Lloyd A. Greene

Release, storage and uptake of catecholamines by a clonal cell line of nerve growth factor (NGF) responsive pheochromocytoma cells

Release, storage and uptake of catecholamines have been studied in cultures of a clonal line of rat pheochromocytoma cells designated as PC12. The PC12 line has previously been shown to respond to nerve growth factor (NGF) by extending neuronal-like processes and to synthesize and contain norepinephrine (NE) and dopamine (DA). In the present experiments, upon exposure to51.5mMK+, PC12 cells released a substantial proportion (ca. 30% and 12% for NGF-untreated and -treated cells, respectively) of their endogenous NE and DA. The release was dependent on the presence of Ca2+ and was inhibited by excess Mg2+. Veratridine, an alkaloid which activates Na+ action potential ionophores, was also effective in releasing DA and NE. Exposure of PC12 cells (NGF-treated and -untreated) to reserpine (10−5 M) resulted in depletion of intracellular DA and NE levels by over 90% in 21 h. PC12 cells were also found to take up NE from the external medium by means of a saturable transport mechanism which follows Michaelis-Menten kinetics (apparentKm≈ 2 μM), is energy and sodium dependent and which is blocked by low concentrations of cocaine and desmethylimipramine. Such findings indicate that PC12 cells (a) can release catecholamines by means of an exocytotic secretion mechanism, (b) store most of their endogenous catecholamines in chromaffin granules and/or vesicles and (c) have an uptake1-type transport system for norepinephrine. PC12 cells thus appear to express a number of differentiated properties characteristic of sympathetic neurons and adrenal chromaffin cells and may be a useful system in which to study catecholamine metabolism.

Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons: I. Sympathetic neurons

Abstract Quantitative studies on the nerve growth factor (NGF) requirement of chick embryo sympathetic neurons in dissociated cell culture revealed the following. (i) The minimum concentration of 2.5 S NGF required for survival of maximal numbers of neurons is about 0.5 ng/ml (∼2 × 10−11 M). In culture, this concentration of NGF appears not to be stable for more than 24 hr. Long-term neuronal maintenance with medium changes twice weekly requires a minimum of 5 ng/ml of NGF. (ii) At 24 hr after plating in medium containing 10% fetal bovine serum, neuronal survival is less than optimal at NGF concentrations above 5 ng/ml; in medium with 5% horse serum, survival is constant with up to 5000 ng/ml of NGF. (iii) Survival of neurons after 1 week in culture was less than optimal at NGF concentrations greater than 50 ng/ml, even in medium containing horse serum. (iv) No correlation was observed between the level of NGF (0.5–500 ng/ml) and the estimated neuronal somatic volumes up to 1 month in vitro. (v) Withdrawal of NGF, even after 4 weeks of culture, resulted in degeneration of nerve cell bodies and processes.

NGF stimulates incorporation of fucose or glucosamine into an external glycoprotein in cultured rat PC12 pheochromocytoma cells

Abstract Rat PC12 pheochromocytoma cells respond to Nerve Growth Factor (NGF) by ceasing to undergo mitosis and acquiring neuronal characteristics, including outgrowth of neurites and electrical excitability. We have found that NGF treatment results in no consistent qualitative and only a few minor quantitative changes in the two-dimensional electrophoresis pattern of 14 C-amino acid-labeled proteins synthesized by the cells. On the other hand, NGF stimulates the incorporation of radiolabeled fucose or glucosamine into several components, including one of apparent molecular weight 230,000 daltons on one- and two-dimensional SDS-polyacrylamide gels. This component is removed by mild trypsinization of intact cells and therefore appears to be a glycoprotein (named here NILE) at least partially exposed on the cell surface. Stimulation of NILE glycoprotein labeling can first be detected after 2 days of exposure to NGF and increases progressively with time of treatment. This change is not solely a consequence of the cessation of cell division caused by NGF, since it does not occur in nondividing PC12 populations prepared by cytosine arabinoside treatment. NGF stimulates labeling of NILE glycoprotein even when attachment and process outgrowth are prevented by growing the cells in spinner suspension cultures. The relative rate of labeling attained under these conditions is less, however, than in NGF-treated, substrate-attached cells. Stimulation of NILE glycoprotein labeling by NGF is selectively blocked (as is neurite outgrowth) by camptothecin, an RNA synthesis inhibitor, and thus may require transcription. Despite their similarities in apparent size and exposure on cell surfaces, the NILE and LETS glycoproteins are shown to be immunologically distinct.

Release of (3H)norepinephrine from a clonal line of pheochromocytoma cells (PC12) by nicotinic cholinergic stimulation.

Release experiments were carried out in vitro with a clonal line of rat pheochromocytoma cells (designated PC12) which synthesize and store catecholamines and which, after treatment with nerve growth factor (NGF), cease cell division and extend neuronal-like processes. In the present study, PC12 cells were exposed to [3H]norepinephrine (NE) which they took up and stored in reserpine-sensitive sites. Exposure of such cells to nicotinic cholinergic agonists resulted in release of [3H]NE into the external medium. Release terminated within 1 min and partially returned after 20 min in the absence of agonist. After 1 min of stimulation with nicotine, NGF-treated cells released 5-6% of their contents of [3H]NE while NGF-untreated cells released 1-2%. Release from both NGF-treated and -untreated PC12 cells was inhibited in the absence of Ca2+ or by elevated Mg2+ and was blocked by the nicotinic antagonists D-tubocurarine and mecamylamine (50% inhibition at 0.1 and 0.06 micrometer, respectively). Release was not affected by the presence of tetrodotoxin. Such findings suggest that release of [3H]NE from PC12 cells may be mediated via stimulation of nicotinic acetylcholine receptors and a consequent stimulation-secretion coupling mechanism.

Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons: II. Sensory neurons☆

Abstract Studies were carried out in dissociated cell cultures on the nerve growth factor (NGF) requirement of chick embryo dorsal root ganglionic (DRG) neurons. Findings were: (i) The minimum level of 2.5 S NGF required to sustain the survival of maximal numbers of process-bearing cells derived from 8-day (E8) embryonic DRGs is 0.5 ng/ml (∼2 × 10−11 M). (ii) Cultures derived from chick embryos of increasing ages (E8 to E18) showed a progressive increase in the proportion of process-bearing cells which survived in the absence of NGF. While few process-bearing cells survived in cultures of E8 ganglia in the absence of NGF, survival of neurons in cultures derived from E17 and E18 ganglia was not affected by the absence of the factor. Comparable results were obtained with cultures in which the number of non-neuronal cells was greatly reduced. (iii) Neurons derived from E8 ganglia lost their NGF requirement in culture at a conceptual age similar to that which they appear to do so in vivo. These results are discussed with respect to the role of NGF in development of sensory neurons.

The effects of nerve growth factor on acetylcholinesterase and its multiple forms in cultures of rat PC12 pheochromocytoma cells: increased total specific activity and appearance of the 16 S molecular form.

Abstract The effects of nerve growth factor (NGF) on acetylcholinesterase (AChE) activity and the distribution of this enzyme among its different molecular forms were studied in monolayer cultures of rat PC12 pheochromocytoma cells. It has been previously shown that PC12 cells respond to NGF by ceasing cell division and extending long, branching neurites. In NGF-untreated cultures, the specific activity of AChE was constant over a wide range of cell densities. By 4 days of NGF treatment there was a threefold increase in the activity of AChE per milligram protein. Analysis of the multiple molecular forms of AChE by sucrose gradient sedimentation revealed the 4, 6.5, and 10 S forms of the enzyme in NGF-untreated cultures. After 3 days of exposure to NGF, the 16 S form of the enzyme became apparent; by 7–10 days of treatment this form reached maximal levels (2–3% of total AChE activity). In spinner-suspension cultures, in which PC12 cells do not attach to a substrate and cannot grow neurites, NGF treatment yielded an increase in specific activity of AChE, but did not induce the 16 S form of the enzyme.

SHORT-TERM REGULATION OF CATECHOLAMINE BIOSYNTHESIS IN A NERVE GROWTH FACTOR RESPONSIVE CLONAL LINE OF RAT PHEOCHROMOCYTOMA CELLS

Abstract— A clonal cell line (designated PC12) has been previously established from a transplantable rat adrenal medullary pheochromocytoma. Tissue cultures of PC12 cells synthesize, store, release and take up catecholamines. PC12 cells also respond to nerve growth factor (NGF) protein by cessation of mitosis and extension of neurites. The present studies concern the comparison of several aspects of catecholamine metabolism in PC12 cultures with that in normal noradrenergic tissues. One question was why the ratio of dopamine to norepinephrine in PC12 cultures (in contrast to that in normal noradrenergic tissue) is considerably more than one. The presence of exogenous reduced ascorbate (a cofactor for dopamine‐β‐monooxygenase) enhanced by 5–10‐fold the rate at which PC12 cultures converted [3H]tyrosine to [3H]norepinephrine. Under such conditions, the rate of synthesis of [3H]do‐pamine was unchanged. It was also found that the ratio of norepinephrine to dopamine increased by 10‐fold when the cells were grown in vivo as tumors. Since tissue culture medium is essentially free of reduced ascorbate, it is likely that the absence of this cofactor is responsible for the low norepinephrine to dopamine ratio in PC12 cultures. Experiments were also carried out on short‐term regulation of catecholamine synthesis in PC12 cultures. These studies revealed the following: (1) The rate of conversion of [3H]tyrosine to [3H]catechols was increased 2–3‐fold (as compared with controls) in the presence of depolarizing levels of K+ (51.5 mM), and by 2‐fold in the presence of 0.5–2 mM‐dibutyryl cyclic adenosine 3′, 5’monophosphoric acid (db‐cAMP). (2) Similar increases occurred in cultures which had been treated with (and had responded to) nerve growth factor. (3) The stimulatory effects of 51.5 mM‐K+ rapidly returned toward control levels when the cultures were returned to control medium and (4) required the presence of Ca2+ in the extracellular medium. (5) Stimulation of catechol synthesis by 51.5 mM‐K+ and db‐cAMP also occurred in the presence of an inhibitor of DOPA decar‐boxylase. Thus, the ultimate effects of these agents were probably at the level of conversion of tyrosine to dopa by tyrosine 3‐monooxygenase. (6) Simultaneous exposure of cultures to 51.5 mM‐K+ and mM‐db‐cAMP gave additive levels of stimulation. Such findings demonstrate that catecholamine synthesis in cultures of PC12 cells undergoes short‐term regulation which is similar to that previously demonstrated in normal monoaminergic tissues. As a homogeneous tissue culture line, the PC12 bears certain advantages for studying the primary mechanisms of such effects.

Binding of α-bungarotoxin to chick sympathetic ganglia: properties of the receptor and its rate of appearance during development

Studies were carried out on the binding of [125I]α-bungarotoxin (αBT) to membrane fragments of chick sympathetic ganglia. Specific binding of toxin was saturable with aKDof1.1nM. The rates of association and dissociation of the toxin from ganglionic membranes were4.3 × 104 M−1sec−1and4.6 × 10−5sec−1 (t1/2=4.2h). respectively. Binding was inhibited (by up to 95%) by low concentrations of nicotinic, but not by a muscarinic cholinergic ligand. The properties of the ganglionic binding site for αBT were consistent with its being a nicotinic acetylcholine receptor. n nThe development of toxin receptors in chick ganglia was also studied. From days 7 to 11 in ovo, few receptors were present; from days 12 to 20 in ovo, there was a 10-fold increase in receptor number per ganglion; from hatching to maturaty, the number receptor per ganglion slowly increased and reached a maximum of 14 fmoles. The ontogeny of receptors for αBT in sympathetic appears to correlate with the cytological maturation and innervation of the principal neurons.

Chick sympathetic neurons develop receptors for α-bungarotoxin in vitro, but the toxin does not block nicotinic receptors

Studies were carried out on the development and physiological role of receptors for alpha-bungarotoxin (alphaBT) on chick embryo sympathetic neurons maintained in dissociated cell culture. Neurons from embryos of 13 days incubation (E13) developed alphaBT receptors in vitro with a time course and to a maximum level per cell similar to that previously observed for such neurons in vivo. In vitro receptor development by E11 and E8 neurons was also present, but (in comparison with E13 neurons) reached somewhat lower maximal levels. Receptor development in vitro was not affected by exclusion of non-neuronal cells from the cultures. In the present and in previous studies, binding of alphaBT to chick sympathetic neurons was blocked by a variety of ligands of nicotinic acetylcholine receptors. However, saturating concentrations of toxin were found here to be ineffective in blocking either (a) release of [3H]norepinephrine from the cultured neurons elicited via nicotinic stimulation of acetylcholine receptors or (b) depolarizing responses of the cultured neurons elicited by iontophoretically applied acetylcholine and nicotine. Kinetic studies further revealed that, while the idssociation of alphaBT from the cultured neurons is considerably enhanced in the presence of a cholinergic ligand (100 micrometer nicotine), the rate of this dissociation (t1/2 congruent to 30 min) appears to be too slow to account for the inability of the toxin to block nicotinic responses. Such findings show that chick embryo sympathetic neurons can develop receptors for alphaBT both in vivo and in vitro, but that the toxin does not block activation of their nicotinic acetylcholine receptors. The physiologic nature of specific binding sites for alphaBT on such neurons is thus presently unclear.

The binding properties and regional ontogeny of receptors for α-bungarotoxin in chick brain

Summary The subcellular distribution, binding kinetics, pharmacologic properties and regional ontogeny of binding sites for α-[125I]bungarotoxin (α-[125I]BT) were studied in preparations of chick brain. Almost 95% of the specific binding was found in a 20, 000 × g pellet. Two binding components were found, one with high (half-saturation at 1.5 × 10−9 M) and another with low affinity for the toxin. The rate constants of association and dissociation of the toxin for the high-affinity site were found to be 3.5 × 104 M−1 ·sec−1 and 3.8 × 10−5sec−1 (τ1/2 = 5.1h, respectively). These data yielded a Kd value of 1.1 × 10−9 M. Nicotinic, but not muscarinic ligands were potent inhibitors of toxin binding at the high-affinity site. Thus, as in muscle, the binding site for toxin in chick brain appears to have the properties of a nicotinic acetylcholine (ACh) receptor. The ontogeny of the α-BT/ACh receptor was studied using homogenates of both whole brain and of discrete brain regions. In brain stem, optic lobe and cerebellum specific binding reached maximum levels in ovo and then decreased by maturity to values of 10, 22 and 4.5 fmoles/mg protein, respectively. In contrast, whole brain and cerebral hemispheres reached plateau levels in ovo of 17 and 10 fmoles/mg protein, respectively. Major increases in total receptor number per region appeared in ovo between days 12 and 19 in brain stem and cerebellum, days 12 and 15 in optic lobes, and days 15 and 19 in hemispheres. The hemispheres were the only region to show a continued increase in total binding after hatching (3-fold increase by maturity). These findings are interpreted with respect to the development of chick brain and a model is suggested for the relationship between receptor ontogeny and neuronal differentiation and maturation.