monensin and Pheochromocytoma

Incubation with a K+/H+ ionophore nigericin attenuated the nerve growth factor (NGF)-induced neurite outgrowth in rat pheochromocytoma PC12 cells. However, a Na+/H+ ionophore monensin and a Ca2+ ionophore A23187 did not inhibited the neurite outgrowth. Nigericin also inhibited the NGF-caused induction of acetylcholinesterase and suppression of cell proliferation. These changes were dependent on the amount of the ionophore added to the culture. In addition, a distinct K+ ionophore, valinomycin, similarly inhibited the NGF-induced neuronal differentiation. These results suggest the presence of the K+ ionophore-sensitive mechanism in the NGF-induced differentiation system in PC12 cells.

Topics: Acetylcholinesterase; Adrenal Gland Neoplasms; Animals; Calcimycin; Cell Differentiation; Dose-Response Relationship, Drug; Kinetics; Monensin; Nerve Growth Factors; Neurites; Neurons; Nigericin; PC12 Cells; Pheochromocytoma; Potassium; Rats; Valinomycin

PC12 cells express two atrial-natriuretic-factor-(ANF)-receptor subtypes with molecular masses of 130,000 (B receptor) and 70,000 (C receptor). The B-receptor subtype constitutes 65% of the cell-surface receptor population, and the remaining 35% are C receptors as determined by saturation binding studies in the presence of C-ANF, a C-receptor-selective analogue. ANF-(99-126)-peptide [ANF(99-126)], which can bind to both B- and C-receptor subtypes, was rapidly internalized into the cells after incubation at 37 degrees C. Internalization of 125I-ANF(99-126) was used as an index of the receptor-mediated endocytosis and to quantify receptor internalization. In the presence of a saturating concentration of C-ANF, receptor-mediated internalization of 125I-ANF(99-126) was reduced by 24%, indicating B receptor mediate 76% of ligand internalization. Incubation of cells with 10 microM-ANF at 37 degrees C down-regulated both receptor subtypes as reflected by decreased surface binding. Time-dependent studies suggest that B- and C-receptor subtypes undergo differential down-regulation. Incubation of down-regulated cells for 120 min in ANF-free medium produced a recovery of 35% of the original cell-surface binding. Affinity cross-linking of 125I-ANF to the receptors on the plasma membrane in re-incubated (up-regulated) cells demonstrated expression of predominantly the B-receptor subtype. Monensin blocked 72% of receptor up-regulation, whereas cycloheximide inhibited 43%, suggesting an active recycling mechanism involved in mediating up-regulation of the B receptors. The present study demonstrates a rapid internalization and intracellular recycling mechanism for B receptors in PC12 cells. C receptors also undergo internalization and down-regulation, but recycling of this receptor subtype into the plasma membrane occurs at a lower rate and to a lesser extent than is the case for the B receptor.

Topics: Adrenal Gland Neoplasms; Animals; Atrial Natriuretic Factor; Cell Line; Cell Membrane; Cycloheximide; Cytochalasin B; Down-Regulation; Kinetics; Molecular Weight; Monensin; Peptide Fragments; Pheochromocytoma; Rats; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface

The effects of agents that inhibit receptor-mediated endocytosis on type I (slow or high-affinity) and type II (fast or low-affinity) NGF binding have been examined in rat PC12 cells. Compounds interfering with endocytosis eliminate type I NGF binding; those interfering with acidification of endosomal vesicles cause increased type I binding at the expense of type II binding. Measurement of NGF binding during and after treatment with inhibitors indicates that NGF receptors rapidly cycle from the cell surface into an undefined endocytotic compartment and back to the surface with little degradation of receptor or NGF, consistent with a model in which NGF receptors are rapidly and reversibly endocytosed or sequestered; those receptors free on the surface represent type II NGF receptors, while those in the process of endocytosis represent type I NGF receptors. The type I and type II NGF receptor species can be interconverted by agents that can manipulate the position of the receptor in the internalization cycle.

Topics: Animals; Arsenicals; Chloroquine; Digitonin; Endocytosis; Monensin; Nerve Growth Factors; Pheochromocytoma; Rats; Receptors, Cell Surface; Receptors, Nerve Growth Factor; Tumor Cells, Cultured

Monensin, a monovalent cation ionophore, induced profound release of radiolabeled materials from clonal rat pheochromocytoma cells (PC12h) preloaded with [3H]norepinephrine (NE). The release was suppressed in the absence of external Na+, but was not affected at all in the absence of external Ca2+. Cytosolic free Ca2+ concentration ([Ca2+]i), that was monitored by means of a fluorescent Ca2+ indicator, Quin 2, was temporarily increased upon a depolarizing stimulus of high-K+, which induced the Ca2+-dependent release of [3H]NE from PC12h cells. On the other hand, monensin induced only a slight increase in [Ca2+]i. The radiolabeled materials released by high-K+ treatment were mainly [3H]NE, whereas those by monensin were mainly the metabolites of [3H]NE. Pargyline, a monoamine oxidase inhibitor, suppressed both the degradation of [3H]NE stored in PC12h cells and the monensin-induced release of radiolabeled compounds from them. Monensin decreased the content of [3H]NE in storage granules of pargyline-treated cells. Thus, it is likely that monensin expels NE from the storage vesicles to cytosol and then its metabolites by monoamine oxidase are released in a non-exocytotic manner.

Topics: Adrenal Gland Neoplasms; Aminoquinolines; Animals; Calcium; Cell Line; Clone Cells; Cytosol; Kinetics; Monensin; Norepinephrine; Pheochromocytoma; Potassium Chloride; Rats; Subcellular Fractions; Tritium

Monensin was used to ascertain the location in the biosynthetic pathway where the 77,000-Mr membrane-bound subunit form of dopamine beta-hydroxylase is post-translationally converted to the 73,000-Mr soluble form. Treatment with low concentrations of monensin (less than or equal to 50 nM) completely depleted the cells of the norepinephrine and dopamine, had a small effect on protein synthesis, and enhanced post-translational processing of only dopamine beta-hydroxylase which was previously synthesized and presumably packaged into neurosecretory vesicles. At these low concentrations, exit from the Golgi apparatus did not appear to be blocked since stimulated secretion of a group of high molecular weight [35S]methionine-labeled proteins was not inhibited. Treatment with higher concentrations of monensin (200 nM) prevented the secretion of the [35S] methionine-labeled proteins normally released with a secretagogue, and also prevented the secretion of [3H] mannose-labeled proteins including dopamine beta-hydroxylase. Surprisingly, a group of lower molecular weight [35S]methionine-labeled proteins was now released from monensin-treated cells. Treatment with high concentrations of monensin (greater than or equal to 200 nM) appeared to block the secretory pathway prior to the packaging step, probably in the Golgi apparatus. If the proteins were packaged prior to monensin treatment, they were released upon stimulation with secretagogues. Monensin treatment (200 nM) enabled the post-translational processing of newly synthesized dopamine beta-hydroxylase, from the 77,000-Mr to the 73,000-Mr subunit form, to go to completion. The susceptibility of this 73,000-Mr subunit form to endoglycosidase H digestion was unaltered, suggesting that dopamine beta-hydroxylase from monensin-treated cells may have the same high mannose oligosaccharide content as native dopamine beta-hydroxylase. These experiments indicate that the post-translational processing of dopamine beta-hydroxylase occurs in the Golgi apparatus and may continue in immature granules prior to their acidification.

Topics: Adrenal Gland Neoplasms; Carbohydrates; Catecholamines; Cells, Cultured; Dopamine beta-Hydroxylase; Dose-Response Relationship, Drug; Furans; Humans; Mannose; Methionine; Molecular Weight; Monensin; Nerve Tissue Proteins; Pheochromocytoma; Protein Processing, Post-Translational; Sulfur Radioisotopes

The carboxylic ionophore monensin inhibits the activity of tyrosine 3-monooxygenase and decreases the rate of catecholamine synthesis in pheochromocytoma cells incubated in vitro. The ionophore inhibits dopa production in intact pheochromocytoma cells, but does not itself inhibit tyrosine 3-monooxygenase and does not produce a stable inactivation of the enzyme as assayed in cell-free extracts of the cells. The inhibition of dopa production by monensin is dependent upon extracellular Na+, but does not require extracellular Ca++. This effect of monensin is more pronounced in the presence of pargyline. In the absence of pargyline, monensin also depletes the cells of norepinephrine and increases the accumulation of the deaminated norepinephrine metabolite, dihydroxyphenylglycol. Finally, monensin increases the release of catecholamines from isolated chromaffin granules. These results are consistent with the hypothesis that monensin causes the release of norepinephrine from chromaffin granules into the cytoplasm of pheochromocytoma cells and that the increase in cytoplasmic norepinephrine inhibits tyrosine 3-monooxygenase activity.

Topics: Adrenal Gland Neoplasms; Animals; Catecholamines; Cells, Cultured; Chromaffin Granules; Dihydroxyphenylalanine; Dopamine; Furans; Monensin; Neoplasms, Experimental; Norepinephrine; Pheochromocytoma; Rats; Tyrosine; Tyrosine 3-Monooxygenase

PC12 pheochromocytoma cell store dopamine in chromaffin-type granules. The carboxylic ionophore momensin depletes the cells of dopamine and of granules PC12 cultures incubated for 30 min with 100 nM monensin lose more than 90% of their dopamine. A small fraction of the dopamine that disappears from the cells is converted to dihydroxyphenylacetic acid. The remainder is presumably converted to other, unidentified metabolites. Monensin-treated cells contain many cytoplasmic vesicles. Some of these vesicles contain amorphous, electron-dense material, which may represent the matrix of disrupted granules. There is no morphological evidence of exocytosis. Thus, monensin appears to promote the intracellular lysis of chromaffin-type granules in PC12 cells.

Topics: Adrenal Gland Neoplasms; Adrenal Glands; Animals; Cell Line; Chromaffin Granules; Chromaffin System; Dopamine; Furans; Microscopy, Electron; Monensin; Neoplasms, Experimental; Pheochromocytoma; Rats

Topics: Amiloride; Animals; Cells, Cultured; Monensin; Neoplasms, Experimental; Nerve Growth Factors; Pheochromocytoma; Potassium; Rats; Sodium; Sodium-Potassium-Exchanging ATPase; Stimulation, Chemical

A number of carboxylic ionophores stimulate the secretion of norepinephrine from cell suspensions prepared from a transplantable rat pheochromocytoma. The divalent-cation ionophore ionomycin stimulates catecholamine secretion by a mechanism that is dependent upon the presence of extracellular Ca++. It is likely that ionomycin-induced catecholamine secretion results from the ionophore-mediated entry of Ca++ into the cells. The monovalent-cation ionophore monensin stimulates catecholamine secretion by a mechanism that is independent of extracellular Ca++, but is markedly dependent upon extracellular Na+. Monensin probably transports Na+ into the pheochromocytoma cells and increases the intracellular concentration of Na+ in these cells. This rise in intracellular Na+ may cause the release of Ca++ from some intracellular store. Lasalocid stimulates catecholamine secretion by a mechanism that is independent of extracellular Ca++ and is only slightly dependent upon extracellular Na+. The action of lasalocid, in contrast to the actions of ionomycin and monensin, is potentiated by decreased pH. It is likely that lasalocid enters the cells in its uncharged, protonated form. Once inside the cells, lasalocid may promote the release of intracellular Ca++. Alternatively, lasalocid and monensin may stimulate catecholamine secretion by the process which is independent of Ca++. These experiments show that ionophores can stimulate catecholamine secretion by at least three distinct ionic mechanisms.

Topics: Adrenal Gland Neoplasms; Barium; Calcium; Catecholamines; Ethers; Hydrogen-Ion Concentration; Ionomycin; Ionophores; Lasalocid; Monensin; Neoplasms, Experimental; Norepinephrine; Pheochromocytoma; Sodium