Regulation of cyclic AMP accumulation by lysophosphatidic acid in human airways smooth muscle cells from normal and asthmatic donors

Marie Valente1, Mark R. Dowling2, Christopher E. Brightling3, R.A. John Challiss1. 1Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, LE1 9HN, United Kingdom, 2Novartis Institutes for Biomedical Research, Horsham, West Sussex, RH12 5AB, United Kingdom, 3Department of Respiratory Medicine, Glenfield Hospital, Leicester, LE3 9QP, United Kingdom.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator with demonstrated roles in airway function and disease (Tager et al., 2008). LPA exerts its actions through activation of a family of cognate G protein-coupled receptors, which couple to a variety of G proteins, including Gαi to inhibit adenylate cyclase (AC) activity. Cyclic AMP is a known regulator of airway smooth muscle tone and this second messenger system is a primary target of treatments to alleviate the airway smooth muscle hyper-contractility associated with asthma. In this study, we have asked whether differences exist in the regulation of cyclic AMP accumulation by LPA in low passage (P2-P4) human airway smooth muscle (hASM) cells derived from 6 normal (N) and 10 asthmatic (A) donors.

No significant differences were found in basal cyclic AMP levels in N- and A-derived hASM (N, 17 ± 3; A, 38 ± 8 pmol mg-1 protein (mean ± s.e.m.)). Addition of the AC activator forskolin (10 µM for 10 min) caused large increases in cyclic AMP accumulation (N, 644 ± 75; A, 932 ± 185 pmol mg-1 protein). Pre-addition of LPA (for 5 min) caused concentration-dependent decreases in cyclic AMP accumulation in response to subsequent forskolin addition, with a maximally-effective concentration of LPA (3 µM) causing 56 ± 7% and 50 ± 6% reductions in forskolin-stimulated cyclic AMP accumulation in N- and A-derived hASM cells, respectively. The inhibition caused by LPA could be completely blocked by incubation with the LPA1/3 receptor-selective antagonist, Ki16425 (Ohta et al., 2003). Addition of the β-adrenoceptor agonist, isoprenaline (1 µM for 10 min), resulted in a significantly greater cyclic AMP accumulation in A- versus N-derived hASM cells (N, 504 ± 56; A, 761 ± 115 pmol mg-1 protein; P<0.05, Student’s t-test). Pre-incubation with a range of LPA concentrations (0.1-3000 nM) followed by addition of isoprenaline (1 µM) results in a biphasic response with lower LPA concentrations (1-10 nM) reducing isoprenaline-stimulated responses (by a maximum of (N) 27 ± 9% and (A) 21 ± 5%), but higher LPA concentrations (>10 nM) increasing the response (to (N) 144 ±19% and (A) 137 ± 9%) compared to the effects of isoprenaline alone. Pre-incubation with Ki16425 blocked both the inhibitory and stimulatory phases of the LPA modulation.

The biphasic LPA modulation of the isoprenaline response could be recapitulated in a model cell-line (SW982), which endogenously expresses only the LPA1 receptor subtype. Furthermore, mechanistic studies in the SW982 cell-line have shown that the inhibitory, but not the stimulatory phase of the LPA modulatory effect is pertussis toxin-sensitive, and the modulation is largely insensitive to removal of extracellular Ca2&plus;. While our study has highlighted some significant differences in cyclic AMP turnover in hASM cells derived from normal and asthmatic donors, no differences in the modulatory actions of LPA have been observed.

Ohta H, et al. (2003) Mol Pharmacol 64:994-1005.

Tager AM, et al. (2008) Nat Med 14:45-54.

We thank the BBSRC for PhD CASE studentship funding for M.V.