Brain-derived neurotrophic factor enhances calcium regulatory mechanisms in human airway smooth muscle

Abstract

Neurotrophins (NTs), which play an integral role in neuronal development and function, have been found in non-neuronal tissue (including lung), but their role is still under investigation. Recent reports show that NTs such as brain-derived neurotrophic factor (BDNF) as well as NT receptors are expressed in human airway smooth muscle (ASM). However, their function is still under investigation. We hypothesized that NTs regulate ASM intracellular Ca(2+) ([Ca(2+)](i)) by altered expression of Ca(2+) regulatory proteins. Human ASM cells isolated from lung samples incidental to patient surgery were incubated for 24 h (overnight) in medium (control) or 1 nM BDNF in the presence vs. absence of inhibitors of signaling cascades (MAP kinases; PI3/Akt; NFκB). Measurement of [Ca(2+)](i) responses to acetylcholine (ACh) and histamine using the Ca(2+) indicator fluo-4 showed significantly greater responses following BDNF exposure: effects that were blunted by pathway inhibitors. Western analysis of whole cell lysates showed significantly higher expression of CD38, Orai1, STIM1, IP(3) and RyR receptors, and SERCA following BDNF exposure, effects inhibited by inhibitors of the above cascades. The functional significance of BDNF effects were verified by siRNA or pharmacological inhibition of proteins that were altered by this NT. Overall, these data demonstrate that NTs activate signaling pathways in human ASM that lead to enhanced [Ca(2+)](i) responses via increased regulatory protein expression, thus enhancing airway contractility.

Figure 1. Effect of prolonged exposure to brain-derived neurotrophic factor (BDNF) on intracellular Ca²⁺ ([Ca²⁺]_i) responses to agonist stimulation in human airway smooth muscle (ASM) cells.

Figure shows representative tracings (A, C) and summaries of peak and plateau [Ca²⁺]_i responses (B, D) of ASM cells to 10 µM histamine (A, B) and 1 µM ACh (C, D) following 24 h exposure to vehicle (control), 1 nM BDNF, or 500 nM of the novel agonist 7,8-dihydroxyflavone which specifically activates the high-affinity BDNF receptor tropomyosin related kinase B (TrkB). The enhancing effects of BDNF and 7,8-dihydroxyflavone were comparable, suggesting that BDNF acts via TrkB in mediating effects on [Ca²⁺]_i. Values are means ± SE (n = 4 patients). *Significant difference from control (p<0.05).

Figure 2. Role of the low-affinity neurotrophin receptor p75NTR in BDNF enhancement of [Ca²⁺]_i responses in human ASM cells.

Pre-exposure of ASM cells to 1 µM TAT-pep5, a peptide that blocks p75NTR, had no significant effect on subsequent BDNF enhancement of [Ca²⁺]_i responses to ACh or histamine (A, B). TAT-pep5 also did not affect 7,8-dihydroxyflavone enhancement of [Ca²⁺]_i. These data support the idea that BDNF acts via TrkB in enhancing [Ca²⁺]_i. Values are means ± SE (n = 4 patients). *Significant difference from control (p<0.05).

Figure 3. Effect of prolonged BDNF exposure on store-operated Ca²⁺ entry (SOCE) in human ASM cells.

SOCE was evaluated by depleting sarcoplasmic reticulum stores in the absence of extracellular Ca²⁺, and then rapidly re-introducing extracellular Ca²⁺ in the continued presence of CPA – (A). Compared to vehicle controls, SOCE was significantly greater in cells exposed overnight to BDNF or to 7,8-Dihydroxyflavone. However, TAT-pep5 had no significant effect on either BDNF or 7,8-Dihydroxyflavone enhancement of SOCE (B). Values are means ± SE (n = 4 patients). *Significant difference from control (p<0.05).

Figure 4. Effect of prolonged BDNF exposure on expression of membrane-associated Ca²⁺ regulatory proteins in human ASM cells.

Western blot analysis demonstrated increased expression of the ectoenzyme CD38, and the Ca²⁺ influx regulatory components Orai1 and STIM1, but not TRPC3 (A, B). Inhibition of MAP kinases with PD98059, PI3/Akt with Wortmannin, or NFκB with SN50 had differential blunting effects on BDNF enhancement of CD38 vs. STIM1 vs. Orai1 (A, B; see text for detailed description). Consistent with these results, inhibition of signaling intermediates (see Methods for details) such as ERK, Raf, RSK and IKK blunted the effects of BDNF on CD38, while effects on STIM1 were more affected by IKK inhibition (C, D). In contrast, inhibition of Akt was generally less effective. Protein expression was normalized to GAPDH. *Significant difference from control; #Significant effect of inhibitor. Values are means ± SE (n = 4 patients).

Figure 5. Effect of prolonged BDNF exposure on expression of sarcoplasmic reticulum Ca²⁺ regulatory proteins in human ASM cells.

Western blot analysis demonstrated increased expression of both Ca²⁺ release mechanisms IP₃R and RyR channels, as well as the Ca²⁺ reuptake protein SERCA (A, B). Compared to membrane regulatory proteins (Figure 4), inhibition of MAP kinases, PI3/Akt or NFκB (A, B) or their signaling intermediates (C, D) were equally effective in blunting BDNF enhancement of Ca²⁺ regulatory proteins. Protein expression was normalized to GAPDH. *Significant difference from control; #Significant effect of inhibitor. Values are means ± SE (n = 4 patients).

Figure 6. Functional consequence of BDNF-induced changes in Ca²⁺ regulatory protein expression.

Small interference RNA (siRNA) inhibition of CD38, Orai1 or STIM1 expression substantially reduced peak [Ca²⁺]_i responses to histamine not only in cells exposed to vehicle only (i.e. no BDNF), but also in those exposed for 24 h to BDNF (A). Transfection agent (Lipofectamine) or nonsense siRNA had no effect on [Ca²⁺]_i responses in either group (not shown). In contrast, siRNA inhibition of TRPC3 had substantially less effect on BDNF enhancement of peak [Ca²⁺]_I, suggesting that the other mechanisms were functionally linked to BDNF effects, and consistent with no change in TRPC3 expression. Similarly, in ASM cells exposed to BDNF for 24 h, inhibition of RyR channels (high concentration ryanodine) or IP₃ channels (Xestospongin C, XeC) or of SERCA (with cyclopiazonic acid; CPA) significantly blunted BDNF enhancement of peak [Ca²⁺]_i responses to histamine (B). *Significant difference from control; #Significant effect of inhibitor. Values are means ± SE (n = 4 patients).

Figure 7. Effect of signaling cascade inhibitors on BDNF enhancement of peak [Ca²⁺]_i responses in human ASM cells.

Overnight incubation of ASM cells with either PD98059 or SN50 in the presence of BDNF substantially blunted the enhanced peak [Ca²⁺]_i responses observed with BDNF only for both histamine (A, B) and ACh (C, D). In summary graphs (B, D), *Significant difference from control; #Significant effect of inhibitor. Values are means ± SE (n = 4 patients).

Figure 8. Effect of inhibiting signaling intermediates on BDNF enhancement of peak [Ca²⁺]_i responses in human ASM cells.

Compared to the enhanced [Ca²⁺]_i response to ACh or histamine in BDNF-exposed cells, the presence of inhibitors for intermediates of MAP kinase, PI3/Akt or NFκB pathways resulted in peak [Ca²⁺]_i responses that were substantially blunted. #Significant effect of inhibitor. Values are means ± SE (n = 4 patients).