Molecular mechanisms underlying the regulation of proenkephalin gene expression in cultured spinal cord cells

The regulation of proenkephalin (proENK) mRNA levels by cAMP and protein kinase C (PKC) pathways was studied in cultured rat spinal cord cells in the present study. Spinal cord cells were cultured from 14 day (E 14) embryos of Sprague-Dawley rats. After 7 days in vitro, the spinal cord cells were incubated with either forskolin (5 microM) or phorbol-13-myristate acetate (PMA; 2.5 microM) for 1, 3, 6, 9, 12 or 24 h and total RNA and proteins were isolated for Northern and Western blot analyses. The proENK mRNA level began to increase within an hour, then reached and remained at a peak 3-12 h after stimulation by both forskolin and PMA. The increased proENK mRNA level in forskolin-treated cells was slightly decreased 24 h after the stimulation, whereas the level of proENK mRNA returned to basal levels in PMA-treated cells. A Western blot assay revealed that the intracellular level of proENK protein was not changed by treatment with either forskolin or PMA. Pretreatment of cells with cycloheximide (a protein synthesis inhibitor; 10 microM) did not affect the forskolin- or PMA-induced increase of proENK mRNA. However, pretreatment with nimodipine (an L-type Ca2+ channel blocker; 2 microM), omega-conotoxin (an N-type Ca2+ channel blocker; 1 microM), calmidazolium (a calmodulin antagonist; 1 microM) or KN-62 (a Ca2+/calmodulin-dependent protein kinase II inhibitor; 5 microM) attenuated the forskolin- or PMA-induced increase of proENK mRNA levels. Dexamethasone (1 microM) did not affect the forskolin-induced increase of proENK mRNA levels. Our results suggest that the elevation of proENK mRNA levels in the spinal cord is regulated by both cAMP and PKC pathways. Calcium influx through both L- and N-type calcium channels, calmodulin and Ca2+/calmodulin-dependent protein kinase II appear to be involved in the increase of proENK mRNA levels induced by either forskolin or PMA. Furthermore, ongoing protein synthesis is not required for forskolin- or PMA-induced alterations in proENK mRNA.