Hypercontractility of intestinal longitudinal smooth muscle induced by cytokines is mediated by the nuclear factor-κB/AMP-activated kinase/myosin light chain kinase pathway.
Recent studies have identified AMP-activated kinase (AMPK) as a target of Ca(2+)/calmodulin-dependent kinase kinase (CaMKKβ) and a negative regulator of myosin light-chain (MLC) kinase (MLCK). The present study examined whether a change in expression or activity of AMPK is responsible for hypercontractility of intestinal longitudinal muscle during inflammation or in response to proinflammatory cytokines. In mouse colonic longitudinal muscle cells, acetylcholine (ACh) stimulated AMPK and MLCK phosphorylation and activity and induced MLC20 phosphorylation and muscle contraction. Blockade of CaMKKβ with STO609 (7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate) inhibited AMPK and MLCK phosphorylation and augmented MLCK activity, MLC20 phosphorylation, and smooth muscle cell contraction. In muscle cells isolated from the colon of TNBS (2,4,6-trinitrobenzenesulfonic acid)-treated mice or from strips treated with interleukin-1β or tumor necrosis factor-α, nuclear factor κB was activated as indicated by an increase in p65 phosphorylation and IκBα degradation, and AMPK was phosphorylated at a cAMP-dependent protein kinase (PKA)-specific site (Ser(485)) that is distinct from the stimulatory CaMKKβ site (Thr(172)), resulting in attenuation of ACh-stimulated AMPK activity and augmentation of MLCK activity and muscle cell contraction. Inhibition of nuclear factor-κB activity with MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal Z-LLL-CHO) or PKA activity with myristoylated PKA inhibitor 14-22 amide blocked phosphorylation of AMPK at Ser(485) and restored MLCK activity and muscle cell contraction to control levels. The results imply that PKA released from IκBα complex phosphorylated AMPK at a PKA-specific site and inhibited its activity, thereby relieving the inhibitory effect of AMPK on MLCK and increasing MLCK activity and muscle cell contraction. We conclude that hypercontractility of intestinal longitudinal muscle induced by inflammation or proinflammatory cytokines is mediated by nuclear factor κB/PKA-dependent inhibition of AMPK and activation of MLCK.
Department of Physiology and Biophysics, Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia.
Ancy D
AD
Nalli
Department of Physiology and Biophysics, Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia.
Divya P
DP
Kumar
Department of Physiology and Biophysics, Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia.
Sunila
S
Mahavadi
Department of Physiology and Biophysics, Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia.
Othman
O
Al-Shboul
Department of Physiology and Biophysics, Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia.
Reem
R
Alkahtani
Department of Physiology and Biophysics, Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia.
John F
JF
Kuemmerle
Department of Physiology and Biophysics, Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia.
John R
JR
Grider
Department of Physiology and Biophysics, Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia skarnam@vcu.edu.
Karnam S
KS
Murthy
D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000037:antagonists & inhibitors; D002352:Carrier Proteins / Q000494:pharmacology; D003092:Colitis / Q000139:chemically induced / Q000378:metabolism; D003106:Colon / Q000187:drug effects / Q000378:metabolism*; D016207:Cytokines / Q000494:pharmacology*; D004347:Drug Interactions; D053583:Interleukin-1beta / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008297:Male; D051379:Mice; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D053644:Naphthalimides / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D001562:Benzimidazoles; D002352:Carrier Proteins; D016207:Cytokines; D053583:Interleukin-1beta; D007976:Leupeptins; D018994:Myosin Light Chains; D016328:NF-kappa B; D053644:Naphthalimides; D010446:Peptide Fragments; C458525:STO 609; D014409:Tumor Necrosis Factor-alpha; C477818:myristoylated protein kinase A inhibitor amide 14-22; D014302:Trinitrobenzenesulfonic Acid; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D009219:Myosin-Light-Chain Kinase; D055372:AMP-Activated Protein Kinases; D000109:Acetylcholine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde