Protein Kinase C and the Opposite Regulation of Sodium Channel α‐ and β₁‐Subunit mRNA Levels in Adrenal Chromaffin Cells

Abstract

Our previous [3 H]saxitoxin binding and 22 Na influx assays showed that treatment of cultured bovine adrenal chromaffin cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C (PKC), decreased the number of cell surface Na channels (IC 50 ϭ 19 nM) but did not alter their pharmacological properties; Na channel downregulation developed within 3 h, reached the peak decrease of 53% at 15 h, and was mediated by transcriptional/translational events. In the present study, treatment with 100 nM TPA lowered the Na channel ␣-subunit mRNA level by 34 and 52% at 3 and 6 h, followed by restoration to the pretreatment level at 24 h, whereas 100 nM TPA elevated the Na channel ␤ 1 -subunit mRNA level by 13-61% between 12 and 48 h. Reduction of ␣-subunit mRNA level by TPA was concentration-dependent (IC 50 ϭ 18 nM) and was mimicked by PDBu but not by the biologically inactive 4␣-TPA; it was prevented by H-7, an inhibitor of PKC, but not by HA-1004, a less active analogue of H7, or by H-89, an inhibitor of cyclic AMPdependent protein kinase. Treatment with cycloheximide, an inhibitor of protein synthesis, per se sustainingly increased the ␣-subunit mRNA level and decreased the ␤ 1 -subunit mRNA level for 24 h; also, the TPA-induced decrease of ␣-subunit mRNA and increase of ␤ 1 -subunit mRNA were both totally prevented for 24 h by concurrent treatment with cycloheximide. Nuclear run-on assay showed that TPA treatment did not alter the transcriptional rate of the ␣-subunit gene. A stability study using actinomycin D, an inhibitor of RNA synthesis, revealed that TPA treatment shortened the t 1/2 of ␣-subunit mRNA from 18.8 to 3.7 h. These results suggest that Na channel ␣-and ␤ 1 -subunit mRNA levels are differentially downand up-regulated via PKC; the process may be mediated via an induction of as yet unidentified short-lived protein(s), which may culminate in the destabilization of ␣-subunit mRNA without altering ␣-subunit gene transcription. Key Words: Sodium channel ␣-and ␤ 1 -subunits-Protein kinase C-Down-and up-regulationsNorthern blot-Nuclear run-on assay-mRNA stability.

Destabilization of Nav1.7 Sodium Channel α-Subunit mRNA by Constitutive Phosphorylation of Extracellular Signal-Regulated Kinase: Negative Regulation of Steady-State Level of Cell Surface Functional Sodium Channels in Adrenal Chromaffin Cells

“…Plasmids containing hNE-Na cDNA, and rat brain Na ϩ channel ␤ 1 -subunit cDNA were generously donated by Drs. F. Hofmann (Technischen Universitä t Mü nchen) and Y. Oh (University of Alabama), respectively Yanagita et al, 1999Yanagita et al, , 2000Shiraishi et al, 2001a,b).…”

“…Isolated bovine adrenal chromaffin cells were cultured (4 ϫ l0 6 /dish, Falcon; 35 mm in diameter) under 5% CO 2 /95% air in a CO 2 incubator in Eagle's minimum essential medium containing 10% calf serum and 3 M cytosine arabinoside to suppress the proliferation of nonchromaffin cells (Yanagita et al, , 1999(Yanagita et al, , 2000. The cells were exposed to normal fresh medium or serum-free fresh medium (serum deprivation treatment) or treated without or with PD98059 or U0126 in normal fresh medium for up to 48 h, 3 days after plating.…”

“…32 P-Labeled RNAs (5 ϫ l0 6 cpm/ml) were hybridized overnight at 70°C in Rapid-hyb buffer with nylon membrane immobilizing 10 g of pBII alone, and pBII containing hNE-Na cDNA or GAPDH cDNA. hNE-Na cDNA fragment (nt 1-2253) was liberated by digesting hNE-Na plasmid with KpnI and BglII, and subcloned into pBII (Yanagita et al, 1999). The membrane was sequentially washed in 2ϫ SSC containing 0.1% SDS at 65°C for 15 min, 2ϫ SSC containing 10 g/ml RNase A at 37°C for 10 min, 0.2ϫ SSC containing 0.1% SDS at 65°C for 10 min, and then subjected to autoradiography.…”

“…Calcineurin, a Ca 2ϩ /calmodulin-dependent protein phosphatase 2B, or the FK506 binding protein-and rapamycin-associated protein (FRAP), a serine/threonine protein kinase, up-regulated Na ϩ channels via modulating cell surface externalization and internalization of Na ϩ channels (Shiraishi et al, 2001b). Protein kinase C (PKC) down-regulated Na ϩ channels via PKC isoform-specific mechanisms; conventional PKC-␣ promoted endocytic internalization of Na ϩ channels, whereas novel PKC-⑀ accelerated degradation of ␣-subunit mRNA and decreased its level without altering ␣-subunit gene transcription (Yanagita et al, , 1999(Yanagita et al, , 2000. It is, however, unknown whether mitogen-activated protein kinases (MAPK), a family of serine/threonine protein kinases, modulate density and activity of Na ϩ channels at any given tissue.…”

Myosin II Activity Softens Cells in Suspension

“…ML7 directly targets the myosin light chain kinase pathway by inhibiting its activity (23). Phorbol 12,13-dibutyrate, on the other hand, activates the protein kinase C (24,25) and therefore increases myosin II activity. So far, there exists a range of data on mechanical property measurements of adherent cells treated with these drugs (26)(27)(28)(29), which have shown that inhibition of myosin activity leads to a loss of cell prestress and a subsequent lower measured elastic modulus of the cells.…”