Abstract 483: The Signaling Pathways of Nicotine-induced ERK1/2 Phosphorylation in Rat Mesangial Cells
Tobacco smoking is associated with accelerated progression of chronic kidney disease of different etiologies including diabetes and hypertension. However, the mechanisms involved are not well understood. We have previously reported that nicotine, a biologically active compound present in high concentrations in tobacco, induces cell proliferation and fibronectin production in mesangial cells which are prevented by ERK1/2 inhibition (AJP’05). In these studies we determined whether rat mesangial cells (MC) express nicotine receptors and characterized the signaling pathways that lead to ERK1/2 phosphorylation in response to nicotine. MC were grown in DMEM with 15% FBS in the presence of 0.4 mg/ml G418 and starved for 24 hours in DMEM without FBS before treatment. We first demonstrated that MC are endowed with several nicotinic Ach receptor (nAChR) subunits including α2-7 and β1-4 as assessed by western blot. Treatment of rat MC with nicotine at 10-7M caused a time-dependent ERK1/2 phosphorylation which peaked after 10 min of stimulation( N=3). Several protein kinase inhibitors were then used to identify the upstream kinases that mediate nicotine-induced ERK1/2 phosphorylation. The calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 (10-7M) decreased the ERK1/2 phosphorylation level by ∼57% (0.41 of 0.95) as compared to nicotine. The PKC inhibitor Go6983 at 10-9M, the PKA inhibitor H89 (10-8 M) and the EGFR inhibitor AG 1478 (10-7 M) also inhibited ERK1/2 phosphorylation by 60% (0.38 of 0.95), 48% (0.63 of 1.20) and 68% (0.40 of 1.23) respectively as compared to nicotine. Given the role of the nicotine receptors as agonist-regulated Ca2+ channels, we determined the effects of Ca2+ channel blockade on nicotine induced ERK1/2 phosphorylation. Treatment of MC with the calcium channel Verapamil (10-9 M) resulted in 33% (0.49 of 0.73) inhibition of ERK1/2 phosphorylation as compared to nicotine. In summary, we have determined in these studies that rat MC are endowed with several nAChR subunits and that ERK1/2 phosphorylation in response to nicotine requires CaMK II, PKA, PKC and EGFR. In addition, we have demonstrated that these effects require Ca2+ consistent with the role of the nAChR as agonist-regulated Ca2+ channels in MC.
- © 2012 by American Heart Association, Inc.