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.
Department of Internal Medicine, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1083, USA.
Qingjie
Q
Li
Sushil K
SK
Sarna
D000818:Animals; D020746:Calcium Channels, L-Type / Q000096:biosynthesis*; D003106:Colon / Q000502:physiology*; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000502:physiology; D000071679:Glycogen Synthase Kinase 3 beta; D018021:Lithium Chloride / Q000494:pharmacology; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D009638:Norepinephrine; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D021122:Protein Subunits / Q000096:biosynthesis; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects; D015398:Signal Transduction / Q000502:physiology; D013315:Stress, Psychological / Q000503:physiopathology*; D054875:Ubiquitination / Q000187:drug effects; D015854:Up-Regulation
D020746:Calcium Channels, L-Type; D015852:Eukaryotic Initiation Factor-2; D021122:Protein Subunits; D012333:RNA, Messenger; D011942:Receptors, Adrenergic, alpha; C493217:calcium channel, l type beta2c subunit, rat; D019869:Phosphatidylinositol 3-Kinases; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; D018021:Lithium Chloride; D009638:Norepinephrine