Protein kinase C (PKC) has been implicated in the regulation of smooth muscle cell (SMC) contraction and may contribute to airway hyperresponsiveness. Here, we combined optical and biochemical analyses of mouse lung slices to determine the effects of PKC activation on Ca2+ signaling, Ca2+ sensitivity, protein phosphorylation, and contraction in SMCs of small intrapulmonary airways. We found that 10 µM phorbol-12-myristate-13-acetate or 1 µM phorbol 12,13-dibutyrate induced repetitive, unsynchronized, and transient contractions of the SMCs lining the airway lumen. These contractions were associated with low frequency Ca2+ oscillations in airway SMCs that resulted from Ca2+ influx through L-type voltage-gated Ca2+ channels and the subsequent release of Ca2+ from intracellular stores through ryanodine receptors. Phorbol ester stimulation of lung slices in which SMC intracellular Ca2+ concentration ([Ca2+]i) was “clamped” at a high concentration induced strong airway contraction, indicating that PKC mediated sensitization of the contractile response to [Ca2+]i. This Ca2+ sensitization was accompanied by phosphorylation of both the PKC-potentiated PP1 inhibitory protein of 17 kD (CPI-17) and the regulatory myosin light chain. Thrombin, like the phorbol esters, induced a strong Ca2+ sensitization that was inhibited by the PKC inhibitor GF-109203X and also potentiated airway contraction to membrane depolarization with KCl. In conclusion, we suggest that PKC activation in small airways leads to both the generation of Ca2+ oscillations and strong Ca2+ sensitization; agents associated with airway inflammation, such as thrombin, may activate this pathway to sensitize airway smooth muscle to agonists that cause membrane depolarization and Ca2+ entry and induce airway hyperresponsiveness.
Enhanced airway smooth muscle (ASM) contraction is an important component in the
pathophysiology of asthma. We have shown that ligand gated chloride channels modulate ASM
contractile tone during the maintenance phase of an induced contraction, however the role
of chloride flux in depolarization-induced contraction remains incompletely understood. To
better understand the role of chloride flux under these conditions, muscle force (human
ASM, guinea pig ASM), peripheral small airway luminal area (rat ASM) and airway smooth
muscle plasma membrane electrical potentials (human cultured ASM) were measured. We found
ex vivo guinea pig airway rings, human ASM strips and small peripheral airways in rat
lungs slices relaxed in response to niflumic acid following depolarization-induced
contraction induced by K+ channel blockade with tetraethylammonium chloride
(TEA). In isolated human airway smooth muscle cells TEA induce depolarization as measured
by a fluorescent indicator or whole cell patch clamp and this depolarization was reversed
by niflumic acid. These findings demonstrate that ASM depolarization induced contraction
is dependent on chloride channel activity. Targeting of chloride channels may be a novel
approach to relax hypercontractile airway smooth muscle in bronchoconstrictive
disorders.
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