Inhibition of vasoactive intestinal peptide (VIP) binding on human melanoma cells IGR39 by nitric oxide: cGMP is not involved.

Nitric oxide (NO) and the NO generating agent nitroprusside (SNP), inhibit the binding of [125I] vasoactive intestinal peptide (VIP) to its receptor at the surface of IGR39 human melanoma cells. Cysteine (10 mM) increases the sensitivity of the system to SNP while N-acetylcysteine (10 mM) decreases it. The NO gas as well as SNP inhibits the [125I]VIP binding capacity. These observations sustain an effect of SNP-generated NO rather than an effect of the SNP molecule per se or the cyanoferrate portion of the molecule. The inhibitory effect of NO is time and concentration dependent and is fully reversible. Affinity constants of high and low affinity VIP receptors of SNP-treated IGR39 cells are not modified while maximal binding capacity (Bmax) of both receptor types are decreased to the same extent. Production of cGMP by SNP-treated cells is time and concentration dependent and the maximum amount of cGMP obtained reaches 13 times the basal level. The cAMP production is not affected by SNP. However, the SNP effects on the [125I]VIP binding are not mimicked by the membrane permeant cGMP analogs dibutyryl cGMP and 8-bromo cGMP even at concentrations as high as 0.5 mM. Taken altogether, these data demonstrate a regulatory action of NO on VIP binding capacity of IGR39 melanoma cells which is not cGMP mediated. They also evidence a new step which could be involved in the NO-VIP interaction.

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.

Thyroid lysosomes: the stability of the lysosomal membrane.

To determine the integrity of lysosomes during their isolation from rat thyroid glands and their subsequent incubation at 37 degrees C, the sedimentability of lysosomal acid phosphatase and thyroglobulin (amount of undisrupted lysosomes) and the latency of sedimentable acid phosphatase (permeability of undisrupted lysosomes) were measured concomitantly. The following results were obtained: (a) During isolation of lysosomes in 0.25 M sucrose medium, mild homogenization of thyroid tissue or cholesterol addition did not modify the amount of undisrupted lysosomes but reduced their permeability. Homogenization in 0.5 M sucrose decreased both the amount and the permeability of undisrupted lysosomes. It also reduced their content of recently iodinated thyroglobulin (Tg). Cholesterol addition had no effect in 0.5 M sucrose medium. (b) During incubations at 37 degrees C of lysosomes, the amount of undisrupted lysosomes decreased progressively while their permeability increased. According to the incubation pH, the permeability of lysosomes prepared in 0.25 M sucrose was either more (pH 8) or less (pH 6) extensively increased than that of lysosomes prepared in 0.5 M sucrose. From these results, we concluded: (a) that isolation and incubation of the thyroid lysosomal fraction induce increased permeability of lysosomes prior to their complete disruption: (b) that recently formed lysosomes (high content of recently iodinated Tg) and aged lysosomes (low content of recently iodinated Tg) differ significantly. Recently formed lysosomes are more permeable, are stabilized by cholesterol and are more extensively disrupted in 0.5 M sucrose medium. During incubations, the permeabilities of these two classes of lysosomes are also differently affected by external pH.

Intralysosomal hydrolysis of thyroglobulin: specific and cAMP-mediated activation by TSH.

Pig thyroid slices, pre-labelled with [125I]iodide, were incubated with or without thyrotropin (TSH), dibutyryl cyclic AMP (dbcAMP) or forskolin. After lysosome isolation, intralysosomal thyroglobulin (Tg) hydrolysis was determined by the increment in trichloroacetic acid (TCA)-soluble radioactivity. TSH stimulated the intralysosomal Tg hydrolysis. This stimulation was time and concentration dependent and was mimicked by forskolin or dbcAMP. When endocytosis and protein synthesis were blocked by inhibitors (nocodazole and puromycin) the stimulatory effect was still maintained. We conclude that TSH increases quickly and specifically, via a cAMP-mediated process, intralysosomal Tg hydrolysis, independent of its effects on endocytosis of Tg and lysosomal protease synthesis.

Ribavirin-induced resistance to heat shock, inhibition of the Ras-Raf-1 pathway and arrest in G(1).

Ribavirin [1-(beta-D-ribofuranosyl)1,2,4-triazole-3-carboxamide (virazole)], a specific inhibitor of inositide 5'-monophosphate dehydrogenase (IMPDH), induces a strong depletion of GTP pools in IGR39 cells. After a 3-day treatment, the cell cycle was reversibly arrested in G(0)/G(1), suggesting the involvement of GTP in the cell cycle process. The reduction of the GTP cell content modified the appearance of the microtubule network, as examined using immunofluorescence. However, the dynamics of repolymerisation were not altered. When arrested in G(0)/G(1), cells displayed a surprising resistance to a 3-h period of heat shock at 45 degrees C. Considering the lack of coimmunoprecipitation of p21ras with Raf-1, the reduction of the level of GTP-associated p21ras and the decrease of the activation of the extracellular signal-regulated protein kinases (ERK), also known as mitogen-activated protein (MAP) kinase, in ribavirin-treated cells, we suggest a possible relationship between the expression of heat-shock proteins and the change, in GTP-depleted cells, of the regulation of Raf kinase by ras protein.

Evidence for an ATP-driven proton pump in rat thyroid phagolysosomes. Effects of protonophores and ionophores.

During incubations at 37 degrees C in appropriate media (buffered 0.25 M sucrose) isolated thyroid phagolysosomes degrade the thyroglobulin they contain (labelled with 131I in vivo) giving rise to trichloroacetic-acid-soluble radio-iodine. Thyroglobulin-degradation is unaffected by external pH (7 or 8) or by 20-40 mM external NaCl or KCl, while it is strongly inhibited by ionophores and protonophores. As a consequence, thyroglobulin degradation can be used as an index of the intralysosomal pH which appears to be powerfully maintained in basal conditions (no ionophore and no protonophore) by the strong impermeability of the lysosomal membranes to various compounds including ionic species MgATP which does not modify basal proteolysis prevents or minimizes the alkalinizing effects of both ionophores and protonophores. ATP can thus be concluded to promote a protonic flux inward thyroid lysosomes via the activity of a lysosomal ATP-driven proton pump regulated by the magnitude of the intralysosomal pH.

Identification of glycosylphosphatidylinositol-specific phospholipases C in mouse brain membranes.

Using the membrane form of variant surface glycoprotein from Trypanosoma equiperdum labelled with [3H]myristate as a substrate, we identified two glycosylphosphatidylinositol phospholipase C enzymic activities in mouse brain. These activities were associated with particulate membrane fractions. They were characterized by their pH activity maxima and sensitivity to activators and ion chelators. One of the activities was maximal at acidic pH, stimulated by butanol, sensitive to cation chelator and insensitive to manganese. The activity of the other was maximal at neutral pH, stimulated by the detergent deoxycholate and independent of the presence of cation chelator or calcium. On membrane subfractionation, the acidic butanol-stimulated activity was found mainly associated with the lysosomal compartment, whereas the neutral deoxycholate-stimulated activity sediments with the myelin and plasma membrane compartment. These activities could be differentiated from particulate phosphatidylinositol phospholipases C, whose acidic lysosomal form is sensitive to manganese and insensitive to cation chelator or butanol, whereas the deoxycholate-activated enzymes are Ca2(+)-dependent.

Intralysosomal hydrolysis of thyroglobulin. II. Different fates of poorly and fully iodinated Tg and specific activation by TSH of the degradation of fully iodinated Tg.

Degradations of recently (1 h) iodinated thyroglobulin (Tg) and of Tg iodinated for 48 h (fully iodinated molecule) inside thyroid phagolysosomes have been measured in vitro, at different pH, in basal (lysosomes from normal rats) and stimulated (lysosomes from TSH-treated rats) conditions. In basal conditions, recently iodinated Tg was shown to be preferentially degraded at pH 5 but not at pH 8. Mercaptoethanol (ME) was found to preferentially stimulate the degradation of 48 h-iodinated Tg at pH 5 but not at pH 8. In stimulated conditions and in the presence of ME, no preferential hydrolysis of recently iodinated Tg was observed, whatever the pH. Since the degradation of Tg and its stimulation by ME are known to depend on the mean iodine content of the molecule, it was concluded that: 1) the recently iodinated Tg population contains both poorly and fully iodinated molecules, the degradations of which are faster or similar to that of 48 h-iodinated Tg, respectively, 2) among thyroid phagolysosomes, only those which exhibit a pH-dependent proteolytic activity contain poorly iodinated molecules while others, which are functional at either pH, contain solely fully iodinated molecules (1 and 48 h labelled), 3) reduction of the disulphide bridges of fully iodinated Tg, mediated by ME and probably a lysosomal transhydrogenase, is a prerequisite for its optimal degradation by lysosomal proteinases, 4) in addition, TSH, which preferentially stimulates the degradation of fully iodinated Tg, was concluded to interfere directly upon the intralysosomal hydrolytic process, independently of its effect upon Tg endocytosis. This effect of TSH, similar and additive to that of ME suggests that the hormone might activate a lysosomal transhydrogenase.

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.

ATP-induced stimulation of the intralysosomal hydrolysis of thyroglobulin. Evidence for an ATP-driven proton pump in thyroid lysosomes.

It is commonly admitted that the intralysosomal hydrolysis of thyroglobulin, which promotes thyroid hormonal secretion, requires an acidic pH. As a consequence the mechanisms by which such an acidic pH is set-up and maintained present an acute physiological interest. In the present work the extent of the intralysosomal hydrolysis of thyroglobulin (amount of TCA-soluble radioiodine formed during incubations of isolated thyroid lysosomes) has been retained as an index for the intralysosomal pH. Incubations in highly concentrated (250 mM) alkaline buffer, preincubations at 0 degree C in the presence of alkaline buffers or of methylamine, and incubations in the presence of FCCP (protonophore which dissipates proton-gradients) induced alkalinization of the lysosomal matrix (significant inhibition of thyroglobulin degradation). Addition of ATP to the incubation medium was shown to prevent these alkalinizations and to re-establish the acidic intralysosomal pH (normal thyroglobulin degradation) every time it had been experimentally increased. These results argue in favour of the existence of an ATP-driven proton pump in the membranes of thyroid lysosomes.

Intralysosomal hydrolysis of thyroglobulin. I. Modulation by lysosomal membrane permeability and exogenous factors.

Intralysosomal hydrolysis of endogenous (125I in vivo labelled) thyroglobulin (Tg) inside thyroid phagolysosomes and its modulation by exogenous factors (external pH, activators and inhibitors of lysosomal proteinases) were studied during in vitro incubations. Tg degradation was followed as the time-dependent increase in TCA-soluble radioiodine (free iodoaminoacids). The results demonstrate that this hydrolysis is only partially pH-dependent, one component being abolished by pH 8 and a cathepsin B inhibitor (zinc) whereas the residual activity was pH-independent and insensitive to zinc. It would seem therefore that the total thyroid phagolysosome population contains lysosomes permeable to both incubation buffer and zinc and lysosomes impermeable to these compounds. Both classes are operational (degrade Tg) at acidic or neutral pHs, while only impermeable organelles function when incubated at pH 8. These impermeable lysosomes have an acidic internal pH (about pH 5) and resist alkalinization very efficiently due to the high impermeability of their membranes. Additional experiments are in progress to determine the physiological significance of these two classes of lysosomes which might be related to a functional maturation of thyroid phagolysosomes.

Hydrolysis of thyroglobulin inside isolated rat thyroid and liver lysosomes.

In vitro, poorly iodinated thyroglobulin (Tg) is hydrolysed at the same rate whether it is enclosed in thyroid or in liver lysosomes, while fully iodinated Tg is degraded faster inside liver lysosomes. After in vivo TSH administration, thyroid lysosomes hydrolyse fully iodinated Tg as fast as do liver lysosomes.

Glycosylphosphatidylinositol is involved in the membrane attachment of proteins in granules of chromaffin cells.

Incubation at 37 degrees C or treatment of granule membranes of chromaffin cells with Staphylococcus aureus phosphatidylinositol-specific phospholipase C converted from an amphiphilic to a hydrophilic form two proteins with molecular masses of 82 and 68 kDa respectively. Their release is time- and enzyme-concentration-dependent. We showed that they were immunoreactive with an anti-(cross-reacting determinant) antibody known to be revealed only after removal of a diacylglycerol anchor. Furthermore, the action of HNO2 suggests the presence of a non-acetylated glucosamine residue in the determinant. This is one of the first reports suggesting that a glycosylphosphatidylinositol anchor might exist in membranes other than the plasma membrane. We showed that the 68 kDa protein is probably not the subunit of dopamine (3,4-dihydroxyphenethylamine) beta-hydroxylase, an enzyme present in granules in both soluble and membrane-associated forms.

The effects of ribavirin on the GTP level and the VIP receptor dynamic of human IGR39 cells.

GTP is one of the major cellular molecules involved in fundamental functions of cell life. Ribavirin, and antiviral and antitumoral agent, the primary site of action of which is the IMP deshydrogenase, was used in order to depress the intracellular GTP level. Consequential effects were tested on the property and dynamic of the VIP receptor on human melanoma IGR 39 cells. A concentration of 100 microM of Ribavirin reduced the intracelluar GTP level by more than 60% and induced a reversible growth arrest. Nevertheless this drug displayed no effect on: i) the VIP binding parameters (Kd and Bmax) of both high and low affinity receptors; ii) the cycling of the VIP receptor; iii) the based and VIP-stimulated cAMP production and iv) the subcellular GTP distribution. We show that Ribavirin, in the range of concentrations used, is very efficient to inhibit GTP synthesis in the human melanoma cell line IGR 39 and its growth, without affecting VIP receptor functions.

Role of endoproteolytic processing in the adhesive and signaling functions of alphavbeta5 integrin.

Some integrin alpha subunits undergo a post-translational cleavage in their extracellular domain. However, the role of this cleavage in integrin function is unclear. Enzymes involved in this maturation belong to the subtilisin-like endoprotease family (convertases). To understand the role of the alpha subunit cleavage in integrin function, we have designed stable transfectants (PDX39P cells) expressing alpha(1)-PDX, a convertase inhibitor. Immunoprecipitation of cell surface proteins from PDX39P showed that alpha(3), alpha(6) and alpha(v) integrins lack endoproteolytic cleavage. We have compared adhesion between PDX39P cells and mock-transfected cells on different extracellular matrix proteins. No difference in adhesion could be observed on laminin-1 and type I collagen, while attachment of PDX39P cells to vitronectin (ligand of the alpha(v)beta(5) integrin) was dramatically reduced. The reduced adhesion of PDX39P cells was not due to changes in integrin affinity as determined by solid-phase receptor assay in a cell-free environment. Intracellular signaling pathways activated by alpha(v) integrin ligation were also affected in PDX39P cells. It thus seems that the absence of endoproteolytic cleavage of alpha(v) integrins has important consequences on signal transduction pathways leading to alterations in integrin function such as cell adhesion.