Modifications of the binding properties of the human VIP receptor of IGR39 cells by sulfhydryl reagents.

The effects of specific sulfhydryl reagents, N-ethylmaleimide (NEM), p-chloromercuribenzoic acid (PCMB) and 5-5'-dithiobis(2-nitrobenzoic acid) (DTNB), were tested on the vasoactive intestinal peptide (VIP) receptor binding capacity of the human superficial melanoma-derived IGR39 cells. On intact cell monolayers NEM and PCMB inhibit the specific [125I]VIP binding in a time and dose-dependent manner while DTNB has no effect at any concentration tested. Inhibitory effects of NEM and PCMB on high and low affinity VIP receptor are not identical. With NEM-treated cells, only low affinity sites remained accessible to the ligand. Their affinity constant is not modified. With PCMB-treated cells, the binding capacity of high affinity sites is reduced by 56% while the binding capacity of low affinity sites is not significantly affected. For both types of binding sites, the affinity constants remain in the same range of that of untreated cells. On cells made permeable by lysophosphatidylcholine, DTNB is able to inhibit the specific [125I]VIP binding in a time and dose-dependent manner. The three sulfhydryl reagents stabilize the preformed [125I]VIP receptor complex whose dissociation in the presence of native VIP is significantly reduced. Labeling of free SH groups with tritiated NEM after preincubation of cells with DTNB and VIP made possible the characterization of reacting SH groups which probably belong to the receptor. Taken together, these data allow us to define three classes of sulfhydryl groups. In addition, it is shown that high and low affinity sites have different sensibility to sulfhydryl reagents.

Adrenoceptors and adenyl cyclase in gills.

The content of cyclic 3',5'-adenosine monophosphate (cAMP) in the gills of the mullet (Mugil capito) has been measured. The basal level was found to be 2.9+/-0.3 pmol/mg wet weight. When fish were first anaesthetized with MS222 (tricaine) and the gills perfused to remove blood, the concentration of cAMP was raised to 14.3+/-1.9 pmol/mg wet weight.Adrenaline caused a significant increase in cAMP content, an effect that was blocked by propranalol, a beta-adrenoceptor antagonist. In the presence of the alpha-adrenoceptor block- ing agent phentolamine, adrenaline still caused an increase in nucleotide content.The results are discussed in relation to adrenaline and the adenyl cyclase system.

[Separation of hemoglobins F, Fac, S, C, A1c and determination of hemoglobin F using high performance liquid chromatography]. / Séparation des hémoglobines F, Fac, S, C, A1c et dosage de l'hémoglobine F par chromatographie liquide haute performance.

Separation of hemoglobins F, Fac, S, C and A1c was performed using high performance liquid chromatography with a cation exchange Polycat A column in a 15-minute assay. HbF titration results were well correlated with those of the reference alkali denaturation technique for values below 12% (r = 0.95; P < 0.001). This technique may be used as a confirmation test for neonatal screening of sickle cell disease.

Microtubules in the "chloride cell" of the gill and disruptive effects of colchicine on the salt balance of the sea water adapted Mugil capito.

Electron-microscopic examination of the gill of the grey mullet, Mugil capito adapted to sea water, reveals the presence of numerous microtubules in the apical region of the mitochondria-rich cells. No microtubules are found in other types of epithelial cell. Exposure of the fish to colchicine (10(-4)M) for four hours induces a 20% increase of plasma Na and Cl. Colchicine leaves the water permeability of the gill unchanged but Cl and Na exchange fluxes are inhibited by 30-50% and the gill potential pattern is altered. Salt gain replaces salt excretion across the gill. The K dependent Na and Cl efflux components, independent of the gill potential shift produced by K, are totally inhibited by colchicine. Exposure to lumicolchicine is not followed by a significant change of these Na and Cl efflux-components and potential pattern. The possibility that microtubules intervene in the salt excretion process across the chloride cells is discussed.

[Origin of ammonia excreted by trout gill (Salmo gairdneri)]. / Origine de l'ammonium excrété par les branchies chez la Truite (Salmo gairdneri).

In the perfused head of Trout the gills are the site of ammonia clearance which corresponds exactly to the appearance rate of ammonia in the external medium. Thus metabolic production of ammonia by the gill apparently does not participate to its excretion. Under ammonia-free Ringer perfusion however, endogenous production is observed and ammonia is excreted in both external and internal media.

Thyrotropic hormone and thyroidal function initiation in fetal rat.

In rat fetus thyrotropic hormone (TSH) is present in the blood as early as day 16 and reaches maximum on day 18. On day 21, TSH level falls off to an intermediate value. Thyroxine is detected in the plasma of fetuses from day 18; the subsequent increase in T4 concentration until day 21, probably generates the negative feedback controlling TSH secretion. The observed increase and maximum in plasma TSH are concomitant with the highest concentrations of cAMP in the thyroid. There may be a causal relationship behind this concomitance since TSH stimulates in vitro the accumulation of cAMP in explanted thyroid from 17 days-old fetuses. The implications of these findings on the role of TSH in the developing rat thyroid are discussed.

Changes in the cAMP synthesis by fetal thyroid during the 5 last days of the intrauterine life in the rat.

The cAMP synthesis by fetal rat thyroids is stimulated in vitro by thyrotropin (TSH) or forskolin during the 5 last days of intrauterine life. The effects are TSH- or forskolin-dose-dependent with equal responses to 20 mIU.ml-1 TSH or to 1 microM forskolin. The magnitude of the responses to TSH or to forskolin decreases significantly as the fetus is ageing. Since forskolin effects bypass hormone receptors, the adenylate cyclase activity can be concluded to decrease during the end of gestation probably in relation with thyroid iodine accumulation. The responses to TSH and forskolin becoming synergic between 19 1/2 and 21 1/2 days indicate modifications of adenylate cyclase properties probably related to some maturation of the enzyme.

Cyclic AMP as the second hormonal messenger in the in vitro folliculogenesis of the foetal thyroid gland of the rat.

Thyroid-stimulating hormone, the catecholamine isoproterenol, and prostaglandins E1 and E2, all substances known to increase cAMP concentration in thyroid tissue, accelerate the formation of follicular cavities in explanted thyroid of 15-day-old rat foetuses. Dibutyryl-cAMP added to the medium, but not sodium fluoride, also stimulates the folliculogenesis. Since fluoride stimulates membrane adenylate cyclase but does not increase the intracellular cAMP level, these results show that cAMP is involved as a second messenger in the activation of foetal thyroid morphogenesis induced by hormones. They indicate also that the thyroid gland of the foetal rat is capable of responding to hormonal stimulation as early as the 15th day of pregnancy; this implies that on day 15, the foetal thyroid possesses receptors not only for the thyroid-stimulating hormone, but also for catecholamines and prostaglandins.

Effect of inhibiting N-glycosylation or oligosaccharide processing on vasoactive intestinal peptide receptor binding activity and structure.

We used inhibitors of four steps of the glycosylation pathway to examine the contribution of carbohydrate moieties to the ligand-binding activity, cell-surface expression and apparent molecular mass of the human vasoactive intestinal peptide (VIP) receptor. Human melanoma IGR 39 cells, incubated for 60 h with the inhibitors tunicamycin, castanospermine, swainsonine or deoxymannojirimycin, under conditions where cell viability and protein synthesis were not affected, expressed VIP receptor species with different VIP-binding properties. The most pronounced effects on VIP binding were obtained with tunicamycin and deoxymannojirimycin, which respectively caused 80% and 67% inhibition. Treatment with either swainsonine or castanospermine resulted in only a 25-32% decrease in VIP specific binding. Based on Scatchard analyses of data from competition experiments, the decrease in VIP-binding activity in either swainsonine- or deoxymannojirimycin-treated cells was due to a decrease in ligand affinity; the cell-surface number of VIP-binding sites remained unchanged. In contrast, tunicamycin and castanospermine caused decreases in the cell-surface number of functional VIP receptors without affecting affinity. Besides, the drug-treated cells produced VIP-binding proteins with different molecular masses and endoglycosidase H (Endo H) sensitivities. When compared with their counterpart synthesized in control cells, VIP-binding proteins produced by deoxymannojirimycin- or swainsonine-treated cells were smaller in size and exhibited the expected sensitivity to Endo H. No modification in the apparent molecular mass was observed in the presence of either castanospermine or tunicamycin. In addition, after Endo F digestion, all of the deglycosylated proteins migrated with the same electrophoretic mobility. Finally, processing in the presence of castanospermine led to an Endo H-resistant receptor species which showed an unexpected neuraminidase-sensitivity, indicating that, as in control cells, these receptors carry V-linked oligosaccharides with terminal sialic acid residues.

TSH stimulation of iodine organification in early foetal rat thyroid in vitro.

Thyroid glands from 15 day-old rat foetuses were incubated in Eagle's medium containing Na 125I and supplemented, or not, with TSH for 4 or 24 hours. Electron microscopic radioautographic study shows silver grains mainly in follicular cavities only in the thyroids submitted to TSH during 24 hr. A functional differentiation must therefore take place in thyroid cells under TSH stimulation.

Fetal hypophysis as the main source of serum TSH in fetal rat.

Decapitation performed at days 17-18 leads to a drastic drop (82%) in blood TSH of 19 and 21-day-old rat fetuses below the mother's level. 125I-TSH injected at 21 days into the mother's bloodstream is not found in fetal blood. The fetal hypophysis is the main source of fetal plasmatic TSH.

Branchial effects of epinephrine in the seawater-adapted mullet. II. Na+ and Cl- extrusion.

Injection of epinephrine into Mugil capito adapted to seawater is followed by a 40-60% inhibition of the Na and Cl effluxes. Simultaneously the Na influx is decreased by 30%, the overall result being a reduction of the net sodium extrusion rate by the gill. The change in Na influx is in part explained by a 75-80% decrease of the oral ingestion of seawater. This branchial adrenergic response is sensitive to alpha-blockade by phentolamine and tolazoline and insensitive to beta-blockade by propranolol. Both alpha-blockers are ineffective when injected alone. Propranolol injected alone mimics epinephrine while simultaneous injection of phentolamine blocks the response to propranolol. Rapid transfer experiments suggest that epinephrine inhibits the branchial Cl pump and its associated Na/K exchange mechanism. The leak pathway for these ions remains insensitive to epinephrine.

A human melanoma-derived cell line (IGR39) with a very high number of vasoactive-intestinal-peptide (VIP) receptors. 2. Effect of VIP on cAMP production and on cell-surface VIP-binding sites.

Vasoactive intestinal peptide (VIP) stimulated in a dose-dependent manner the accumulation of cAMP in human melanoma-derived cell line IGR39. The maximal effect (about 100 times the basal level) was observed with 10 nM VIP. Half-maximum cAMP production was obtained at 0.78 nM VIP. VIP-related peptides were also potent in stimulating the cAMP production in IGR39 cells. The order of potency was VIP much greater than peptide histidine-methioninamide greater than human growth-hormone-releasing factor(1-44) greater than secretin greater than glucagon. Using the same conditions, IGR37 cells, a metastasic counterpart of IGR39 cells, displayed a weak stimulation of cAMP production. After exposure of IGR39 cells to 10 nM VIP, the cAMP response to a new stimulation by VIP was strongly reduced. This desensitization of IGR39 cells to VIP was rapid (t1/2 less than 2 min) and homologous. Preincubation of IGR39 cells in the presence of native VIP induced disappearance of the VIP-binding sites at the cell surface. This phenomenon was dependent on time and VIP concentration. Maximum effect (loss of 80% of binding capacity) was obtained after exposure of the cells at 37 degrees C with a VIP concentration of 1 microM. The t1/2 of maximum disappearance was less than 2 min and the concentration of VIP giving half-maximum decrease in binding of mono[125I]iodinated VIP (125I-VIP) was 8 nM. This phenomenon was also reversible since 85% of the VIP-binding capacity could be restored in less than 1 h by incubating IGR39 cells in a VIP-free medium. The IGR39 cell line should be a useful model for further study of the structure and function of the human VIP receptor.

Suramin-induced differentiation of the human colic adenocarcinoma cell clone HT29-D4 in serum-free medium.

The clonal cell line HT29-D4 was able to grow in a completely defined medium containing EGF, selenous acid, and transferrin in the presence of the anti-helminthic drug suramin. In the absence of suramin, the kinetics of cell growth and the cell density obtained were dependent on the external EGF concentration. In the presence of suramin, cell density reached a plateau independent of EGF concentration above 50 ng/ml. At the morphological level, suramin allowed hemicyst formation in the cell monolayer. The cells were polarized with a well-ordered brush border facing the culture medium and mature junctional complexes that divided the cell membrane in two distinct domains. The carcinoembryonic antigen was found to be restricted to the apical membrane domain while the major histocompatibility molecules HLA-ABC were segregated within the basolateral domain. The electrical parameters of suramin-treated cells grown on permeable filters were measured and demonstrated that the cell monolayer was electrically active. These properties were never found in the absence of the drug. Moreover, the vasoactive intestinal polypeptide (VIP) was able to induce a dramatic increase in cAMP only when it was added, in agreement with the localization of the VIP receptor, in the lower compartment of the culture chamber. In conclusion we described for the first time conditions allowing the growth of functionally differentiated human colic cell monolayers in chemically defined medium. This model will contribute to a better understanding of suramin action and of the mechanisms involved in cell polarization.

Forskolin stimulates cAMP production and the onset of the functional differentiation in the fetal rat thyroid in vitro.

Forskolin, a diterpen stimulating the adenylcyclase induces in vitro an accumulation of cAMP in thyroid glands explanted from 17 day-old rat fetuses. Thyroid glands from 15 day-old fetuses exposed to forskolin exhibit, within 24 hr, an active folliculogenesis and 125I fixation in follicular cavities. The results indicate that cAMP probably activates the onset of both morphological and physiological maturation in the fetal rat thyroid.