On the role of Ca^(2+) in the transmitter choice made by cultured sympathetic neurons

Abstract

Depolarization or neuronal activity influences the differentiation of neonatal rat sympathetic neurons in dissociated cell culture by reducing their ability to respond to a cholinergic factor in conditioned medium (CM), allowing adrenergic differentiation to proceed (Walicke, P., R Campenot, and P. Patterson (1977) Proc. Natl. Acad. Sci. U. S. A. 74: 5767-5771). The present study analyzes the role of Ca2+ in the mechanism of this effect of activity. Addition of the Ca2 + influx inhibitors, MgCb, D600, diphenylhydantoin (DPH), or EGTA to the growth medium overcomes the developmental effect of depolarization. Elevation of the CaCb level in the medium, or addition of BaCh, slightly enhances the effect of depolarization. In non-depolarized cultures, Ba2+ potently inhibits cholinergic differentiation and the additional Ca2+ has a similar, though smaller, effect. Chronic depolarization of the neurons with either elevated K+ or veratridine leads to an increase in cyclic AMP (cAMP) content, and this elevation is blocked, along with the effect on transmitter choice, by MgCb and D600. On the other hand, EGTA (ethylene glycol bis(,8-aminoethyl ether)-N,N'-tetraacetic acid) and DPH, which also favor cholinergic differentiation, have little effect on the depolarization- induced elevation in cAMP. Exogenous addition of cyclic nucleotide derivatives or prostaglandin Ei, like depolarization, decrease cholinergic induction. Addition of EGTA, however, decreases the developmental effects of these agents while not interfering in their ability to increase cAMP levels. Thus, there are several ways cAMP levels can be uncoupled from the transmitter choice. The ability of the Ca2+ influx inhibitors to counteract the effect of depolarization on development and cAMP levels probably does not reflect the blockade of an action of an interposed neurotransmitter, since addition of phentolamine, phenoxybenzamine, adenosine deaminase, or alloxazine have no effect on transmitter choice or cAMP levels. Although calcium and cAMP undoubtedly have complex interactions in these neurons, calcium appears to play a major role in the developmental effects of depolarization and in the intracellular events governing transmitter choice.

Files

Additional details