concanavalin-a and oxophenylarsine

The agonist-induced internalization of several G protein-coupled receptors is an obligatory requirement for their activation of MAPKs. Studies on the relationship between endocytosis of the angiotensin II (Ang II) type 1 receptor (AT1-R) and Ang II-induced ERK1/2 activation were performed in clone 9 (C9) rat hepatic cells treated with inhibitors of endocytosis [sucrose, phenylarsine oxide (PAO), and concanavalin A]. Although Ang II-induced endocytosis of the AT1-R was prevented by sucrose and PAO, and was partially inhibited by concanavalin A, there was no impairment of Ang II-induced ERK activation. However, the specific epidermal growth factor receptor (EGF-R) kinase inhibitor, AG1478, abolished Ang II-induced activation of ERK1/2. Sucrose and PAO also inhibited EGFinduced internalization of the EGF-R in C9 cells, and the inability of these agents to impair EGF-induced ERK activation suggested that the latter is also independent of receptor endocytosis. In COS-7 cells transiently expressing the rat AT1A-R, Ang II also caused ERK activation through EGF-R transactivation. Furthermore, a mutant AT1A-R with truncated carboxyl terminus and impaired internalization retained full ability to activate ERK1/2 in response to Ang II stimulation. These findings demonstrate that Ang II-induced ERK1/2 activation in C9 hepatocytes is independent of both AT1-R and EGF-R endocytosis and is mediated by transactivation of the EGF-R.

Topics: Angiotensin II; Animals; Arsenicals; Cell Line; Concanavalin A; COS Cells; Endocytosis; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Hepatocytes; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Quinazolines; Rats; Receptor, Angiotensin, Type 1; Receptors, Angiotensin; Sucrose; Transfection; Tyrphostins

Previous studies have demonstrated that an increase in the activity of protein-tyrosine kinase (PTK) is involved in the down-regulation of the activity of apical small conductance K(+) (SK) channels in the cortical collecting duct (CCD) from rats on a K(+)-deficient diet (). We used the patch clamp technique to investigate the role of protein-tyrosine phosphatase (PTP) in the regulation of the activity of SK channels in the CCD from rats on a high K(+) diet. Western blot analysis indicated that PTP-1D is expressed in the renal cortex. Application of 1 microm phenylarsine oxide (PAO) or 1 mm benzylphosphonic acid, agents that inhibit PTP, reversibly reduced channel activity by 95%. Pretreatment of CCDs with PAO for 30 min decreased the mean NP(o) reversibly from control value 3.20 to 0.40. Addition of 1 microm herbimycin A, an inhibitor of PTK, had no significant effect on channel activity in the CCDs from rats on a high K(+) diet. However, herbimycin A abolished the inhibitory effect of PAO, indicating that the effect of PAO is the result of interaction between PTK and PTP. Addition of brefeldin A, an agent that blocks protein trafficking from Golgi complex to the membrane, had no effect on channel activity. Moreover, application of colchicine, a microtubule inhibitor, or paclitaxel, a microtubule stabilizer, had no effect on channel activity. In contrast, PAO still reduced channel activity in the presence of brefeldin A, colchicine, or paclitaxel. Furthermore, the effect of PAO on channel activity was absent when the tubules were bathed in 16% sucrose-containing bath solution or treated with concanavalin A. We conclude that PTP is involved in the regulation of the activity of SK channels and that inhibition of PTP may facilitate the internalization of the SK channels.

Topics: Animals; Arsenicals; Benzoquinones; Brefeldin A; Colchicine; Concanavalin A; Diet; Enzyme Inhibitors; Female; Intracellular Signaling Peptides and Proteins; Kidney Tubules, Collecting; Lactams, Macrocyclic; Male; Paclitaxel; Patch-Clamp Techniques; Potassium; Potassium Channels; Potassium Channels, Calcium-Activated; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; Quinones; Rats; Rats, Sprague-Dawley; Rifabutin; Small-Conductance Calcium-Activated Potassium Channels

Mice homozygous for the gld (generalized lymphoproliferative disease) mutation develop both lymphadenopathy and autoimmune disease. CD4-CD8- (double negative, DN) T cells comprise the major population of T cells in mature C3H-gld/gld peripheral lymphoid tissues. These DN T cells are unresponsive to many forms of stimuli and have previously been shown to exhibit abnormally elevated levels of membrane phosphotyrosine phosphatase (PTPase) activity. In the present study, we demonstrate that IP3 production in response to mitogenic stimulation with Con A or anti-CD3 mAb (145-2CII) is significantly diminished in C3H-gld/gld lymphocytes when compared to that in congenic C3H(-)+/+ cells. The capacity to produce this second-messenger can be restored by pretreating C3H-gld/gld cells with the PTPase inhibitor, phenylarsine oxide (PAO). Although the inhibition of PTPase activity by treatment with PAO did restore C3H-gld/gld cell ability to produce IP3, the signal did not lead to lymphocyte proliferation, but instead to cell death. Our results suggest that the altered phosphoinositide hydrolysis observed in the mutant cells is related to their elevated membrane PTPase activity and that the anergy in these cells is at least in part related to the abnormally high activity of endogenous PTPases.

Topics: Animals; Arsenicals; Cell Survival; Clonal Anergy; Concanavalin A; Dose-Response Relationship, Immunologic; Hydrolysis; Inositol 1,4,5-Trisphosphate; Lymph Nodes; Lymphocyte Activation; Lymphocytes; Mice; Mice, Inbred C3H; Mice, Mutant Strains; Phosphatidylinositols; Protein Tyrosine Phosphatases

Phorbol myristate acetate (PMA) plus ionomycin induces the tyrosine phosphorylation of several cytotoxic T lymphocyte (CTL) substrates, including one with an apparent molecular weight of 100,000 (pp100) in cloned murine CTL. cis-Unsaturated fatty acids and low concentrations of phenylarsine oxide specifically inhibit the tyrosine phosphorylation of pp100. Genistein also inhibits tyrosine phosphorylation of pp100, but not with the same specificity as cis-fatty acids or low concentrations of phenylarsine oxide. Degranulation triggered by PMA plus ionomycin is inhibited by cis-fatty acids, low concentrations of phenylarsine oxide, and genistein, under the same conditions that these agents inhibit tyrosine phosphorylation of pp100. Depleting CTL of protein kinase C (PKC) activity by prolonged exposure to PMA eliminates the increase in tyrosine phosphorylation when challenged by PMA plus ionomycin, but not when these PKC-depleted CTL are activated by cognate target cells, immobilized anti-T cell receptor (TCR) antibodies, or concanavalin A. Tyrosine phosphorylation of pp100 triggered by TCR engagement in PKC-depleted cells is inhibited by cis-fatty acids and phenylarsine oxide, indicating that the inhibitory mechanism of the tyrosine phosphorylation of pp100 is independent of PKC. Furthermore, because all three tyrosine phosphorylation inhibitors are unlikely to inhibit PKC, these results suggest that, in addition to PKC activation and a rise in intracellular Ca2+, CTL degranulation requires the tyrosine phosphorylation of a CTL substrate(s), in addition to phospholipase C, and the present results are consistent with pp100 as that substrate. Taken together with previous studies, these results suggest that tyrosine phosphorylation of pp100 may play a central role in CTL function.

Topics: Animals; Arsenicals; Calcium; Clone Cells; Concanavalin A; Fatty Acids, Nonesterified; Genistein; Ionomycin; Isoflavones; Kinetics; Lymphocyte Activation; Mice; Molecular Weight; Oleic Acid; Oleic Acids; Phosphoproteins; Phosphotyrosine; Protein Kinase C; Protein-Tyrosine Kinases; T-Lymphocytes, Cytotoxic; Tetradecanoylphorbol Acetate; Tyrosine

Bradykinin-induced activation of peripheral nociceptors has been studied in an isolated spinal cord/tail preparation from the neonatal rat. Prolonged administration of bradykinin consistently produced a selective desensitization which could be prevented by concanavalin A but not by succinyl concanavalin A or phenylarsine oxide. These data indicate that mannose-containing glycoproteins occur in or close to the bradykinin receptor site. In addition the desensitization observed under the present conditions, did not involve the internalization of bradykinin receptors.

Topics: Animals; Animals, Newborn; Arsenicals; Bradykinin; Concanavalin A; In Vitro Techniques; Membrane Potentials; Nerve Fibers; Nociceptors; Rats; Spinal Cord

Human A431 and rat glioma C6 cells exposed to isoproterenol underwent a time- and dose-dependent loss of isoproterenol-stimulated adenylate cyclase activity. Desensitization was accompanied by sequestration of beta-adrenergic receptors, which became less accessible to the hydrophilic antagonist 3H-labeled 4-(3-tert-butylamino-2-hydroxypropoxy)benzimidazole-2-one hydrochloride ([3H]CGP-12177) and redistributed from the heavier density plasma membrane fraction to a lighter density membrane fraction. Prior treatment of the cells with concanavalin A or phenylarsine oxide blocked sequestration of the receptors but not desensitization of the agonist-stimulated adenylate cyclase. The membranes from such pretreated cells were exposed to alkali to inactivate adenylate cyclase, and the receptors were transferred to a foreign adenylate cyclase by membrane fusion with polyethylene glycol. beta receptors from desensitized cells exhibited a reduced ability to maximally stimulate the foreign adenylate cyclase, but remained accessible to [3H]CGP-12177 in the fused membranes. When isoproterenol-treated cells were washed free of agonist, there was a time-dependent recovery of agonist responsiveness and [3H]CGP-12177-binding sites. Using the fusion technique, the receptors recovered their functional activity in the resensitized cells. In concanavalin A-treated cells, desensitization and resensitization appeared to occur in the absence of receptor sequestration. Finally, membranes from desensitized cells pretreated with concanavalin A were fused with polyethylene glycol and assayed for agonist-stimulated adenylate cyclase. There was no reversal of the desensitized state. Thus, the primary, essential step in the desensitization process is a reduction in functional activity of the beta-adrenergic receptor. In contrast, sequestration of the receptors is not a prerequisite, but a secondary event during desensitization.

Topics: Adenylyl Cyclases; Animals; Arsenicals; Cell Line; Concanavalin A; Dose-Response Relationship, Drug; Humans; Isoproterenol; Propanolamines; Rats; Receptors, Adrenergic, beta; Time Factors