Ca2+ oscillations, Ca2+ sensitization, and contraction activated by protein kinase C in small airway smooth muscle

Abstract

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.

Selective targeting of the α5-subunit of GABAA receptors relaxes airway smooth muscle and inhibits cellular calcium handling

“…07069). Mouse lung slices were prepared as previously described (5,34). Briefly, male C3H mice (8 -12 wk) were killed with pentobarbital (40 mg/kg ip) and the chest cavity was opened to allow for cannulation of the trachea.…”

“…Measurements of changes in small-airway cross-sectional lumen area (lumen area hereafter) and the fluorescent measurements of [Ca 2ϩ ]i in ASM cells were performed as previously described (34). Briefly, lung slices were mounted in a custom-made perfusion chamber and airways were visualized by use of a ϫ10 objective.…”

“…The lumen area was normalized to the area before stimulation. In some experiments lung slices were loaded with Oregon green 488 BAPTA-1 acetoxymethyl ester (Invitrogen) for the intracellular imaging of Ca 2ϩ as previously described (34). Changes in fluorescence intensity were analyzed by selecting regions of interest (ROI) ranging from 25 to 49 square pixels.…”

Active Components of Ginger Potentiate β-Agonist–Induced Relaxation of Airway Smooth Muscle by Modulating Cytoskeletal Regulatory Proteins

“…The potentiation observed by Boterman and colleagues and Nakahara and colleagues could be attributed to decreased CPI-17 phosphorylation downstream of PKC or Rho kinase inhibition. Recently, Mukherjee and colleagues (44) found that PKC activation in the airway leads to CPI-17 phosphorylation and increases in MLC 20 phosphorylation. Here, we have shown that 6-gingerol, 8-gingerol, and 6-shogaol prevent ACh-induced phosphorylation of CPI-17.…”

Changes in Phosphoinositide Turnover in Airway Smooth Muscles and Blood Lymphocytes in Ova Sensitized Guinea Pig Model of Asthma

“…19 In another study, inhibition of PKC by calphostin-C, prevented proliferation of bovine tracheal smooth muscle cells following activation of mannose receptors by β-hexosaminidase and also abolished Ca 2+ -dependent and -independent PKC activity 20 which suggests involvement of different isoenzymes of PKC.…”