tetrodotoxin and Pheochromocytoma

Pheochromocytoma PC12 cells incubated with and without nerve growth factor were investigated using the patch-clamp technique in the whole-cell recording mode, and the concentration clamp method in the rat. On the fourth day of incubation with nerve growth factor, sodium potential-activated ionic currents appeared in the membranes of the most morphologically differentiated cells. At the same period a three-fold increase of acetylcholine-activated current density, compared with the cells incubated without nerve growth factor, was observed. Thus, the qualitative and quantitative changes in membrane properties can be a result of metabolic reorganization in PC12 cells induced by nerve growth factor and accompanied by morphological differentiation according to neuronal phenotype.

Topics: Acetylcholine; Adrenal Gland Neoplasms; Animals; Dose-Response Relationship, Drug; Evoked Potentials; Ion Channels; Kinetics; Nerve Growth Factors; PC12 Cells; Pheochromocytoma; Potassium Channels; Sodium Channels; Tetrodotoxin; Time Factors

cAMP-dependent protein kinase (PKA) and phospholipid-dependent protein kinase (PKC) play a role in nerve growth factor (NGF)-mediated differentiation. In PC12 cells, NGF causes neurite outgrowth and increases the number of voltage-gated Na+ channels. Neurite outgrowth involves in part activation of PKC. How NGF regulates Na+ channel number is unknown. Using patch-clamp techniques, we find that agents activating PKC, including phorbol esters and a ras oncogene product (p21) that induces neurites, caused little increase in channel number. In contrast, agents increasing intracellular cAMP were as effective as NGF. A specific protein inhibitor of the PKA catalytic subunit blocked increases by NGF or cAMP. Thus, NGF increases Na+ channel number in PC12 cells in part by activating PKA but apparently not PKC.

Topics: 1-Methyl-3-isobutylxanthine; Adrenal Gland Neoplasms; Animals; Bucladesine; Cell Differentiation; Cell Line; Colforsin; Cyclic CMP; Dibutyryl Cyclic GMP; Dimethyl Sulfoxide; Electric Conductivity; Electrophysiology; Gene Expression; Genes, ras; Kinetics; Nerve Growth Factors; Pheochromocytoma; Protein Kinases; Rats; Sodium Channels; Tetrodotoxin

Rat pheochromocytoma (PC12) cells, persistently infected with mumps virus (MV), failed to generate full-sized stimulus-evoked action potentials (SEAPs) when examined by intracellular electrophysiological recording techniques. Application of tetrodotoxin (TTX) had little or no effect on MV-reduced SEAPs, indicating that the number of functional voltage-gated Na+ channels was decreased or their operation was blocked by the virus. In contrast, MV-infected cells generated normal Ca2+ spikes when bathed in a solution containing TTX, tetraethylammonium ions and a high concentration (20 mM) of Ca2+. In addition, when infected cells bathed in TTX were superfused with Co2+ the SEAP profile reverted to that typical of PC12 cells with functional voltagegated K+ channels only. These observations indicate that MV affects voltage-gated Na+ channels, but spares voltage-gated Ca2+ and K+ channels of persistently infected cells.

Topics: Adrenal Gland Neoplasms; Animals; Calcium Channels; Cell Line; Evoked Potentials; Mumps; Pheochromocytoma; Rats; Sodium Channels; Tetrodotoxin; Tumor Cells, Cultured

The PC12 clone is a line of rat pheochromocytoma cells that undergoes neuronal differentiation in the presence of NGF protein. In the absence of NGF, PC12 cells are electrically inexcitable, while after several weeks of NGF treatment they develope Na+ action potentials. Past estimates made by measuring binding of 3H-saxitoxin (STX) indicate that NGF treatment brings about a large increase in Na channel density that is of sufficient magnitude to account for the induction of excitability. We have now used 22Na uptake to measure the Na permeability of PC12 cells before and after long-term NGF treatment. Treatment with NGF does not change the resting Na+ permeability. The alkaloid toxins veratridine and batrachotoxin (BTX) and scorpion toxin were used to activate Na channels. Such studies demonstrate that these toxins induce TTX-sensitive Na uptake in both NGF-treated and untreated cells and reveal differences in functional Na channel numbers per cell and per unit of membrane area that are similar to those found in the STX binding studies. On the other hand, affinities for drugs that activate these channels are not affected by NGF treatment. We also find that NGF-treated PC12 cells contain a population of Na channels with low affinity for TTX. These channels account for 5-20% of total BTX or veratridine-stimulated flux. Thus, NGF has 2 effects regarding the Na channels of PC12 cells: it increases the number of functional Na channels that otherwise behave similarly to those present before NGF treatment, and it induces the presence of TTX-resistant Na channels. These findings indicate that the PC12 model system may serve to study the developmental regulation of Na channel expression and properties.

Topics: Animals; Batrachotoxins; Cell Line; Drug Resistance; Ion Channels; Nerve Growth Factors; Neurons; Permeability; Pheochromocytoma; Scorpion Venoms; Stimulation, Chemical; Tetrodotoxin; Time Factors; Veratridine

The role of calcium currents in the regulation of neurite outgrowth is still rather speculative. As a contribution to this field, macroscopic voltage dependent calcium currents were investigated in relation to the nerve growth factor (NGF)-induced outgrowth of neurites in PC 12 cells. Calcium currents were recorded in isolated growth cones of PC 12 cells using the whole cell patch clamp method. The currents were activated at high voltages and only slightly inactivated with time. The currents were identical to those found in the cell soma of PC 12 cells and similar to the classical high-voltage-activated calcium current found in many neuronal cells. The peak current density in the growth cones was in the same range as in the cell somata. The calcium currents of the cell somata were not modified during the early phase of NGF application, despite the occurrence of NGF-induced soma growth and outgrowth of neurites. The current density at this time was therefore lower in NGF-treated cells than in untreated cells. In a later phase, maximal current amplitudes of NGF-treated cells were higher than in untreated cells indicating an increase in current density to values similar to that found in the untreated cells. In addition, the calcium current inactivation was found to be more pronounced in the NGF-treated cells by that time. The results are discussed with regard to a possible role of calcium currents in the regulation of NGF-induced neurite outgrowth in these cells.

Topics: Adrenal Gland Neoplasms; Animals; Calcium; Cell Division; Electrophysiology; Nerve Growth Factors; Pheochromocytoma; Rats; Tetraethylammonium Compounds; Tetrodotoxin

Effects of maitotoxin, the most potent marine toxin, were studied using a rat pheochromocytoma cell line, PC12h. A low concentration (10(-8) g/ml) of maitotoxin induced a profound increase in CA2+ influx into PC12h cells and the Ca2+-dependent release of [3H]norepinephrine from them. The effects of maitotoxin were not affected by treatment with tetrodotoxin (10(-6) M) and were observed even in the absence of external Na+. Furthermore, these effects were markedly inhibited or abolished by treatment with verapamil (30-300 microM), Mn2+ (5 mM), or tetracaine (1 mM). These results suggest that maitotoxin activates the voltage-dependent calcium channels of PC12h cells.

Topics: Adrenal Gland Neoplasms; Animals; Calcium; Cell Line; Ion Channels; Kinetics; Manganese; Marine Toxins; Neoplasms, Experimental; Oxocins; Pheochromocytoma; Rats; Tetracaine; Tetrodotoxin; Verapamil

1. The pathways for Ca entry during stimulus-secretion coupling were studied by measuring carbamylcholine and KCl activated dopamine (DA) release from PC12, a clonal cell line originated from a rat pheochromocytoma. Various conditions were used to establish the existence of two independent pathways for Ca entry, i.e. a voltage dependent Ca channel and the acetylcholine (ACh) receptor linked channel. 2. DA release from PC12 was stimulated by activation of nicotinic ACh receptors with carbamylcholine. Release was dependent on external Ca, but not Na, and was insensitive to tetrodotoxin. 3. High concentrations of external KCl (15--56 mM) also stimulated the release of dopamine from PC12. This release required external Ca, but not Na, was insensitive to tetrodotoxin and was diminished by high concentrations of Ca. 4. Ni, Co and, to a lesser extent, Mg inhibited both the carbamylcholine and KCl stimulated release of DA in normal and Na-free media. 5. In the absence of Ca and Na, Mn supported DA release stimulated by either carbamylcholine or KCl. 6. The divalent cation ionophore A23187 stimulated DA release when Ca or Mn, but not Co, Ni or Mg, was the only divalent cation present in the medium. 7. Conclusions can be summarized as follows, (a) KCl stimulates DA release by activation of voltage dependent Ca channels. (b) Carbamylcholine probably stimulates DA release by Ca influx through both the ACh channel and voltage dependent Ca channels. (c) Mn ion is a suitable substitute for Ca ion as regards permeation of the Ca and the ACh channels and activation of the secretory process. (d) Na flux through voltage dependent Na channels is not necessary for stimulation of release by either KCl or carbamylcholine.

Topics: Adrenal Gland Neoplasms; Animals; Calcium; Carbachol; Cations, Divalent; Cell Line; Clone Cells; Dopamine; Ion Channels; Neurons; Pheochromocytoma; Rats; Receptors, Cholinergic; Secretory Rate; Tetrodotoxin