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Regulator of G-Protein Signaling Protein 2 Modulates Purinergic Calcium and Ciliary Beat Frequency Responses in Airway Epithelia

Division of Pulmonary and Critical Care Medicine, Department of Molecular and Cellular Pharmacology, and Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida

Address correspondence to: Dr. Matthias Salathe, Division of Pulmonary and Critical Care Medicine (R-47), University of Miami School of Medicine, 1600 NW 10th AVE, RMSB 7063, Miami, FL 33136. E-mail: msalathe{at}miami.edu

In ciliated airway epithelial cells, purinergic stimulation increases both intracellular calcium ([Ca²⁺]_i) and ciliary beat frequency (CBF). Because regulator of G-protein signaling protein 2 (RGS2) terminates Gq-mediated phospholipase C activation, we examined its role in regulating purinergic signaling in human and ovine airway epithelial cells. RT-PCR of both human and ovine epithelial cell RNA yielded fragments of expected size ( 491 bp) and sequence, confirming RGS2 message. Immunofluorescence demonstrated RGS2 protein expression in cultured airway epithelial cells of both species. Overexpression of an EGFP-RGS2 fusion protein (increasing RGS2 protein levels 1.8 times control, n = 28 cells) resulted in a reduced [Ca²⁺]_i and CBF response to 10 µM ATP (human: 58 ± 9% and 49 ± 8% lower, respectively; n = 8 measurements, 4 cells; ovine: 56 ± 12% and 53 ± 16% lower, respectively; n = 5 measurements, 4 cells). Reducing RGS2 protein levels using antisense oligonucleotides increased the response of both [Ca²⁺]_i and CBF to ATP in human cells by 57 ± 10% and 47 ± 11%, respectively (n = 10 measurements, 6 cells), and in ovine cells by 88 ± 13% and 48 ± 9%, respectively (n = 10 measurements, 5 cells). These data provide functional evidence that RGS2 modulates purinergic signaling in human and ovine ciliated airway epithelial cells.

Abbreviations: air–liquid interface, ALI • ciliary beat frequency, CBF • Dulbecco's modified Eagle's medium, DMEM • enhanced green fluorescent protein, EGFP • Hanks' balanced salt solution, HBSS • intracellular calcium concentration, [Ca²⁺]_I • phospholipase C-ß, PLC-ß • regulators of G-protein signaling, RGS • region of interest, ROI • tetramethylrhodamine isothiocyanate, TRITC

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