Chronic stress targets posttranscriptional mechanisms to rapidly upregulate α1C-subunit of Cav1.2b calcium channels in colonic smooth muscle cells.

Chronic stress elevates plasma norepinephrine, which enhances expression of the α(1C)-subunit of Ca(v)1.2b channels in colonic smooth muscle cells within 1 h. Transcriptional upregulation usually does not explain such rapid protein synthesis. We investigated whether chronic stress-induced release of norepinephrine utilizes posttranscriptional mechanisms to enhance the α(1C)-subunit. We performed experiments on colonic circular smooth muscle strips and in conscious rats, using a 9-day chronic intermittent stress protocol. Incubation of rat colonic muscularis externa with norepinephrine enhanced α(1C)-protein expression within 45 min, without a concomitant increase in α(1C) mRNA, indicating posttranscriptional regulation of α(1C)-protein by norepinephrine. We found that norepinephrine activates the PI3K/Akt/GSK-3β pathway to concurrently enhance α(1C)-protein translation and block its polyubiquitination and proteasomal degradation. Incubation of colonic muscularis externa with norepinephrine or LiCl, which inhibits GSK-3β, enhanced p-GSK-3β and α(1C)-protein time dependently. Using enrichment of phosphoproteins and ubiquitinated proteins, we found that both norepinephrine and LiCl decrease α(1C) phosphorylation and polyubiquitination. Concurrently, they suppress eIF2α (Ser51) phosphorylation and 4E-BP1 expression, which stimulates gene-specific translation. The antagonism of two upstream kinases, PI3K and Akt, inhibits the induction of α(1C)-protein by norepinephrine. Cyanopindolol (β(3)-AR-antagonist) almost completely suppresses and propranolol (β(1/2)-AR antagonist) partially suppresses norepinephrine-induced α(1C)-protein expression, whereas phentolamine and prazosin (α-AR and α(1)-AR antagonist, respectively) have no significant effect. Experiments in conscious animals showed that chronic stress activates the PI3K/Akt/GSK-3β signaling. We conclude that norepinephrine released by chronic stress rapidly enhances the protein expression of α(1C)-subunit of Ca(v)1.2b channels by concurrently suppressing its degradation and enhancing translation of existing transcripts to maintain homeostasis.