Pabelick, C.M.

Abstract Rationale: Within the asthmatic lung, inflammatory signaling alters the contractile capacity of airway smooth muscle (ASM). In chronic inflammation, ASM become hypercontractile and are a key contributor toward airway hyperresponsiveness (AHR). As a heterogenous disorder, several asthmatic endotypes have been defined. A severe ASM endotype is partly defined as being glucocorticoid insensitive. We have reported the role of Kisspeptin (Kp)/KISS1R signaling in the lung in the context of airway fibrosis. In this study, KISS1R and Kp-10 were investigated for a potential interactions with intracellular calcium [Ca2+]i signaling and ASM contractility in the in vitro setting of severe asthmatic endotypes. Methods: Primary ASM were obtained from non-pathological samples from the Mayo Clinic. ASM cells were treated with inflammatory cytokines (TNFα 20 ng/ml, or IL-13 50 ng/mL)± Kp-10 (1 μM). The steroid-insensitive asthma endotype was investigated using pro-inflammatory cytokines (TNFα 20 ng/ml and IFN-γ 25 ng/mL) ± a corticosteroid (Fluticasone Propionate 100 nM). Β2-Adrenergic Receptor (β2-AR) desensitized endotypes were developed by treating cells with a β2-AR agonist (Salbutamol 1 μM) for 24 hours prior to and along with cytokine treatments (TNFα 20 ng/ml and IFN-γ 25 ng/mL or IL-13 50 ng/mL) ± Kp-10. [Ca2+]i was measured via Fura-2AM (4 μM) labeling on an Olympus Fluoview 300 microscope. Myosin light chain (MLC) phosphorylation studies were used to quantify downstream mechanics of ASM contractility. Results: KISS1R activation in IL-13 treated cells reduced [Ca2+]i in response to histamine. In contrast, TNFα treated cells were responsive to Kp-10 for acute treatments, however this effect was lost after 24 hours of Kp-10 treatment. TNFα and IFN-γ treated cells showed a robust [Ca2+]i response to histamine, and both fluticasone (∼20% reduction) and Kp-10 (∼10% reduction) alone had a minimal effect in reducing [Ca2+]i. Fluticasone and Kp-10 co-exposure significantly reduced [Ca2+]i and significantly reduced the effect of TNFα/IFN-γ (∼45% reduction). In B2-AR desensitized cells, Kp-10 exposure maintained the effect of reducing [Ca2+]i, suggesting Kp-10/KISS1R signaling works through an alternate or independent pathway. Kp-10 treatments on cytokine exposed cells reduced p-MLC in response to histamine, suggesting a role in ASM contractility. Conclusion: The increase of [Ca2+]i in inflamed cells is associated with AHR; Kp-10/KISS1R signaling showed varying efficacy in reducing [Ca2+]i within inflammatory endotypes. These results also highlight that Kp-10 acts synergistically with the current therapeutics available for asthma.