Human bronchial epithelial cells express K_Ca3.1 mRNA and protein, and K_Ca3.1 expression is upregulated in the asthmatic bronchial epithelium.

<p><b>(A)</b> K_Ca3.1 mRNA (predicted PCR product size: 130 bp) was detected in monolayers of primary HBECs isolated from both patients with asthma (n = 10; denoted with “A”) and non-asthmatic healthy controls (n = 5; denoted with “NA”), alongside the housekeeping gene β-actin (predicted PCR product size: 146 bp). (<b>B</b>) qPCR revealed that K_Ca3.1 mRNA was expressed at similar levels in primary HBECs isolated from patients with asthma (n = 10) and healthy controls (n = 5). (<b>C</b>) An immunoreactive band of the appropriate size (K_Ca3.1: 48 kDa; β-actin: 42 kDa) was detected in lysates of primary HBECs isolated from two patients with asthma and one healthy control. (<b>D</b>) Quantification of K_Ca3.1 immunostaining by threshold analysis revealed that K_Ca3.1 expression was significantly elevated in asthmatic bronchial epithelium (*P = 0.007). (<b>E</b>) K_Ca3.1 immunostaining was significantly increased in severe asthma compared to mild asthma (**P = 0.008) and compared with healthy controls (*P = 0.002). (<b>F</b>) K_Ca3.1 and MUC5AC immunostaining co-localised in bronchial epithelial cells of sequential sections of biopsies from patients with asthma and healthy controls. (<b>G</b>) Quantification of MUC5AC immunostaining by threshold analysis revealed that MUC5AC expression was significantly increased in asthmatic bronchial epithelium (*P = 0.030), and (<b>H</b>) this was driven by a significant difference between the severe asthma and healthy control groups (*P = 0.034). (<b>I</b>) A significant correlation was found between K_Ca3.1 and MUC5AC immunostaining across the different severities of asthma and the healthy control groups (P < 0.001; r_s = 0.608). All data are plotted as median ± interquartile range; horizontal bars represent medians.</p

The K_Ca3.1 channel does not regulate epithelial mucus production or secretion.

<p>(<b>A</b>) Recombinant human amphiregulin (rh-AR) upregulated MUC5AC mRNA expression in H292 cells cultured in 6 well plates in a concentration-dependent manner after 24 h (*P = 0.010; **P = 0.028; n = 3 individual experiments). (<b>B</b>) Pre-treatment of H292 cells with the K_Ca3.1 blocker TRAM-34 (200 nM) for 30 min prior to stimulation with 10 ng/ml rh-AR for 24 h did not prevent rh-AR-induced MUC5AC mRNA expression (*P = 0.008, **P = 0.042; n = 6 individual experiments). rh-AR dose-dependently increased the mucin content of (<b>C</b>) H292 ALI culture lysates (*P = 0.029; n = 3 individual experiments) and (<b>D</b>) H292 ALI culture apical washes (*P = 0.009; n = 4 individual experiments) after 24 h, analysed by ELLA. Pre-treatment of H292 ALI cultures with 200 nM TRAM-34 did not prevent rh-AR-induced upregulation of mucin content within (<b>E</b>) H292 lysates (*P = 0.019; n = 6 individual experiments) or (<b>F</b>) apical washes (*P = 0.027; n = 3 individual experiments). Data are expressed as mean ± SEM.</p

Asthmatic HBECs exhibit significantly larger K_Ca3.1 currents than healthy HBECs.

<p>(<b>A</b>) Current-voltage curves demonstrating that baseline whole cell membrane currents recorded from asthmatic and healthy primary HBECs were similar. (<b>B</b>) The 1-EBIO-dependent (1-EBIO minus baseline) currents recorded from asthmatic HBECs (n = 29 cells from 8 donors) were significantly larger than those from healthy controls (n = 16 cells from 5 donors); *P = 0.006 at +40 mV. (<b>C</b>) Raw 1-EBIO-dependent current trace demonstrating characteristic features of K_Ca3.1. The voltage protocol is shown inset. The K_Ca3.1 channel blocker TRAM-34 (200 nM) inhibited currents induced by 1-EBIO in both (<b>D</b>) asthmatic HBECs (n = 19 cells from 8 donors) and (<b>F</b>) healthy HBECs (n = 14 cells from 5 donors). Data are presented as mean ± SEM.</p

Inhibition of the K_Ca3.1 channel attenuates features of TGFβ1-dependent EMT.

<p>(<b>A</b>) Cell elongation, quantified by a ratio of cell length:cell width of vimentin-stained BEAS-2B cells after 72 h, was increased by TGFβ1 or TGFβ1+DMSO compared to untreated cells (0.1% PBS/BSA; *P < 0.001). Pre-treatment of BEAS-2B cells with 200 nM TRAM-34 (#P < 0.001) or 100 nM ICA-17043 (##P = 0.027) significantly inhibited TGFβ1-induced cell elongation compared to TGFβ1+DMSO control. In contrast, TRAM-7, an inactive analog of TRAM-34, did not inhibit TGFβ1-induced cell elongation (**P = 0.022 compared to TRAM-34). Data are presented as mean ± SEM from 6 individual experiments. (<b>B</b>) BEAS-2B cells treated with 10 ng/ml TGFβ1 or TGFβ1+DMSO for 72 h exhibited a loss of E-cadherin expression in comparison to untreated cells (0.1% PBS/BSA; *P < 0.001, **P = 0.001). Pre-treatment with TRAM-34 (200 nM; #P = 0.013) or ICA-17043 (100 nM; ##P = 0.002) attenuated the TGFβ1-dependent loss of E-cadherin compared to TGFβ1+DMSO. TRAM-7 (200 nM) did not prevent TGFβ1-induced loss of E-cadherin immunostaining (***P = 0.014 compared to TRAM-34). Data are presented as mean ± SEM from 6 individual experiments. (<b>C</b>) BEAS-2B cells treated with 10 ng/ml TGFβ1 or 10 ng/ml TGFβ1+DMSO control for 72 h displayed an increase in collagen-1 expression in comparison to untreated cells (0.1% PBS/BSA) (*P = 0.005; **P = 0.028 respectively). However, pre-treatment with TRAM-34 (200 nM; #P = 0.021) or ICA-17043 (100 nM; ##P = 0.024) significantly inhibited this effect compared to TGFβ1+DMSO. TRAM-7 (200 nM) did not inhibit TGFβ1-induced upregulation of collagen-1 immunostaining (***P = 0.021 compared to ICA-17043). Data are presented as mean ± SEM from 5 individual experiments.</p

The K_Ca3.1 channel does not regulate airway epithelial ciliary beat frequency.

<p>(<b>A</b>) Ciliary beat frequency of epithelial cells from healthy controls treated with either TRAM-34 or DMSO (n = 3). (<b>B</b>) Ciliary beat frequency of epithelial cells from asthmatic subjects treated with either TRAM-34 or DMSO (n = 3). *P = 0.001; data are expressed as mean ± SEM.</p

Prevalence of cough in different age groups in the entire cohort, and separately for children with and without wheeze.

<p>Prevalence of cough in different age groups in the entire cohort, and separately for children with and without wheeze.</p

Prevalence of coughing more than peers, cough with colds, cough apart from colds and night cough in the entire cohort, and among children with wheeze and non-wheezers, for different age groups.

<p>Prevalence (%) with 95% confidence intervals.</p

Characteristics of the study population in 1998 (N = 6808).

<p>Characteristics of the study population in 1998 (N = 6808).</p

International comparison of age-specific prevalence of cough, assessed by different questions.

<p>International comparison of age-specific prevalence of cough, assessed by different questions.</p

Prevalence of cough triggered by exercise, house dust, pollen, pets, food and laughter or crying in the entire cohort, and among children with wheeze and non-wheezers, for different age groups.

<p>Prevalence (%) with 95% confidence intervals.</p