sq-23377 and Pheochromocytoma

Neuronal calcium sensor-1 (NCS-1), the mammalian orthologue of frequenin, belongs to a family of EF-hand-containing Ca(2+) sensors. NCS-1/frequenin has been shown to enhance synaptic transmission in PC12 cells and Drosophila and Xenopus, respectively. However, the precise molecular mechanism for the enhancement of exocytosis is largely unknown. In PC12 cells, NCS-1 potentiated exocytosis evoked by ATP, an agonist to phospholipase C-linked receptors, but had no effect on depolarization-evoked release. NCS-1 also enhanced exocytosis triggered by ionomycin, a Ca(2+) ionophore that bypasses K(+) and Ca(2+) channels. Overexpression of NCS-1 caused a shift in the dose-response curve of inhibition of ATP-evoked secretion using phenylarsine oxide, an inhibitor of phosphatidylinositol 4-OH kinase (PI4K). Plasma membrane phosphatidylinositol 4,5-bisphosphate pools were increased upon NCS-1 transfection as visualized using a phospholipase C-delta pleckstrin homology domain-green fluorescent protein construct. NCS-1-transfected cell extracts displayed increased phosphatidylinositol-4-phosphate biosynthesis, indicating an increase in PI4K activity. Mutations in NCS-1 equivalent to those that abolish the interaction of recoverin, another EF-hand-containing Ca(2+) sensor, with its downstream target rhodopsin kinase, lost their ability to enhance exocytosis. Taken together, the present data indicate that NCS-1 modulates the activity of PI4K, leading to increased levels of phosphoinositides and concomitant enhancement of exocytosis.

Topics: 1-Phosphatidylinositol 4-Kinase; Adenosine Triphosphate; Animals; Arsenicals; Calcium Signaling; Calcium-Binding Proteins; Drosophila; Enzyme Inhibitors; Exocytosis; Ionomycin; Membrane Potentials; Neuronal Calcium-Sensor Proteins; Neuropeptides; Neurosecretory Systems; PC12 Cells; Pheochromocytoma; Rats; Type C Phospholipases; Xenopus

Phospholipase A2 has recently been proposed as the effector enzyme involved in the receptor-mediated release of arachidonic acid (AA). Released AA and its metabolites have been demonstrated to play an important role in the regulation of cell functions. [3H]AA release from prelabeled PC12 cells was stimulated by a Ca ionophore such as ionomycin or A23187. Although ATP and its effective analog, adenosine 5'-O-(3-thiotrisphosphate) (ATP gamma S), 2-methylthio ATP and 3'-O-(4-benzoyl)benzoyl ATP, did not stimulate [3H]AA release on their own, they did enhance Ca ionophore-stimulated [3H]AA release. The effect of ATP analogs was dose-dependent. ADP, UTP, GTP, ITP, alpha beta-methylene ATP, beta gamma-methylene ATP and 8-bromo ATP showed no effect or very limited effect. The effect of ATP gamma S was antagonized by suramin, a putative P2Y receptor antagonist. The effective ATP analogs also increased [Ca2+]i (cytosolic free Ca2+ concentration) via Ca2+ influx. However, the addition of 50 mM KCl or 10 microM bradykinin, which are well-known to increase [Ca2+]i by different pathways, did not stimulate [3H]AA release, either with or without the Ca ionophore. The addition of phorbol 12-myristate 13-acetate, an activator of protein kinase C, showed no effect on [3H]AA release, either with or without the Ca ionophore. These data suggest that 1) ATP increased Ca ionophore-stimulated AA release via a P2Y-like ATP receptor, and that 2) the elevation of [Ca2+]i by ATP does not quantitatively explain the ATP-stimulated AA release in PC12 cells.

Topics: Adenosine Triphosphate; Animals; Arachidonic Acid; Bradykinin; Calcium; Dose-Response Relationship, Drug; Fura-2; Ionomycin; PC12 Cells; Pheochromocytoma; Rats; Receptors, Purinergic P2

The introduction of impermeant probes such as antibodies and other proteins into living cells without compromising physiological function is an important approach for studying cellular regulatory mechanisms. Many techniques including direct microinjection, liposome-mediated delivery, fusion of red cell ghosts, and osmotic lysis of pinocytic vesicles have been used to introduce proteins into intact cells. We have used a modification of the voltage-discharge technique to introduce antibodies and other proteins into living physiologically responsive pheochromocytoma and other cultured cells. In this technique, called electroinjection, a single discharge of relatively low field strength is used to transiently permeabilize the plasma membrane. Our experiments demonstrate that electroinjection permits the introduction of large amounts (microM) of probe into 2-5 x 10(6) cells simultaneously without compromising cell viability or physiological responsiveness when performed under carefully defined conditions. They also demonstrate that electroinjection results in a single population of loaded cells and that protein incorporation is a function of field strength, capacitance, molecular weight of the protein, and the concentration of the protein in the electroinjection buffer. Interestingly, a significant fraction of the protein electroinjected into cells is trapped in the plasma membrane when cells are shocked at high capacitance. These results demonstrate that electroinjection appears to be an efficient method for loading exogenous proteins into cells while maintaining the integrity of the physiological properties of the cell.

Topics: Adrenal Gland Neoplasms; Animals; Antibodies; Catecholamines; Cell Line; Cell Survival; Electrophysiology; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluoresceins; Ionomycin; Microinjections; Pheochromocytoma; Rats; Serum Albumin, Bovine

Maitotoxin (MTX) activates calcium channels and stimulates phosphoinositide breakdown in pheochromocytoma PC12 cells, while having no effect on basal levels of the cyclic nucleotides cAMP and cGMP. Atrial natriuretic factor (ANF) induces a dose-dependent accumulation of cGMP in PC12 cells through the activation of a membrane bound guanylate cyclase. Effects of ANF on cGMP are independent of extracellular concentrations of calcium. Since agents that activate phosphoinositide breakdown can indirectly affect cyclic nucleotide formation, the effects of MTX on ANF-mediated accumulation of cGMP was studied. MTX induces a dose-dependent inhibition of ANF-mediated accumulation of cGMP. The inhibition by MTX requires the presence of extracellular calcium, but is unaffected by the calcium channel blocker nifedipine. The inhibitory effect of MTX is not mimicked by the calcium ionophore ionomycin. A phorbol ester, PMA, which stimulates protein kinase C, also inhibits ANF-mediated accumulation of cGMP. Sodium nitroprusside induces large accumulations of cGMP in PC12 cells through the stimulation of a soluble guanylate cyclase. Neither MTX nor PMA inhibit nitroprusside-mediated accumulation of cGMP. The results indicate that in PC12 cells, protein kinase C activation, either directly with PMA, and indirectly with MTX through phosphoinositide breakdown and formation of diacylglycerol, leads to inhibition of ANF-mediated, but not nitroprusside-mediated accumulation of cGMP.

Topics: Atrial Natriuretic Factor; Calcium; Cyclic GMP; Dose-Response Relationship, Drug; Ionomycin; Marine Toxins; Nifedipine; Nitroprusside; Oxocins; Pheochromocytoma; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Depolarization of plasma membrane potential has a potent inhibitory effect on divalent cation influx catalyzed by the carboxylic ionophores ionomycin and A23187. This effect is observed in different cell models and does not depend on either inhibition of Ca2+-activated cation channels or activation of Ca2+ extrusion mechanisms as suggested previously. A dependence of divalent cation influx on the magnitude of membrane potential is observed also in artificial liposomes. The inhibition of ionophore-dependent divalent cation transport by membrane potential depolarization can be modified varying the ionophore concentration and the external pH. These findings suggest that both neutral and positively charged ionophore-cation complexes can cross the plasma membrane and that their contribution to the overall transport process can be varied according to the experimental conditions.

Topics: Adrenal Gland Neoplasms; Animals; Benzofurans; Calcimycin; Calcium; Calcium Channels; Cations, Divalent; Cations, Monovalent; Cell Line; Chlorides; Fluorescent Dyes; Fura-2; Gramicidin; Ionomycin; Kinetics; Liposomes; Manganese; Manganese Compounds; Mathematics; Membrane Potentials; Models, Theoretical; Pheochromocytoma; Rats

The role of various intracellular signals and of their possible interactions in the control of neurotransmitter release was investigated in PC12 cells. To this purpose, agents that affect primarily the cytosolic concentration of Ca2+, [Ca2+]i (ionomycin, high K+), agents that affect cyclic AMP concentrations (forskolin; the adenosine analogue phenylisopropyladenosine; clonidine) and activators of protein kinase C (phorbol esters) were applied alone or in combination to either growing chromaffin-like PC12-cells, or to neuron-like PC12+ cells differentiated by treatment with NGF (nerve growth factor). In addition, the release effects of muscarinic-receptor stimulation (which causes increase in [Ca2+]i, activation of protein kinase C and decrease in cyclic AMP) were investigated. Two techniques were employed to measure catecholamine release: static incubation of [3H]dopamine-loaded cells, and perfusion incubation of unlabelled cells coupled to highly sensitive electrochemical detection of released catecholamines. The results obtained demonstrate that: (1) release from PC12 cells can be elicited by both raising [Ca2+]i and activating protein kinases (protein kinase C and, although to a much smaller extent, cyclic AMP-dependent protein kinase); and (2) these various control pathways interact extensively. Activation of muscarinic receptors by carbachol induced appreciable release responses, which appeared to be due to a synergistic interplay between [Ca2+]i and protein kinase C activation. The muscarinic-induced release responses tended to become inactivated rapidly, possibly by feedback desensitization of the receptor mediated by protein kinase C. Muscarinic inactivation was prevented (or reversed) by agents that increase, and accelerated by agents that decrease, cyclic AMP. Agents that stimulate release primarily through the Ca2+ pathway (ionomycin and high K+) were found to be equipotent in both PC12- and PC12+ cells, whereas the protein kinase C activator 12-O-tetradecanoyl-phorbol 13-acetate was approx. 10-fold less potent in PC12+ cells, when administered either alone or in combination with ionomycin. In contrast, the cell binding of phorbol esters was not greatly modified by NGF treatment. Thus control of neurotransmitter release from PC12 cells is changed by differentiation, with a diminished role of the mechanism mediated by protein kinase C.

Topics: Adrenal Gland Neoplasms; Animals; Carbachol; Catecholamines; Cell Differentiation; Clonidine; Colforsin; Cyclic AMP; Dopamine; Ethers; Ionomycin; Nerve Growth Factors; Pheochromocytoma; Phorbol Esters; Potassium; Rats; Receptors, Muscarinic; Tumor Cells, Cultured

The properties of the various types of voltage-sensitive Ca++ channels (VSCC) are becoming increasingly well characterized, but the mechanisms which control the number and types of channels expressed by cells are virtually unknown. To study the regulation of VSCC in neuronal cells we have used PC12 pheochromocytoma cells. Binding of [3H]nitrendipine was used to determine the number of dihydropyridine-sensitive channels, and the uptake of 45Ca++ was used to determine the functional state of VSCC on the cell surface. Prolonged depolarization by elevation of extracellular K+ caused concomitant time and concentration-dependent decreases in both [3H]nitrendipine binding and depolarization-dependent uptake of 45Ca++. Changes in binding and ion flux plateaued at about a 50% decrease with 3 days of depolarization and an extracellular K+ concentration of 50 mM. Return of the cells to normal K+ caused the recovery of both [3H]nitrendipine binding and 45Ca++ uptake within 24 hr. Measurements of the intracellular free Ca++ concentration determined that it remained elevated for several hours with K+ depolarization, but returned to normal within 15 hr. Growth of the cells with a concentration of ionomycin, which caused a similar increase in intracellular free Ca++, also caused a loss of [3H]nitrendipine binding sites. Thus, it appears that the number of functional VSCC can be regulated by changes in intracellular Ca++ such as those associated with prolonged depolarization. However, because Ca++ channel number remained depressed while intracellular free Ca++ returned to normal, other mechanisms controlling channel number also must be involved.

Topics: Adrenal Gland Neoplasms; Animals; Calcium; Calcium Channels; Ethers; Ionomycin; Membrane Potentials; Nitrendipine; Pheochromocytoma; Potassium; Rats; Tumor Cells, Cultured

Incubation of rat pheochromocytoma PC12 cells with 4 beta-phorbol-12 beta-myristate-13 alpha-acetate (PMA), an activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), or forskolin, an activator of adenylate cyclase, is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase. Neither the activation nor increased phosphorylation of tyrosine hydroxylase produced by PMA is dependent on extracellular Ca2+. Both activation and phosphorylation of the enzyme by PMA are inhibited by pretreatment of the cells with trifluoperazine (TFP). Treatment of PC12 cells with 1-oleoyl-2-acetylglycerol also leads to increases in the phosphorylation and enzymatic activity of tyrosine hydroxylase; 1,2-diolein and 1,3-diolein are ineffective. The effects of forskolin on the activation and phosphorylation of the enzyme are independent of Ca2+ and are not inhibited by TFP. Forskolin elicits an increase in cyclic AMP levels in PC12 cells. The increases in both cyclic AMP content and the enzymatic activity and phosphorylation of tyrosine hydroxylase following exposure of PC12 cells to different concentrations of forskolin are closely correlated. In contrast, cyclic AMP levels do not increase in cells treated with PMA. Tryptic digestion of the phosphorylated enzyme isolated from untreated cells yields four phosphopeptides separable by HPLC. Incubation of the cells in the presence of the Ca2+ ionophore ionomycin increases the phosphorylation of three of these tryptic peptides. However, in cells treated with either PMA or forskolin, there is an increase in the phosphorylation of only one of these peptides derived from tyrosine hydroxylase. The peptide phosphorylated in PMA-treated cells is different from that phosphorylated in forskolin-treated cells. The latter peptide is identical to the peptide phosphorylated in dibutyryl cyclic AMP-treated cells. These results indicate that tyrosine hydroxylase is activated and phosphorylated on different sites in PC12 cells exposed to PMA and forskolin and that phosphorylation of either of these sites is associated with activation of tyrosine hydroxylase. The results further suggest that cyclic AMP-dependent and Ca2+/phospholipid-dependent protein kinases may play a role in the regulation of tyrosine hydroxylase in PC12 cells.

Topics: Adrenal Gland Neoplasms; Animals; Cell Line; Colforsin; Cyclic AMP; Diglycerides; Enzyme Activation; Ethers; Ionomycin; Peptide Mapping; Pheochromocytoma; Phosphopeptides; Phosphorylation; Rats; Tetradecanoylphorbol Acetate; Time Factors; Tyrosine 3-Monooxygenase

Ca2+ homoeostasis was investigated in pheochromocytoma neurosecretory (PC12) cells both before and after treatment with nerve growth factor, which induces a neuronal-like differentiation accompanied by a large increase in the number of muscarinic receptors. The resting concentration of free cytosolic Ca2+, [Ca2+]i, measured by the quin2 technique, was found to be higher and more variable in differentiated cells. Moreover, the [Ca2+]i rises induced by the Ca2+ ionophore ionomycin and by depolarizing concentrations of KC1 were greater and more transient. Exposure to carbachol induced modest, but long-lasting, [Ca2+]i rises, which were faster and greater in differentiated than in non-differentiated cells. These effects were due to the activation of the muscarinic receptor, because they were unaffected by nicotinic blockers (hexamethonium and D-tubocurarine) and completely eliminated by low concentrations of the muscarinic antagonists atropine and pirenzepine [IC50 (concn. causing 50% inhibition) = 2 and 60 nM respectively]. The muscarinic-receptor-dependent [Ca2+]i rises were the result of two concomitant processes: (1) redistribution of Ca2+ from cytoplasmic stores to the cytosol, possibly mediated by generation of inositol 1,4,5-trisphosphate as a consequence of the muscarinic-receptor-coupled hydrolysis of polyphosphoinositides, and (2) increased Ca2+ influx through a pathway of the plasmalemma insensitive to verapamil and thus different from the voltage-dependent Ca2+ channel. The existence of this second process was documented: (a) by the difference of the [Ca2+]i responses brought about by carbachol in Ca2+-containing and Ca2+-free media; (b) by the occurrence of [Ca2+]i rise and increased 45Ca accumulation in cells exposed to 1 mM-CaCl2 after having been treated for 2 min with carbachol in Ca2+-free medium; (c) by typical differences in the quin2 signal kinetics observed in parallel samples of PC12 cells loaded with different concentrations of the dye.

Topics: Adrenal Gland Neoplasms; Animals; Biological Transport; Calcium; Carbachol; Cell Line; Ethers; Ionomycin; Ionophores; Pheochromocytoma; Rats; Receptors, Muscarinic; 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