Selective inhibition of histamine-evoked Ca2+ signals by compartmentalized cAMP in human bronchial airway smooth muscle cells

Abstract

Intracellular Ca²⁺ and cAMP typically cause opposing effects on airway smooth muscle contraction. Receptors that stimulate these pathways are therapeutic targets in asthma and chronic obstructive pulmonary disease. However, the interactions between different G protein-coupled receptors (GPCRs) that evoke cAMP and Ca²⁺ signals in human bronchial airway smooth muscle cells (hBASMCs) are poorly understood. We measured Ca²⁺ signals in cultures of fluo-4-loaded hBASMCs alongside measurements of intracellular cAMP using mass spectrometry or [³H]-adenine labeling. Interactions between the signaling pathways were examined using selective ligands of GPCRs, and inhibitors of Ca²⁺ and cAMP signaling pathways. Histamine stimulated Ca²⁺ release through inositol 1,4,5-trisphosphate (IP₃) receptors in hBASMCs. β₂-adrenoceptors, through cAMP and protein kinase A (PKA), substantially inhibited histamine-evoked Ca²⁺ signals. Responses to other Ca²⁺-mobilizing stimuli were unaffected by cAMP (carbachol and bradykinin) or minimally affected (lysophosphatidic acid). Prostaglandin E₂ (PGE₂), through EP₂ and EP₄ receptors, stimulated formation of cAMP and inhibited histamine-evoked Ca²⁺ signals. There was no consistent relationship between the inhibition of Ca²⁺ signals and the amounts of intracellular cAMP produced by different stimuli. We conclude that β-adrenoceptors, EP₂ and EP₄ receptors, through cAMP and PKA, selectively inhibit Ca²⁺ signals evoked by histamine in hBASMCs, suggesting that PKA inhibits an early step in H₁ receptor signaling. Local delivery of cAMP within hyperactive signaling junctions mediates the inhibition.

Keywords: Airway smooth muscle; Ca(2+) signaling; Cyclic AMP; Histamine; Protein kinase A; Spatial organization.

Graphical abstract

Fig. 1

GPCRs stimulate increases in [Ca²⁺]_i in hBASMCs through activation of PLC and IP₃Rs. A, Populations of fluo-4-loaded hBASMCs in 384-well plates were stimulated with the indicated drug concentrations in HBSS. Peak increases in [Ca²⁺]_i are shown (Δ[Ca²⁺]_i) as means ± SEM for cells from donors 1, 2 and 3 (n = 4, 3 and 3, respectively). B, Effects of histamine, bradykinin and carbachol on Δ[Ca²⁺]_i and the sensitivity to each (pEC₅₀) in either HBS or Ca²⁺-free HBS (2.5 mM BAPTA added 37 s before the stimulus). Cells were from donor 1 for histamine and bradykinin (n = 3) and from donor 2 for carbachol (n = 4). C, D, Effects of nimodipine (10 μM, 5 min), trans Ned-19 (1 μM, 5 min) or ryanodine (50 μM, 5 min) on the Ca²⁺ signals evoked by the indicated stimuli in HBSS. Results (B-D) show means ± SEM, n = 7 (histamine, donor 1), n = 4 (bradykinin, donor 1) and n = 3 (carbachol, donor 2). ^*P < 0.05, one-way repeated ANOVA with Dunnett’s test (C) or paired two-tailed Student’s t-test (D), each relative to control. E, Effects of pre-incubation (30 min) with the indicated concentrations of edelfosine on basal [Ca²⁺]_i and the peak increases in [Ca²⁺]_i evoked by carbachol (10 μM). Results (mean ± SEM, n = 3) are from donor 2. F, Effects of the indicated concentrations, of 2-APB added 5 min before histamine (10 μM, n = 7), carbachol (10 μM, n = 3) or bradykinin (1 nM, n = 4) in HBSS, or to ionomycin (1 μM, n = 7) added in Ca²⁺-free HBSS to determine the Ca²⁺ content of the intracellular stores. Results are from donors 1 and 2.

Fig. 2

Pertussis toxin selectivity attenuates the Ca²⁺ signals evoked by LPA and carbachol. A-D, Effects of pre-treatment with pertussis toxin (PTX, 100 ng·mL⁻¹, 24 h) on the peak increases in [Ca²⁺]_i evoked by the indicated stimuli. Results are from cells derived from donors 1 and 2 (n = 3 for B and C; n = 4 for A and D). E, F, Summary results. ^*P < 0.05, paired Student’s t-test, relative to control.

Fig. 3

Inhibition of histamine-evoked Ca²⁺ signals by isoproterenol. A, Effects of isoproterenol (5 min) on intracellular cAMP concentrations in hBASMCs. Results are from MS analyses, n = 3. B, Typical traces from populations of fluo-4-loaded hBASMCs stimulated in HBS with histamine alone (10 μM, black trace) or after pre-incubation with isoproterenol (10 μM, 5 min, red trace) (n = 6). C, Summary results from similar experiments performed in HBSS (n = 7) show Δ[Ca²⁺]_i evoked by histamine alone or after treatment with isoproterenol. D, Concentration-dependent effects of isoproterenol (added 5 min before histamine) on Δ[Ca²⁺]_i evoked by histamine (10 μM) in HBSS. Results, are expressed as percentages of the matched control response without isoproterenol (n = 4). Results are from donor 1 (A, C and D) or donors 1 and 2 (B).

Fig. 4

Isoproterenol inhibits histamine-evoked Ca²⁺ signals through cAMP. A, Peak increases in [Ca²⁺]_i evoked by histamine (10 μM) after pre-treatment with the indicated concentrations of 8-Br-cAMP or 8-Br-cGMP (20 min) (n = 4). B, Effects of the indicated concentrations of IBMX (20 min) on the intracellular concentration of cAMP (measured by MS) and the peak increase in [Ca²⁺]_i evoked by histamine (10 μM). Results are expressed as percentages of the Δ[Ca²⁺]_i evoked by histamine alone (n = 6, donor 1) or as percentages of the cAMP concentration determined with the maximal concentration of IBMX (1 mM) (n = 5, donor 1). C, Effects of pre-incubation (30 min) with isoproterenol (10 μM), forskolin (10 μM) or both on the peak increase in [Ca²⁺]_i evoked by histamine in HBSS, and their sensitivity to histamine (pEC₅₀) (donor 1, n = 4). Parallel experiments show effects of the same treatments on intracellular cAMP accumulation determined after ³H-adenine-labeling of cells in HBS (donor 1, n = 3). ^*P < 0.05, one-way repeated measures ANOVA with Dunnett’s test, relative to response evoked in the presence of isoproterenol. D, Peak increases in [Ca²⁺]_i evoked by the indicated concentrations of histamine in HBS after pre-treatment (5 min) with solvents (DMSO or EtOH), isoproterenol (10 μM), NKH477 (10 μM), TCS 2510 (1 μM) or butaprost (10 μM) (n = 4). E, Intracellular cAMP accumulation in hBASMCs stimulated for 5 min in HBS with NKH477 (10 μM), forskolin (10 μM), isoproterenol (10 μM), PGE₂ (10 μM), TCS 2510 (1 μM) or butaprost (10 μM). Results ([³H]-cAMP, %, see Methods) are from donor 1 (n = 6-8), but were confirmed in donor 2. ^*P < 0.05, one-way repeated measures ANOVA with Dunnett’s test, relative to basal. F, Effects of ESI-05 (25 μM, 30 min) in HBS on the Ca²⁺ signals evoked by histamine (10 μM) added 1 min after the indicated concentrations of isoproterenol. Results are expressed as percentages of matched responses to histamine in the absence of isoproterenol (donors 1 and 2, n = 9).

Fig. 5

Compartmentalized cAMP inhibits histamine-evoked Ca²⁺ signals. A, Effects of pre-treatment (30 min) with the indicated inhibitors and then isoproterenol (1 min) on the Ca²⁺ signals evoked by histamine (10 μM). Results (donors 1 and 2, n = 9) show the peak Ca²⁺ signals (as percentages of matched responses to histamine without isoproterenol) and their sensitivity to inhibition by isoproterenol (pIC₅₀). ^*P < 0.05, one-way repeated measures ANOVA with Dunnett’s test, relative to control. B, Effects of varying the duration of the incubation with isoproterenol (10 μM) on cAMP accumulation (i) and the peak Ca²⁺ signals evoked by histamine (10 μM) (ii and iii). Accumulation of intracellular cAMP was measured after ³H-adenine labeling ([³H]-cAMP, %). Results for Δ[Ca²⁺]_i show the peak response as a percentage of that evoked by histamine alone (ii) and the pIC₅₀ value for isoproterenol (iii). (n = 4). C, Relationship between intracellular cAMP (determined by MS) and the inhibition of Ca²⁺ signals evoked by histamine (10 μM) in cells where the increase in cAMP was evoked by incubation with different concentrations of IBMX (20 min) or isoproterenol (5 min). Each point includes data from 5 (IBMX) or 3 (isoproterenol) MS determination of cAMP associated with 6 (IBMX) or 4 (isoproterenol) measurements of [Ca²⁺]_i. Results (B and C) are from cells from donor 1.

Fig. 6

Ca²⁺ signals evoked by different GPCRs differ in their susceptibility to inhibition by cAMP. A, Peak increases in [Ca²⁺]_i evoked by isoproterenol (10 μM), PGE₂ (10 μM) or forskolin (10 μM) in Ca²⁺-free HBS (n = 7 from donors 1 and 2). ^*P < 0.05, one-way ANOVA with Dunnett's test, relative to control. B, Peak increases in [Ca²⁺]_i evoked by PGE₂ or sulprostone in Ca²⁺-free HBS (BAPTA added 37 s before the stimuli) (n = 3 from donors 1 and 2). C-F, hBASMCs in HBS were pre-treated (5 min) with isoproterenol (10 μM), forskolin (10 μM), butaprost (10 μM), TCS2510 (1 μM) or solvents, and then stimulated with the indicated concentrations of histamine (C), bradykinin (D), carbachol (E) or LPA (F). The code in C applies to panels C-F. Results show peak increases in [Ca²⁺]_i evoked by the final stimulus from 6 independent experiments from donors 1 and 2 (C, D and F), and from 3 independent experiments with donor 2 (E). (G, H) Similar analyses of cells in HBS after treatment with pertussis toxin (PTX, 100 ng·mL⁻¹, 24 h). The code in G applies also to H. Results are from 3 independent experiments from donor 2 (G) and donors 1 and 2 (H).

Fig. 7

Selective inhibition of histamine-evoked Ca²⁺ signals by hyperactive cAMP junctions in human airway smooth muscle. A, Histamine (Hist), bradykinin (BK), carbachol (CCh) and LPA through their respective GPCRs stimulate PLCβ entirely through G_q/ll or with some contribution from G_i. IP₃ then stimulates Ca²⁺ release through IP₃Rs within the sarcoplasmic reticulum. β₂-adrenoceptors or receptors for PGE₂ (EP₂ and EP₄) stimulate AC and thereby PKA, which selectively inhibits the Ca²⁺ signals evoked by histamine, perhaps through phosphorylation of H₁ histamine receptors by PKA. B, Cyclic AMP may be delivered to PKA within ‘hyperactive’ signaling junctions, such that activation of a junction provides more than enough local cAMP to saturate the associated PKA. The junction thereby functions as a robust on-off switch. Concentration-dependent responses to β₂-agonists are due to recruitment of these digital junctions.