Abcejo, Amard J.

Neurotrophins (NTs), which play an integral role in directing proper neuronal development and function, have been found in non‐neuronal tissue (including lung), but their role is still under investigation. We previously found NTs such as brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT3) and their receptors to be expressed in human airway smooth muscle (ASM). In this study, we hypothesized that NTs contribute to regulation of ASM function via regulation of mechanisms that control intracellular Ca 2+ ([Ca 2+ ] i ). Human ASM cells isolated from lung samples incidental to patient surgery were incubated for 24 or 48 h in medium (control), 10 nM BDNF or NT3 in the presence or absence of inhibitors of signaling pathways such as MAP kinases (PD98059), PI3/Akt (Wortmannin) and NFκB (SN50). In fluo‐4 loaded cells, [Ca 2+ ] i responses to ACh and histamine were significantly higher following BDNF and NT3 exposure: effects that were blunted by SN50 and PD98059, but not Wortmannin. Western analysis of cell lysates showed higher expressions of Ca 2+ regulatory proteins such as Orai1, TRPC3, and SERCA with 1 or 10nM BDNF exposure, but decreased expression by NT3. These data show that NTs activate signaling pathways in human ASM that lead to enhanced [Ca 2+ ] i responses, and could thus contribute to airway hyperresponsiveness. Supported by NIH grants HL088029, HL090595 and FAMRI.

Diseases such as asthma are characterized by airway hyperresponsiveness. Enhanced airway smooth muscle (ASM) intracellular Ca2+ ([Ca2+]i) response to agonist stimulation leading to increased airway constriction has been suggested to contribute to airway hyperresponsiveness. Caveolae are flask-shaped plasma membrane invaginations that express the scaffolding protein caveolin and contain multiple proteins important in [Ca2+]i signaling (e.g., agonist receptors, ion channels). We recently demonstrated that caveolae and caveolin-1 are important in [Ca2+]i regulation in human ASM. Proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-13 modulate [Ca2+]i in ASM. We hypothesized that cytokine upregulation of caveolar signaling in ASM contributes to enhanced agonist-induced [Ca2+]i in inflammation. Enzymatically dissociated human ASM cells were exposed to medium (control), 20 ng/ml TNF-α, or 50 ng/ml IL-13 for 24 h. Caveolae-enriched membrane fractions displayed substantial increase in caveolin-1 and -2 expressions by TNF-α and IL-13. Transfection with caveolin-1-mRed DNA substantially accelerated and increased plasma membrane caveolin-1 expression by TNF-α and to a lesser extent by IL-13. Caveolin-1 enhancement was inhibited by nuclear factor-κB and mitogen-activated protein kinase inhibitors. In fura 2-loaded ASM cells, [Ca2+]i responses to 1 μM ACh, 10 μM histamine, or 10 nM bradykinin were all exaggerated by TNF-α as well as IL-13 exposure. However, disruption of caveolae using caveolin-1 suppression via small-interfering RNA resulted in significant blunting of agonist-induced [Ca2+]i responses of vehicle and TNF-α-exposed cells. These functional data were correlated to the presence of TNFR1 receptor (but not the IL-4/IL-13 receptor) within caveolae. Overall, these results indicate that caveolin-1 plays an important role in airway inflammation by modulating the effect of specific cytokines on [Ca2+]i.

Neurally derived tachykinins such as substance P (SP) play a key role in modulating airway contractility (especially with inflammation). Separately, the neurotrophin brain-derived neurotrophic factor (BDNF; potentially derived from nerves as well as airway smooth muscle; ASM) and its tropomyosin-related kinase receptor, TrkB, are involved in enhanced airway contractility. In this study, we hypothesized that neurokinins and neurotrophins are linked in enhancing intracellular Ca2+ concentration ([Ca2+]i) regulation in ASM. In rat ASM cells, 24 h exposure to 10 nM SP significantly increased BDNF and TrkB expression ( P < 0.05). Furthermore, [Ca2+]i responses to 1 μM ACh as well as BDNF (30 min) effects on [Ca2+]i regulation were enhanced by prior SP exposure, largely via increased Ca2+ influx ( P < 0.05). The enhancing effect of SP on BDNF signaling was blunted by the neurokinin-2 receptor antagonist MEN-10376 (1 μM, P < 0.05) to a greater extent than the neurokinin-1 receptor antagonist RP-67580 (5 nM). Chelation of extracellular BDNF (chimeric TrkB-Fc; 1 μg/ml), as well as tyrosine kinase inhibition (100 nM K252a), substantially blunted SP effects ( P < 0.05). Overnight (24 h) exposure of ASM cells to 50% oxygen increased BDNF and TrkB expression and potentiated both SP- and BDNF-induced enhancement of [Ca2+]i ( P < 0.05). These results suggest a novel interaction between SP and BDNF in regulating agonist-induced [Ca2+]i regulation in ASM. The autocrine mechanism we present here represents a new area in the development of bronchoconstrictive reflex response and airway hyperreactive disorders.