Acetylcholine Activates Protein Kinase C-α in Pulmonary Venous Smooth Muscle

Abstract

Background The authors investigated whether acetylcholine-induced contraction in pulmonary venous smooth muscle (PVSM) is associated with the activation of specific protein kinase C (PKC) isoforms. Methods Isolated canine pulmonary venous rings without endothelium were suspended in modified Krebs-Ringer's buffer for measurement of isometric tension. The effects of nonspecific PKC inhibition (bisindolylmaleimide I; 3 x 10 m) and conventional PKC isoform inhibition (Gö7936 10 m) on the acetylcholine dose-response relation were assessed. The expression of conventional PKC isoforms (alpha, beta, gamma), novel PKC isoforms (delta, epsilon, theta), and atypical PKC isoforms (zeta, iota, mu) was measured in PVSM cells by Western blot analysis. The immunofluorescence technique and confocal microscopy were used to localize the cellular distribution of PKC isoforms before and after the addition of acetylcholine. Results Acetylcholine caused dose-dependent contraction in E-pulmonary veins. Pretreatment with bisindolylmaleimide I or Gö7936 attenuated acetylcholine contraction. PKC-alpha, -iota, -mu, and -zeta were expressed, whereas PKC-beta, -gamma, -delta, -epsilon;, and -theta were not expressed in PVSM cells. Immunofluorescence staining for PKC isoforms showed that in unstimulated cells, PKC-alpha and PKC-mu were detected only in the cytoplasm. PKC-iota and PKC-zeta also exhibited a cytoplasmic immunofluorescence pattern, which was especially abundant in the perinuclear zone. Activation with acetylcholine induced translocation of PKC-alpha from cytoplasm to membrane, whereas acetylcholine had no effect on the other PKC isoforms. Translocation of PKC-alpha in response to acetylcholine was blocked by the muscarinic receptor antagonist, atropine. Conclusion Acetylcholine contraction is attenuated by PKC inhibition in PVSM. Acetylcholine induces translocation of PKC-alpha from cytoplasm to membrane in PVSM. These results suggest that PKC-dependent acetylcholine contraction in PVSM may involve activation and translocation of PKC-alpha.