Characterization of the Signaling Pathways Activated by KCl-Induced RTK Stimulation in Guinea Pig Airways

TL;DR

This study explores how KCl-induced depolarization activates signaling pathways in guinea pig airway smooth muscle, revealing roles for RTKs, MEK-ERK, and ROCK in contraction.

Contribution

The study identifies RTKs and downstream pathways as key mediators of KCl-induced airway smooth muscle contraction, offering new insights into airway physiology.

Findings

Pharmacological inhibition of RTKs significantly reduced 20 mM KCl-induced airway smooth muscle contraction.

MEK and ERK inhibitors decreased ERK phosphorylation and altered contraction in response to 20 mM KCl.

ROCK inhibition significantly reduced KCl-induced contraction and MYPT1 phosphorylation.

Abstract

Airway smooth muscle (ASM) adjusts airway diameter in response to an ample variety of stimuli; however, the physical causes remain poorly understood. To fully comprehend complex pathological states like asthmatic ASM and its relation to airway hyperresponsiveness, basic ASM physiology needs to be thoroughly understood. Agonists (histamine, acetylcholine, hydroxitriptamine, etc.) activate well known cell signaling, while KCl is used because of its capacity to depolarize the ASM membrane and induce contraction by bypassing agonist-induced second messenger signaling cascades. In vitro, KCl-induced ASM depolarization activates receptor tyrosine kinases (RTKs). It is worth mentioning that this approach emulates airway smooth muscle signaling pathways activated by physical circumstances (mucus osmolarity, tidal volume, PM2.5). It is known that RTKs are single subunit receptors, and ligands binding to their extracellular domain induce receptor dimerization, initiating downstream signal transduction pathways such as the MAP kinase signaling cascade. In this sense, KCl-induced RTK stimulation is a useful strategy to dissect the signaling pathways involved in ASM contraction. The study of these pathways might contribute to uncovering a potential therapeutic approach for chronic respiratory diseases like asthma. Therefore, we theorized that, in ASM, a low concentration (20 mM) of KCl activates RTKs and their downstream signaling cascades (i.e., MAPK-ERK and ROCK) and studied whether these cascades participate in KCl-induced ASM contraction.

We found that, in guinea pig airway smooth muscle, the pharmacological inhibition of RTKs significantly decreased the contraction induced by 20 mM KCl. We observed that MEK pharmacological inhibitors diminished the contraction induced by 20 mM KCl, but not that induced by 60 mM. On the other hand, ERK inhibitors also altered the contraction generated by 20 mM KCl. When a ROCK inhibitor was tested, we found that it significantly inhibited the KCl-induced contraction. These results were complemented with Western blot experiments, and a decrease in ERK phosphorylation was noticed when the RTKs were inhibited. When MEK and ERK inhibitors were used, we also observed a decrease in ERK phosphorylation. In the case of MYPT1, its phosphorylation decreased when RTK, MEK, and ROCK inhibitors were used. In conclusion, we found that, in guinea pig airway smooth muscle, the contraction induced by 20 mM KCl includes the activation of RTKs and, in turn, MEK-ERK and ROCK.

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