thapsigargin and oxophenylarsine

1 Ca2+ imaging was used to investigate interactions between responses induced by N-methyl-D-aspartate (NMDA; 15 microm) and (RS)-3,5-dihydroxyphenyl-glycine (DHPG; 30 microm) in human embryonic kidney (HEK) 293 cells, transiently transfected with rat recombinant NR1a, NR2A and mGlu5a cDNA. 2 Responses to NMDA were reversibly depressed by DHPG from 244+/-14 to 194+/-12% of baseline. Treatment with thapsigargin (1 microm, 10 min) prevented this effect. 3 After thapsigargin pretreatment, repeated applications of NMDA showed a gradual rundown in amplitude over a period of several hours, and were unaffected by DHPG. 4 Continuous perfusion with staurosporine (0.1 microm), after thapsigargin pretreatment, converted the run-down to a small increase in NMDA responses to 123+/-6 % of baseline. DHPG induced a further and sustained potentiation of NMDA responses to 174+/-12% of the initial baseline. 5 The protein tyrosine kinase (PTK) inhibitors genistein (50 microm) and 3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2; 1 microm) inhibited the staurosporine- and DHPG-induced potentiation of NMDA responses. 6 The protein phosphatase (PTP) inhibitors orthovanadate (100 microm) and phenyl arsine oxide (PAO, 1 microm) facilitated the staurosporine-evoked potentiation of NMDA responses and occluded DHPG-induced potentiation. 7 In conclusion, complex interactions can be demonstrated between mGlu5 and NMDA receptors expressed in HEK293 cells. There is a negative inhibitory influence of Ca2+ release and PKC activation. Inhibition of these processes reveals a tonic, mGlu5 receptor and PTK-dependent potentiation of NMDA receptors that can be augmented by either stimulating mGlu5 receptors or by inhibiting PTPs.

Topics: Animals; Arsenicals; Cell Line; Drug Synergism; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Gene Expression; Genistein; Glycine; Humans; N-Methylaspartate; Phosphorylation; Protein-Tyrosine Kinases; Rats; Receptor Cross-Talk; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Recombinant Fusion Proteins; Resorcinols; Staurosporine; Thapsigargin; Transfection; Tyrosine; Vanadates

The endogenous divalent cations, Ca(2+) and Zn(2+), are both highly toxic upon excessive glutamate triggered intracellular accumulation. Given apparent parallels in their neurotoxic mechanisms, the present study aimed to explore interactions between these cations, by examining effects of moderate intracellular Zn(2+) loading on responses to subsequent Ca(2+) influx. Cortical cultures were briefly exposed to high-K(+) buffer in the presence or absence of Zn(2+) (50-100 microM), to activate and permit a modestly toxic amount of Zn(2+) to enter through VSCC. After 1 h, the cultures were loaded with fluorescent probes, and 2 h after the Zn(2+) exposure, imaged before and after induction of Ca(2+) entry or addition of other drugs. In Zn(2+) preexposed cultures loaded with the Zn(2+) probe, Newport Green, induction of Ca(2+) entry through either VSCC or NMDA channels induced cytoplasmic release of sequestered Zn(2+). The source of this Ca(2+) dependent intracellular Zn(2+) release appears largely to be mitochondria, as indicated by the ability of the mitochondrial protonophore, FCCP, the mitochondrial uncoupler, dinitrophenol with the K(+) ionophore, valinomycin, or the inducer of mitochondrial permeability transition (mPT), phenylarsine oxide (PAO), to substitute for NMDA in triggering Zn(2+) release. Suggesting functional consequences of mitochondrial Zn(2+) uptake, Zn(2+) preexposures resulted in long-lasting mitochondrial depolarization (assessed with rhodamine 123), and reduced mitochondrial reactive oxygen species generation (assessed with hydroethidine) in response to subsequent NMDA triggered Ca(2+) influx.

Topics: Animals; Arsenicals; Calcium; Calcium Channels; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Compartmentation; Cerebral Cortex; Cytosol; Dinitrophenols; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Intracellular Membranes; Ion Channel Gating; Ion Transport; Ionophores; Membrane Potentials; Mice; Mitochondria; Nerve Tissue Proteins; Neurons; Potassium; Reactive Oxygen Species; Receptors, N-Methyl-D-Aspartate; Synaptic Vesicles; Thapsigargin; Uncoupling Agents; Valinomycin; Zinc

Agonists elevate the cytosolic calcium concentration in human platelets via a receptor-operated mechanism, involving both Ca(2+) release from intracellular stores and subsequent Ca(2+) entry, which can be inhibited by platelet inhibitors, such as prostaglandin E(1) and nitroprusside which elevate cAMP and cGMP, respectively. In the present study we investigated the mechanisms by which cAMP and cGMP modulate store-mediated Ca(2+) entry. Both prostaglandin E(1) and sodium nitroprusside inhibited thapsigargin-evoked store-mediated Ca(2+) entry and actin polymerization. However, addition of these agents after induction of store-mediated Ca(2+) entry did not affect either Ca(2+) entry or actin polymerization. Furthermore, prostaglandin E(1) and sodium nitroprusside dramatically inhibited the tyrosine phosphorylation induced by depletion of the internal Ca(2+) stores or agonist stimulation without affecting the activation of Ras or the Ras-activated phosphatidylinositol 3-kinase or extracellular signal-related kinase (ERK) pathways. Inhibition of cyclic nucleotide-dependent protein kinases prevented inhibition of agonist-evoked Ca(2+) release but it did not have any effect on the inhibition of Ca(2+) entry or actin polymerization. Phenylarsine oxide and vanadate, inhibitors of protein-tyrosine phosphatases prevented the inhibitory effects of the cGMP and cAMP elevating agents on Ca(2+) entry and actin polymerization. These results suggest that Ca(2+) entry in human platelets is directly down-regulated by cGMP and cAMP by a mechanism involving the inhibition of cytoskeletal reorganization via the activation of protein tyrosine phosphatases.

Topics: Actins; Adenosine Diphosphate; Alprostadil; Arsenicals; Blood Platelets; Calcium; Carbazoles; Cyclic AMP; Cyclic GMP; Egtazic Acid; Enzyme Activation; Enzyme Inhibitors; Humans; In Vitro Techniques; Indoles; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nitroprusside; Phosphorylation; Platelet Activation; Platelet Aggregation Inhibitors; Protein Tyrosine Phosphatases; Pyrroles; Thapsigargin; Thrombin; Vanadates

Although many antipsychotics have affinities for sigma receptors, the transportation pathway of exogenous sigma(1) receptor ligands to intracellular type-1 sigma receptors are not fully understood. In this study, sigma(1) receptor ligand uptakes were studied using primary cultured neuronal cells. [(3)H](+)-pentazocine and [(3)H](R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate (MS-377), used as a selective sigma(1) receptor ligands, were taken up in a time-, energy- and temperature-dependent manner, suggesting that active transport mechanisms were involved in their uptakes. sigma(1) receptor ligands taken up into primary cultured neuronal cells were not restricted to agonists, but also concerned antagonists. The uptakes of these ligands were mainly Na(+)-independent. Kinetic analysis of [(3)H](+)-pentazocine and [(3)H]MS-377 uptake showed K(m) values (microM) of 0.27 and 0.32, and V(max) values (pmol/mg protein/min) of 17.4 and 9.4, respectively. Although both ligands were incorporated, the pharmacological properties of these two ligands were different. Uptake of [(3)H](+)-pentazocine was inhibited in the range 0.4-7.1 microM by all the sigma(1) receptor ligands used, including N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a selective sigma(1) receptor ligand. In contrast, the inhibition of [(3)H]MS-377 uptake was potently inhibited by haloperidol, characterized by supersensitivity (IC(50), approximately 2 nM) and was inhibited by NE-100 with low sensitivity (IC(50), 4.5 microM). Moreover, kinetic analysis revealed that NE-100 inhibited [(3)H]MS-377 uptake in a noncompetitive manner, suggesting that NE-100 acted at a site different from the uptake sites of [(3)H]MS-377. These findings suggest that there are at least two uptake pathways for sigma(1) receptor ligands in primary cultured neuronal cells (i.e. a haloperidol-sensitive pathway and another, unclear, pathway). In addition, pretreatment of cells with a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), a myosin light chain kinase inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)homopiperazine (ML-9), or microsomal Ca(2+)-ATPase inhibitors resulted in a reduction of the amount of sigma receptor ligand uptake. These findings suggest that the Ca(2+) pump on the endoplasmic reticulum and/or calmodulin-related events might be involved in the regulation of the uptake of sigma receptor lig

Topics: Animals; Anisoles; Arsenicals; Biological Transport; Calcium; Calcium-Transporting ATPases; Calmodulin; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Haloperidol; Hydroquinones; Kinetics; Ligands; Neurons; Ouabain; Pentazocine; Piperazines; Propylamines; Pyrrolidines; Rats; Receptors, sigma; Sigma-1 Receptor; Sodium; Sulfonamides; Tartrates; Thapsigargin; Time Factors; Tritium

Proteinase-activated receptor-2 (PAR-2) is a G-protein-coupled receptor that is expressed by intestinal epithelial cells, which are episodically exposed to pancreatic trypsin in the intestinal lumen. Trypsin cleaves PAR-2 to expose a tethered ligand, which irreversibly activates the receptor. Thus, PAR-2 may desensitize and resensitize by novel mechanisms. We examined these mechanisms in kidney epithelial cells, stably expressing human PAR-2, and intestinal epithelial cells, which naturally express PAR-2. Trypsin stimulated a prompt increase in [Ca2+]i, due to mobilization of intracellular Ca2+, followed by a sustained plateau, due to influx of extracellular Ca2+. Repeated application of trypsin caused marked desensitization of this response, which is due in part to (a) irreversible cleavage of the receptor by trypsin and (b) protein kinase C-mediated termination of signaling. Trypsin exposure resulted in internalization of PAR-2 into early endosomes and then lysosomes; but endocytosis was not the mechanism of rapid desensitization. Thus, activated PAR-2 is endocytosed and degraded. The Ca2+ response to trypsin resensitized by 60-90 min. Brefeldin A, which disrupted Golgi stores of PAR-2, and cycloheximide, which inhibited protein synthesis, markedly attenuated resensitization. Thus, PAR-2-mediated Ca2+ mobilization desensitizes by irreversible receptor cleavage, protein kinase C-mediated termination of signaling, and PAR-2 targeting to lysosomes. It resensitizes by mobilization of large Golgi stores and synthesis of new receptors.

Topics: Arsenicals; Bradykinin; Calcium; Calcium-Transporting ATPases; Cell Line; Endocytosis; Extracellular Space; Fluorescent Antibody Technique; Humans; Interleukin-8; Intestinal Mucosa; Kidney; Lysosomes; Microscopy, Confocal; Protein Kinase C; Receptor, PAR-2; Receptors, Cell Surface; Terpenes; Thapsigargin; Trypsin; Type C Phospholipases

In the rat isolated main pulmonary artery, we investigated the effect of a tyrosine kinase inhibitor (genistein) and that of a tyrosine phosphatase inhibitor (phenylarsine oxide) on agonist-induced contraction. Genistein (10 microM) reduced the amplitude of the contraction evoked by noradrenaline (0.1-10 microM) or angiotensin II (1-100 nM). Phenylarsine oxide (0.5 microM) increased the amplitude of the contraction evoked by these agonists. The effects of genistein and phenylarsine oxide on agonist-induced contractions were also observed in the presence of verapamil (10 microM). Thapsigargin (0.5 microM) increased the amplitude of the contraction induced by noradrenaline (1-10 microM) or angiotensin II (10-100 nM). Subsequent addition of genistein counteracted the effect of thapsigargin on noradrenaline- and angiotensin II-induced contraction. Dantrolene alone (100 microM) reduced noradrenaline- and angiotensin II- but not KCI-induced contraction. In the presence of dantrolene, genistein and phenylarsine oxide failed to modify noradrenaline- and angiotensin II-induced contraction. Finally, in beta-escin skinned preparations, genistein (10-20 microM) and phenylarsine oxide (0.5-1 microM) did not alter Ca(2+)-induced contraction. These results suggest that a tyrosine kinase activity is involved in the vasoconstrictor action of noradrenaline and angiotensin II in the pulmonary circulation. The stimulation of the tyrosine kinase activity appears to be linked to the depletion of an intracellular Ca2+ store.

Topics: Adrenergic alpha-Agonists; Angiotensin II; Animals; Arsenicals; Calcium; Dantrolene; Enzyme Inhibitors; Escin; Genistein; In Vitro Techniques; Intracellular Fluid; Isoflavones; Kinetics; Male; Muscle Contraction; Muscle, Smooth, Vascular; Norepinephrine; Protein-Tyrosine Kinases; Pulmonary Artery; Rats; Rats, Wistar; Thapsigargin; Vasoconstrictor Agents

Phenylarsine oxide (PAO), an inhibitor of tyrosine phosphatases, has been found to inhibit the early elevation in cytosolic Ca2+ concentration ([Ca2+]i), related to the CD3 activation pathway in Jurkat T cells. This inhibition was dose-dependent, consistent with previously reported effects of PAO on tyrosine phosphatases, and reversed by dimercaptopropanol. By contrast, okadaic acid, an inhibitor of serine/threonine phosphatases, had no effect on CD3-induced Ca2+ flux. PAO was compared with phorbol 12-myristate 13-acetate (PMA), which caused a similar, although less potent, inhibition as previously described. The two reagents produced additive inhibition of the CD3-induced [Ca2+]i rise, but did not affect thapsigargin- or ionomycin-driven Ca2+ flux in Jurkat cells. PAO and PMA prevented cells from complete depletion of intracellular Ca2+ stores by an anti-CD3 monoclonal antibody (mAb) and restored, at least partially, the ionomycin-sensitive pool, when added after anti-CD3 mAb. Moreover, the CD3-induced inhibition of phosphatidylserine synthesis, due to depletion of internal Ca2+ stores, is reversed by PAO and PMA. Anti-phosphotyrosine immunoblot analysis show that these effects cannot be accounted for by an inhibition of CD3-induced tyrosine phosphorylations. We propose that PAO and, to a lesser extent, PMA allow the refilling of internal compartments by Ca2+, which consequently abrogates a capacitative entry of external Ca2+.

Topics: Arsenicals; Biological Transport; Calcium; CD3 Complex; Cell Line; Cytosol; Ethers, Cyclic; Humans; Ionomycin; Okadaic Acid; Phosphatidylserines; Phosphorylation; T-Lymphocytes; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Tyrosine