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

1 Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York; and 2 Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Submitted 16 August 2007 ; accepted in final form 15 January 2008 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 1 R, NK 2 R, and NK 3 R) classically couple to G q protein-mediated inositol 1,4,5-trisphosphate (IP 3 ) synthesis and liberation of intracellular Ca 2+ , 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 {NK 1 R, [Sar 9 ,Met(O 2 ) 11 ]-substance P; NK 2 R, [β-Ala 8 ]-neurokinin A(4–10); NK 3 R, senktide} stimulated inositol phosphate synthesis and increased intracellular Ca 2+ concentration ([Ca 2+ ] i ) in native HASM cells and in HASM cells transfected with each NKR subtype. These effects were blocked by NKR-selective antagonists (NK 1 R, L-732138; NK 2 R, GR-159897; NK 3 R, SB-222200). The initial transient and sustained phases of increased [Ca 2+ ] i were predominantly inhibited by the IP 3 receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) or the store-operated Ca 2+ channel antagonist SKF-96365, respectively. These results show that all three subtypes of NKRs are expressed in native HASM cells and that IP 3 levels are the primary mediators of NKR-stimulated initial [Ca 2+ ] i increases, whereas store-operated Ca 2+ channels mediate the sustained phase of the [Ca 2+ ] i increase. intracellular calcium; ryanodine; actin; myosin; lentivirus Address for reprint requests and other correspondence: C. W. Emala, Dept. of Anesthesiology, College of Physicians and Surgeons of Columbia Univ., 630 W. 168th St. P&S Box 46, New York, NY 10032 (e-mail: cwe5{at}columbia.edu )