Involvement of Ras/MAP Kinase in the Regulation of Ca²⁺Channels in Adult Bullfrog Sympathetic Neurons by Nerve Growth Factor

Abstract

The cellular mechanisms that underlie nerve growth factor (NGF) induced increase in Ca(2+)-channel current in adult bullfrog sympathetic B-neurons were examined by whole cell recording techniques. Cells were maintained at low density in neuron-enriched, defined-medium, serum-free tissue culture for 6 days in the presence or absence of NGF (200 ng/ml). The increase in Ba2+ current (IBa) density induced by NGF was attenuated by the RNA synthesis inhibitor cordycepin (20 microM), by the DNA transcription inhibitor actinomycin D (0.01 microgram/ml), by inhibitors of Ras isoprenylation (perillic acid 0.1-1.0 mM or alpha-hydroxyfarnesylphosphonic acid 10-100 microM), by tyrosine kinase inhibitors genistein (20 microM) or lavendustin A (1 microM), and by PD98059 (10-100 microM), an inhibitor of mitogen-activated protein kinase kinase. Inhibitors of the phosphatidylinositol 3-kinase (PI3K) pathway (wortmannin, 100 nM, or LY29400, 100 microM) were ineffective as were inhibitors of phospholipase C gamma (U73122 or neomycin, both 100 microM). The effect of NGF persisted in Ca(2+)-free medium that contained 1.8 mM Mg2+ and 2 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. It was mimicked by a Trk antibody that was capable of inducing neurite outgrowth in explant cultures of bullfrog sympathetic ganglion. Antibodies raised against the low-affinity p75 neurotrophin receptor were ineffective in blocking the effect of NGF on IBa. These results suggest that NGF-induced increase in Ca2+ channel current in adult sympathetic neurons results, at least in part, from new channel synthesis after Trk activation of Ras and mitogen activated protein kinase by a mechanism that is independent of extracellular Ca2+.

Differential Neurotrophic Regulation of Sodium and Calcium Channels in an Adult Sympathetic Neuron

“…This substance, which prevents the NGF-induced increase in I Ba in BFSG neurons (Lei et al 1998), also blocked the LHRH-induced increase in current density (P Ͻ 0.05) (Fig. 1, D and E; Table 1).…”

“…Although the PI3K inhibitor wortmannin failed to prevent LHRH or NGF-induced increases in I Ba (see Table 1 and Lei et al 1998), it was highly effective in inhibiting NGF-induced increases in total I Na . Relevant sample recordings are shown in Fig.…”

“…Although biochemical, electrophysiological, and pharmacological experiments were carried out throughout the year, relatively few experiments were done in May and June when animals were frequently unavailable from the supplier (Anilab, Quebec City, PQ, Canada). Isolation, dissociation, and culture of BFSG neurons in defined medium, serum-free culture were carried out as described by Lei et al (1998). Neurons were dissociated by incubation with trypsin (Sigma) and type 1A collagenase (Sigma) for 42-45 min, at 37°C.…”

“…Various neurotrophic factors have therefore been studied as important determinants of ion channel expression in adult neurons and in cell lines (Chalazonitis et al 1987;Fanger et al 1997;Khorkova and Golowasch 2007;Levine et al 1995;Pollock and Rane 1996;Pollock et al 1990). Target-derived nerve growth factor (NGF) is responsible for the maintenance of tetrodotoxin (TTX)-sensitive and TTX-insensitive sodium currents (I Na ) and of N-and L-type calcium currents (I Ca,N and I Ca,L ) in B-neurons of adult bullfrog sympathetic ganglia (BFSG) (Lei et al 1997(Lei et al , 1998(Lei et al , 2001Petrov et al 2001). In serum-free, defined medium culture, NGF-induced increases in I Ca are initiated via the mitogen-activated protein kinase (MAPK) pathway (Lei et al 1998), whereas the transduction process underlying the NGF-induced increase in I Na remains to be determined.…”

Neuropeptide Y modulates calcium channels in hamster submandibular ganglion neurons

“…MAPK tyrosine kinase inhibitor, 10 μM for 2 min) (Lei et al, 1998) did not attenuate the NPY-induced facilitation of IBa. These results suggest that NPY facilitates VDCCs involving AC and PKA pathways in the SMG neurons ( Fig.…”