Effect of phorbol esters on Ca²⁺ sensitivity and myosin light-chain phosphorylation in airway smooth muscle

Abstract

We studied in β-escin-permeabilized canine tracheal smooth muscle (CTSM) the effect of the protein kinase C (PKC) agonist phorbol 12,13-dibutyrate (PDBu) on isometric force at a constant submaximal Ca2+ concentration (i.e., the effect on Ca2+ sensitivity) and regulatory myosin light-chain (rMLC) phosphorylation. PDBu increased Ca2+sensitivity, an increase associated with a concentration-dependent, sustained increase in rMLC phosphorylation. PDBu altered the relationship between rMLC phosphorylation and isometric force such that the increase in isometric force was less than that expected for the increase in rMLC phosphorylation observed. The effect of four PKC inhibitors [calphostin C, chelerythrine chloride, a pseudosubstrate inhibitor for PKC, PKC peptide-(19—31) (PSSI), and staurosporine] on PDBu-induced Ca2+ sensitization as well as the effect of calphostin C and PSSI on rMLC phosphorylation were determined. Whereas none of these compounds prevented or reversed the PDBu-induced increase in Ca2+sensitivity, the PDBu-induced increase in rMLC phosphorylation was inhibited. We conclude that PDBu increases rMLC phosphorylation by activation of PKC but that the associated PDBu-induced increases in Ca2+ sensitivity are mediated by mechanisms other than activation of PKC in permeabilized airway smooth muscle.

Modulation of the Ca2+ sensitivity of airway smooth muscle cells in murine lung slices

“…This relaxing effect of caffeine was also observed in model or normal airways in the absence of MCh. -induced airway contraction in the presence of GPCR agonist such as ACh, carbachol and endothelin-1 has also been previously observed in permeabilized tracheal SMCs from pig, dog, rabbit, rat, and mouse as well as surgically resected human bronchial SMCs (6,7,10,23,25,32). However, this vital role of GPCR agonists in establishing the sensitivity to Ca 2ϩ for the maintenance of intrapulmonary airway contraction was not previously recognized.…”

Mechanisms involved in carbachol‐induced Ca2+ sensitization of contractile elements in rat proximal and distal colon

“…Chelerythrine inhibited protein kinase C activity by interacting with the catalytic domain of the enzyme, whereas it did not inhibit activity of tyrosine protein kinase, calcium/ calmodulin-dependent protein kinase or cyclic AMP-dependent kinase (Herbert et al, 1990). Chelerythrine inhibited PDBu-induced Ca 2 þ sensitization in canine tracheal smooth muscle (Bremerich et al, 1998) and porcine pulmonary artery (Kutz et al, 1998). In the present study, both PKC(19-31) and chelerythrine significantly inhibited PDBu-induced Ca 2 þ sensitization, but not GTP-induced Ca 2 þ sensitization in preparations of both regions of the colon, indicating the specificity of these compounds to protein kinase C. Thus, these T. Takeuchi et al Ca 2 þ sensitization in rat colon 663 results suggested that CCh-induced Ca 2 þ sensitization was solely through the rho pathway in the rat proximal colon.…”

Protein kinase C isoenzymes: a review of their structure, regulation and role in regulating airways smooth muscle tone and mitogenesis

“…Recent studies by Bremerich et al (1998) have con®rmed that PDBu increases LC 20 phosphorylation in b-escinpermeabilized canine trachealis at a constant Ca 2+ concentration. However, these investigators provided two pieces of evidence against the idea that LC 20 phosphorylation accounts for force development under these conditions.…”

“…Recent studies by Bremerich et al . (1998) have confirmed that PDBu increases LC 20 phosphorylation in β‐escin‐permeabilized canine trachealis at a constant Ca 2+ concentration.…”

“…Second, calphostin C and a pseudosubstrate peptide inhibitor of PKC block PDBu‐induced LC 20 phosphorylation but not Ca 2+ sensitization. Collectively, therefore, it has been proposed that at a constant Ca 2+ concentration PDBu increases force in ASM by a PKC‐independent mechanism that is unrelated to its ability to phosphorylate LC 20 ( Bremerich et al ., 1998 ). In this respect PDBu potentiates the effect of endothelin‐1 on ovine tracheal smooth muscle ( Katoch, 1997 ) and carbachol on guinea‐pig ileum ( Sasaguri & Watson, 1989 ) under conditions where PKC is down‐regulated implying that the phorbol diester is acting at a different intracellular locus.…”