 35 pM) and in COS-7 cells (Kd = 320 +/- 20 pM). This probe labels specifically the V1a receptors in an ultraviolet-dependent manner, and binds covalently to about 12% of the receptors with high stability over several days, even in dissociation or solubilization conditions. SDS/PAGE studies and autoradiographic analyses of the photolabeled receptors reveal a single band (49.5 kDa) and two bands (63 kDa and 93.6 kDa) for receptor-probe associations obtained in Sf9 and COS-7 cells respectively. These molecular masses are consistent with non-glycosylated and highly glycosylated forms of the receptor, according to each expression system. In rat liver membranes, we have identified apparent molecular masses of about 32, 45 and more than 67 kDa. We finally demonstrated a proteolysis of the receptor that appeared to be Zn2+ and leupeptin sensitive. The high potency of this ligand is promising for the monitoring of the purification of the V1a receptor and for mapping its antagonist-binding site.
Affinity Labels↗
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.
Actin Cytoskeleton↗
Characterization of SR 121463A, a highly potent and selective, orally active vasopressin V2 receptor antagonist.
SR 121463A, a potent and selective, orally active, nonpeptide vasopressin V2 receptor antagonist, has been characterized in several in vitro and in vivo models. This compound displayed highly competitive and selective affinity for V2 receptors in rat, bovine and human kidney (0.6 < or = Ki [nM] < or = 4.1). In this latter preparation, SR 121463A potently antagonized arginine vasopressin (AVP)-stimulated adenylyl cyclase activity (Ki = 0.26+/-0.04 nM) without any intrinsic agonistic effect. In autoradiographic experiments performed in rat kidney sections, SR 121463A displaced [3H]AVP labeling especially in the medullo-papillary region and confirmed that it is a suitable tool for mapping V2 receptors. In comparison, the nonpeptide V2 antagonist, OPC-31260, showed much lower affinity for animal and human renal V2 receptors and lower efficacy to inhibit vasopressin-stimulated adenylyl cyclase (Ki in the 10 nanomolar range). Moreover, OPC-31260 exhibited a poor V2 selectivity profile and can be considered as a V2/V1a ligand. In normally hydrated conscious rats, SR 121463A induced powerful aquaresis after intravenous (0.003-0.3 mg/kg) or oral (0.03-10 mg/kg) administration. The effect was dose-dependent and lasted about 6 hours at the dose of 3 mg/kg p.o. OPC-31260 had a similar aquaretic profile but with markedly lower oral efficacy. The action of SR 121463A was purely aquaretic with no changes in urine Na+ and K+ excretions unlike that of known diuretic agents such as furosemide or hydrochlorothiazide. In addition, no antidiuretic properties have been detected with SR 121463A in vasopressin-deficient Brattleboro rats. Thus, SR 121463A is the most potent and selective, orally active V2 antagonist yet described and could be a powerful tool for exploring V2 receptors and the therapeutical usefulness of V2 blocker aquaretic agents in water-retaining diseases.
Adenylyl Cyclases↗
Role of Ca2+ in the action of adrenocorticotropin in cultured human adrenal glomerulosa cells.
The present report details the role of Ca2+ in the early events of ACTH action in human adrenal glomerulosa cells. Threshold stimulations of both aldosterone and cAMP production were obtained with a concentration of 10 pM ACTH, an ED50 of 0.1 nM, and maximal aldosterone stimulation (5.5-fold increase over control) at 10 nM ACTH. ACTH also induced a sustained increase of intracellular calcium ([Ca2+]i) with maximal stimulation of 1.6 +/- 0.1-fold over control values. This increase does not involve mobilization of calcium from intracellular pools since no response was observed in Ca2+-free medium or in the presence of nifedipine, suggesting the involvement of Ca2+ influx by L-type Ca2+ channels. This was confirmed by patch clamp studies that demonstrated that ACTH stimulates L-type Ca2+ channels. Moreover, the Ca2+ ion is not required for ACTH binding to its receptor, but is essential for sustained cAMP production and aldosterone secretion after ACTH stimulation. These results indicate that, in human adrenal glomerulosa cells, a positive feedback loop between adenylyl cyclase-protein kinase A-Ca2+ channels ensures a slow but sustained [Ca2+]i increase that is responsible for sustained cAMP production and aldosterone secretion.
Adolescent↗
Absence of calcium channels in neonatal rat aortic myocytes.
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.
Aging↗
Dual effects of fluoroaluminate on activation of calcium influx and inhibition of agonist-induced calcium mobilization in rat glomerulosa cells.
Results presented in this study demonstrate that, in rat glomerulosa cells, fluoroaluminate (AlF4-) alone stimulates both cAMP accumulation (maximal stimulation 10-fold, ED50, 24 mM) and total inositol phosphate accumulation (maximal stimulation 12-fold, ED50 14 mM). Despite a transient accumulation of Ins(1,4,5)P3 after AlF4- stimulation, no rapid and transient intracellular calcium mobilization was observed. In contrast to angiotensin II (Ang II) or vasopressin (AVP), AlF4- induces only a slow and sustained increase in intracellular Ca2+. We demonstrate that this increase results from a Ca2+ influx mediated by cAMP-protein kinase A (PKA) pathway since preincubation with H-89, a potent PKA inhibitor, inhibits this influx. Moreover, a short preincubation (15 min at 37 degrees C) of cells with AlF4- or ACTH prevents the initial release of Ca2+ from intracellular stores induced by Ang II, but does not affect the amount of InsPs accumulated under Ang II stimulation. This rapid inhibition of Ang II action is mediated by ACTH- or AlF4(-)-stimulated cAMP production since pretreatment with H-89 leads to a complete reversal. cAMP most likely acts at the level of Ins(1,4,5)P3 receptors since an increase in intracellular cAMP blunts the calcium response induced by addition of exogenous Ins(1,4,5)P3 to permeabilized cells. These results point out that, in rat glomerulosa cells, activation of the cAMP pathway can induce a rapid desensitization of the phospholipase C pathway by acting downstream of inositol phosphate accumulation.
Adenylyl Cyclases↗
Modulation of phospholipase C pathway and level of Gq alpha/G11 alpha in rat myometrium during gestation.
The regulation of the receptor-G protein-phospholipase C (PLC) cascade was investigated in rat myometrium at midgestation (day 12) and at term (day 21) comparatively to the estrogen-treated tissue (day 0). Carbachol-mediated generation of [3H]inositol phosphates was insensitive to pertussis toxin and was enhanced at days 12 and 21 two- and threefold, respectively, with no alteration of muscarinic sites (M3 subtype). A similar increase could be detected in the production of inositol 1,4,5-trisphosphate, indicating the stimulation of a PLC degrading phosphatidylinositol 4,5-bisphosphate. AlF4- also enhanced PLC activation during gestation, suggesting pregnancy-related regulations that bypass receptor activation. Immunoreactive G proteins, Gq alpha and G11 alpha, and PLC-beta 3 were detected in all myometrial preparations. The amount of PLC-beta 3 was similar in day 0 and day 21 myometrium, although decreasing by 75% at midgestation. Of significance was the increased amount of Gq alpha in day 12 and day 21 myometrium (3- and 2-fold, respectively) which coincided with the enhanced phosphoinositide breakdown. The upregulation of Gq alpha may contribute to the enhanced PLC activity during pregnancy and at term.
Animals↗
Molecular and functional characterization of V1b vasopressin receptor in rat adrenal medulla.
In rat adrenal medulla, PCR experiments reveal the expression of messenger RNA encoding the gene for the V1b vasopressin receptor. Complementary DNA amplified sequences corresponded to the cloned rat pituitary V1b vasopressin receptor. Video microscopy experiments performed on fura-2-loaded adrenal medullary or adrenal glomerulosa cell primary cultures showed that vasopressin dose dependently mobilized intracellular calcium, suggesting that functional vasopressin receptors are expressed in these tissues. The use of d[D-3-Pal]vasopressin, a specific V1b vasopressin agonist, and SR 49059, a specific V1b vasopressin antagonist, revealed that V1b receptors are exclusively expressed in adrenal medulla. Using an indirect immunological approach (plasma membrane localization of dopamine-beta-hydroxylase), we demonstrated that stimulation of rat adrenal medulla V1b receptor leads to catecholamine secretion. More interestingly, PCR experiments performed on rat adrenal medulla RNA revealed that the arginine vasopressin-encoding gene is also expressed in this tissue. In addition, perifusion experiments indicated that [Arg8] vasopressin is released by the adrenal medulla. Together, these data suggest that vasopressin may regulate the adrenal functions by paracrine/autocrine mechanisms involving distinct vasopressin receptor subtypes: V1a in the adrenal cortex and V1b in the adrenal medulla.
Adrenal Medulla↗
Membrane-delimited G protein-mediated coupling between V1a vasopressin receptor and dihydropyridine binding sites in rat glomerulosa cells.
In rat glomerulosa cells, vasopressin stimulates intracellular calcium mobilization via at least two distinct mechanisms: the release of calcium from inositol-1,4,5-P3-sensitive stores and the activation of transmembrane calcium influx. In this study, we focused on the second mechanism through three experimental approaches. By videomicroscopically examining Fura-2-loaded cells, we demonstrate that vasopressin induces a dose-dependent and receptor-mediated calcium influx fully inhibited by either 1 microM nifedipine or a pertussis toxin pretreatment and potentiated by 1 microM BAY K 8644. Patch-clamp experiments also indicate that vasopressin stimulates L-type calcium current by 87% and only weakly inhibits T-type calcium current. To further characterize the coupling between the vasopressin receptor and the dihydropyridine calcium channel, we performed binding studies using tritiated nitrendipine. With this technique, we showed that on intact cells, vasopressin is able to increase the specific binding of tritiated nitrendipine in a dose-dependent manner (Kact = 2 nM). Pharmacological studies using a series of vasopressin analogs revealed that this effect is mediated via a V1a vasopressin receptor subtype. Furthermore, the vasopressin-stimulated nitrendipine binding was sensitive to pertussis toxin pretreatment, which affected only the maximum binding capacity of nitrendipine-binding sites. More interestingly, we demonstrate that vasopressin still increases nitrendipine binding to plasma membrane preparation and that GTP is absolutely necessary for such a hormonal effect. Altogether, these data confirm the existence of a tight and direct coupling between the V1a vasopressin receptor and a dihydropyridine calcium channel via a pertussis toxin-sensitive G protein.
Animals↗
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.
Actins↗
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.
Animals↗