Cholecystokinin Evokes Excitation-Contraction Coupling in Human Airway Smooth Muscle Cells

Abstract

Rationale: Airway smooth muscle (ASM) is the pivotal tissue regulating bronchomotor tone in asthma. Enhanced excitation-contraction (EC) coupling in human ASM (HASM) cells is the underlying mechanism of airway hyperresponsiveness (AHR) in asthma and obesity. HASM cells express an array of receptors that modulate EC coupling. We previously reported that HASM cells from obese donors show a hyperresponsive phenotype in vitro. Unbiased phosphoproteomic analysis of HASM cells from normal and obese donors identified CCK receptor (CCKR) signaling as a differentially modulated pathway in obesity. We hypothesized that CCK receptors differentially modulate excitation-contraction coupling in HASM cells from obese donors compared to that from non-obese donors. Methods: HASM cells obtained from age, sex and ethnic group-matched non-obese (BMI mean±SD: 23.0±1.0, n=5 donors) and obese (BMI mean±SD:43.0±9.7, n=5 donors) lung donors were used in these experiments. Expression of CCK receptor isoforms A and B were determined by qRT-PCR. Fluo-8-loaded HASM cells were stimulated with CCK (sulfated CCK-8, 0.1-10 nM) and time-dependent fluorescence was measured to determine cytosolic Ca 2+ ([Ca 2+ ] i) levels. Phosphorylation of myosin light chain (MLC) and paxilin were determined following 10 min exposure to CCK (0.1-10 nM) or histamine (1 uM). Results: CCK receptor A (CCKAR) mRNA levels were comparable between non-obese and obese donor-derived HASM cells (CCKAR/cyclophilin C t value ratio mean±SD: non-obese 1.29±0.06 Vs obese 1.29±0.13, n=3 donors/group). However, CCK receptor B (CCKBR) transcripts were undetectable in HASM cells. CCK (0.1-10 nM) elicited robustly increased cytosolic Ca 2+ in HASM cells, accompanied by increased MLC and paxilin phosphorylations.