Ca²⁺‐calmodulin‐dependent protein kinase II‐dependent activation of contractility in ferret aorta

Abstract

The present study was undertaken to determine whether Ca2+‐calmodulin‐dependent protein kinase II (CaMKII) participates in the regulation of vascular smooth muscle contraction, and if so, to investigate the nature of the downstream effectors. The contractility of isolated ferret aorta was measured while inhibiting CaMKII either with antisense oligodeoxynucleotides against CaMKII or with the CaMKII inhibitor KN93. Treatment with antisense oligodeoxynucleotides against CaMKII resulted in, on average, a decrease in protein levels of CaMKII to 56 % of control levels and significantly decreased the magnitude of the contraction in response to 51 mm potassium physiological saline solution (KCl). Contraction in response to the phorbol ester DPBA was not significantly affected. The CaMKII blocker KN93 also resulted in a significant decrease in the force induced by 51 mm KCl but caused no significant change in the contraction in response to DPBA or the α‐adrenoceptor agonist phenylephrine. During contraction with 51 mm KCl, both CaMKII and mitogen‐activated protein kinase (MAPK) activity increased, as determined by phospho‐specific antibodies. The MAPK phosphorylation level was inhibited by KN93, PD098059 (a MAPK kinase (MEK) inhibitor) and calcium depletion. Myosin light chain (LC20) phosphorylation also increased during contraction with KCl and the increase was significantly blocked by PD098059 as well as by both KN93 and antisense oligodeoxynucleotides to CaMKII. The data indicate that CaMKII plays a significant role in the regulation of smooth muscle contraction and suggest that CaMKII activates a pathway by which MAPK activation leads to phosphorylation of LC20 via activation of myosin light chain kinase.

AMP-activated Protein Kinase Phosphorylates and Desensitizes Smooth Muscle Myosin Light Chain Kinase

“…The underlying mechanisms, which were not investigated, are in line with our findings. Ca 2ϩ /CaM-dependent kinase II has also been implicated in the desensitization of MLCK to Ca 2ϩ /CaM stimulation, but the Ca 2ϩ /CaM-dependent kinase II inhibitor KN-93 was shown not to impair phenylephrine-induced contraction (39).…”

The non-excitable smooth muscle: Calcium signaling and phenotypic switching during vascular disease

“…In these cells, CaMKII γ isoforms have been implicated in the control of the differentiated contractile function [71,88,120]. However, cultured VSMC predominantly express the δ 2 (also referred to as δ C ) isoform [124].…”

Differential Association and Localization of Myosin Phosphatase Subunits During Agonist-Induced Signal Transduction in Smooth Muscle

“…13 Protein-matched samples were electrophoresed by SDS-PAGE (ProtoGel, National Diagnostics), transferred to PVDF (Millipore) membranes, and subjected to immunostaining and densitometry as previously described 13 using the appropriate primary antibodies. Equal lane loading was confirmed by inspection of the membrane after Napthol Blue Black staining.…”

“…Tissues were brought to room temperature in acetone/TCA/DTT, then ground with glass pestles, and washed with ether to remove TCA. Tissues were extracted in a urea sample buffer as previously described 13 and run on 10% polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes and subjected to immunoblot with a specific LC20 antibody (1:1500, Sigma).…”