Title : Chronic Nicotine Induces Hypoxia Inducible Factor - 2 α 1 in Perinatal Rat Adrenal Chromaffin Cells : Role in

46 Fetal nicotine exposure causes impaired adrenal catecholamine 47 secretion and increased neonatal mortality during acute 48 hypoxic challenges. Both effects are attributable to 49 upregulation of KATP channels and can be rescued by pre50 treatment with the blocker, glibenclamide. Though use of in 51 vitro models of primary and immortalized, fetal-derived rat 52 adrenomedullary chromaffin cells (i.e. MAH cells) 53 demonstrated the requirements for α7 nAChR stimulation and 54 the transcription factor, HIF-2α, the latter’s role was unclear. 55 Using western blots, we show that chronic nicotine causes a 56 progressive, time-dependent induction of HIF-2α in MAH cells 57 that parallels the upregulation of KATP channel subunit, Kir6.2. 58 Moreover, a common HIF target, VEGF mRNA, was also 59 upregulated after chronic nicotine. All the above effects were 60 prevented during co-incubation with α-bungarotoxin (100 nM), 61 a specific α7 nAChR blocker, and were absent in HIF-2α 62 deficient MAH cells. ChIP assays demonstrated binding of 63 HIF-2α to a putative HRE in Kir6.2 gene promoter. Specificity 64 of this signaling pathway was validated in adrenal glands from 65 pups born to dams exposed to nicotine throughout gestation; 66 the upregulation of both HIF-2α and Kir6.2 was confined to 67 medullary, but not cortical, tissue. This study has uncovered a 68