The alpha subunit of the purified voltage-sensitive sodium channel from rat brain is rapidly phosphorylated to the extent of 3-4 mol phosphate/mol by purified protein kinase C. The alpha subunit of the native sodium channel in synaptosomal membranes is also phosphorylated by added protein kinase C as assessed by specific immunoprecipitation and polyacrylamide gel electrophoresis of labeled membranes. Our results suggest coordinate regulation of sodium channel phosphorylation state by cAMP-dependent and calcium/phospholipid-dependent protein kinases.

Monoamine oxidase activity of the A type was measured in homogenates of cultured human skin fibroblasts. Twenty-four control lines had activities ranging over fifty-fold with an apparent bimodal distribution. Activity in fibroblasts from 20 patients with the Lesch-Nyhan syndrome fell in the low portion of the normal distribution with a mean activity about 50% that of the control mean (p < 0.05). In a subgroup of control and Lesch-Nyhan lines with levels of enzyme activity from 0.9 to 179 pmol/min/mg protein, monoamine oxidase was similar with respect to apparent Km for tryptamine, thermal stability at 56 C, and sensitivity to clorgyline. Thus the lower mean levels of activity observed in the Lesch-Nyhan as compared to control fibroblasts were not associated with other altered properties of the enzyme. The bimodal distribution of enzyme activity suggests that a genetic polymorphism for monoamine oxidase may control levels of activity expressed in fibroblasts.

Neuroblastoma-glioma hybrid cells of line 108CC-5 were found to contain high levels of soluble adenosine 3',5'-cyclic monophosphate (cAMP)dependent protein kinase activity and high levels of two specific cAMP receptor proteins, RI and RI,. Treatment Both cytosol and membranes of mammalian brain contain two types of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (1). As in many other tissues (2), these two types of cAMP-dependent protein kinase differ in their regulatory subunit, the cAMP receptor proteins RI and RI, (1).An increasing amount of evidence indicates that cAMP receptor proteins and cAMP-dependent protein kinases may play an important role in the physiology of nervous tissue (3). Therefore, we were interested in finding model systems for nervous tissue in which to study the regulation of cAMP receptor proteins and cAMP-dependent protein kinases.One model system is provided by cultures of neuroblastoma-glioma hybrid cells (4-10). These hybrid cells were obtained by fusion of mouse neuroblastoma line N18TG2 with rat glioma line C6BU-1. The presence of 1 mM dibutyryl cAMP (Bt2cAMP) in the growth medium causes the cells to reduce their rate of division and to produce long, neurite-like extensions (for a review, see ref. 6). Interestingly, neuroblastoma-glioma hybrid cells, after treatment with Bt2cAMP, are able to form cholinergic synapses with primary and continuous cultures of striated muscle cells (7-9). In the present study, Bt2cAMP treatment of these hybrid cells was examined for possible effects on cAMP receptor proteins and cAMP-dependent protein kinase. MATERIALS AND METHODSThe following materials were used: Bt2cAMP, sodium salt, from JEM Research (Kensington, MD) (before use, Bt2cAMP was ether-extracted at acid pH); prostaglandin E1 (PGEI) from Upjohn; 3-isobutyl-1-methylxanthine (iBuMeXan) (12). Mouse neuroblastoma cell line N-18TG2 is a 6-thioguanine-resistant mutant, derived from clone N-18 of the C1300 tumor (13). The hybrid cell line 108CC-5, obtained by fusion of C6BU-1 and N-18TG2 cells (5), was a kind gift of B. Hamprecht (Martinsried, Germany). Viable cell stocks were stored in the vapor phase of liquid nitrogen in aliquots containing about 4 X 106 cells per ml in medium supplemented with 10% fetal calf serum and 5% dimethyl sulfoxide. For each experiment, one frozen aliquot was thawed and the cells were plated into a 75-cm2 flask and maintained in medium supplemented with 10% fetal calf serum. Cultures were incubated at 370C in a humidified atmosphere of 5% C02/95% air and fed every 2-3 days. Cells were subcultured into dishes (100 mm or 150 mm diameter) containing medium supplemented with 3% fetal calf serum at a density of 5.6 X 103 cells per cm2 of growth area. These cultures were half-fed by replacing one-half of the spent medium with fresh medium containing 3% fetal calf serum every 2-3 days. The hybrid cell line 108CC-5 was used only at subcultures 18-22. Treatment with 1 mM Bt2cAMP was initiated when cultures had reached 60-70% confluency. Unles...

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