Diazepam inhibits calcium, calmodulin-dependent protein kinase in primary astrocyte cultures

The effect of the anticonvulsants diazepam, phenytoin, and valproic acid on calcium, calmodulin-dependent protein phosphorylation in astrocytes was investigated. We found that diazepam inhibited calcium, calmodulin-stimulated phosphorylation in both supernatant and membrane fractions from primary cultures of rat astrocytes, whereas phenytoin and valproic acid (50-500 microM) had little to no effect. Phosphate incorporation in several protein bands, including the major substrates of 59 and 53 kDa, was inhibited by diazepam. A decrease in phosphate incorporation in these crude preparations was observed at 25 microM diazepam and 50% inhibition was attained at about 100 microM. Dibutyryl cyclic AMP-treated astrocytes were enriched in the 59 kDa phosphoprotein; this band was particularly sensitive to diazepam in these cells. These results indicate that diazepam is capable of inhibiting calcium, calmodulin-dependent protein kinase activity in astrocytes, thereby suggesting a possible site of diazepam action and a potential mechanism for a role of astrocytes in epileptogenesis.