Expression and coupling of neurokinin receptor subtypes to inositol phosphate and calcium signaling pathways in human airway smooth muscle cells

Neuropeptide tachykinins (substance P, neurokinin A, and neurokinin B) are present in peripheral terminals of sensory nerve fibers within the respiratory tract and cause airway contractile responses and hyperresponsiveness in humans and most mammalian species. Three subtypes of neurokinin receptors (NK ₁R, NK₂R, and NK₃R) classically couple to G_q protein-mediated inositol 1,4,5-trisphosphate (IP3) synthesis and liberation of intracellular Ca²⁺, which initiates contraction, but their expression and calcium signaling mechanisms are incompletely understood in airway smooth muscle. All three subtypes were identified in native and cultured human airway smooth muscle (HASM) and were subsequently overexpressed in HASM cells using a human immunodeficiency virus-1-based lentivirus transduction system. Specific NKR agonists {NK₁R, [Sar⁹,Met(O ₂)¹¹]-substance P; NK₂R, [β-Ala ⁸]-neurokinin A(4-10); NK₃R, senktide} stimulated inositol phosphate synthesis and increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) in native HASM cells and in HASM cells transfected with each NKR subtype. These effects were blocked by NKR-selective antagonists (NK₁R, L-732138; NK₂R, GR-159897; NK ₃R, SB-222200). The initial transient and sustained phases of increased [Ca²⁺]_i were predominantly inhibited by the IP3 receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) or the store-operated Ca²⁺ channel antagonist SKF-96365, respectively. These results show that all three subtypes of NKRs are expressed in native HASM cells and that IP₃ levels are the primary mediators of NKR-stimulated initial [Ca²⁺]_i increases, whereas store-operated Ca²⁺ channels mediate the sustained phase of the [Ca²⁺]_i increase.