Chronic Nicotine Blunts Hypoxic Sensitivity in Perinatal Rat Adrenal Chromaffin Cells via Upregulation of K_ATPChannels: Role of α7 Nicotinic Acetylcholine Receptor and Hypoxia-Inducible Factor-2α

Abstract

Fetal nicotine exposure blunts hypoxia-induced catecholamine secretion from neonatal adrenomedullary chromaffin cells (AMCs), providing a link between maternal smoking, abnormal arousal responses, and risk of sudden infant death syndrome. Here, we show that the mechanism is attributable to upregulation of K ATP channels via stimulation of ␣7 nicotinic ACh receptors (AChRs). These K ATP channels open during hypoxia, thereby suppressing membrane excitability. After in utero exposure to chronic nicotine, neonatal AMCs show a blunted hypoxic sensitivity as determined by inhibition of outward K ϩ current, membrane depolarization, rise in cytosolic Ca 2ϩ , and catecholamine secretion. However, hypoxic sensitivity could be unmasked in nicotine-exposed AMCs when glibenclamide, a blocker of K ATP channels, was present. Both K ATP current density and K ATP channel subunit (Kir 6.2) expression were significantly enhanced in nicotine-exposed cells relative to controls. The entire sequence could be reproduced in culture by exposing neonatal rat AMCs or immortalized fetal chromaffin (MAH) cells to nicotine for ϳ1 week, and was prevented by coincubation with selective blockers of ␣7 nicotinic AChRs. Additionally, coincubation with inhibitors of protein kinase C and CaM kinase, but not protein kinase A, prevented the effects of chronic nicotine in vitro. Interestingly, chronic nicotine failed to blunt hypoxia-evoked responses in MAH cells bearing short hairpin knockdown (Ͼ90%) of the transcription factor, hypoxia-inducible factor-2␣ (HIF-2␣), suggesting involvement of the HIF pathway. The therapeutic potential of K ATP channel blockers was validated in experiments in which hypoxia-induced neonatal mortality in nicotine-exposed pups was significantly reduced after pretreatment with glibenclamide.

Low glucose sensitivity and polymodal chemosensing in neonatal rat adrenomedullary chromaffin cells

“…2E); in both cases the increased membrane depolarization was sufficient to trigger action potential firing when glibenclamide was present. Previous studies in this laboratory have demonstrated that glibenclamide alone had negligible effect on resting membrane potential and ionic currents in neonatal AMCs under normoxic conditions (6,59). These data suggest that aglycemia activates K ATP channels in these cells favoring membrane hyperpolarization, and this occurs in parallel with the inhibition of other voltage-dependent K ϩ channels and/or activation of channels that promote membrane depolarization.…”

Chronic opioids regulate KATP channel subunit Kir6.2 and carbonic anhydrase I and II expression in rat adrenal chromaffin cells via HIF-2α and protein kinase A

“…We previously demonstrated that exposure of both perinatal rat AMCs and immortalized MAH cells to chronic nicotine causes stabilization of the transcription factor HIF-2␣ that in turn led to the transcriptional upregulation of Kir6.2 subunit and K ATP channel expression (5,28). In other cell types, e.g., neuroblastoma cells, stimulation of -and ␦-opioid receptors can lead to HIF activation under nonhypoxic conditions (8).…”

“…Interestingly, we found that exposure of neonatal rat AMCs to chronic nicotine suppressed only hypoxia chemosensitivity (6), whereas exposure to -and/or ␦-opioid agonists led to the suppression of both hypoxia and hypercapnia sensitivity (30). In both instances, the suppression of hypoxia sensitivity was attributable to the increased expression of functional ATP-sensitive K ϩ (K ATP ) channels, which induce membrane hyperpolarization and decreased excitability during hypoxia (5,30). On the other hand, the suppression of hypercapnia sensitivity in opioid-treated AMCs was associated with decreased expression of the CO 2 -hydrating enzymes carbonic anhydrase (CA) I and II (30).…”

“…As a first step, we considered the potential role of the transcription factor hypoxia-inducible factor (HIF)-2␣, because: 1) HIF-2␣ stabilization in nicotine-treated AMCs mediated the transcriptional upregulation of K ATP channel subunit Kir6.2 (28); and 2) the HIF pathway has previously been implicated in the signaling cascade activated in neuroblastoma cells during chronic opioid exposure (8). To facilitate these studies, we used a fetal-derived, immortalized rat chromaffin cell line, i.e., MAH cells, that is sensitive to both hypoxia and hypercapnia (7, 9) and express Kir6.2 and CAII mRNA and protein (5,27,28). Moreover, the availability of a stable HIF-2␣-deficient (Ͼ90% knockdown) MAH cell line (shMAH) allowed us to investigate the role of HIF-2␣ in the opioid signaling cascade.…”

“…Moreover, the availability of a stable HIF-2␣-deficient (Ͼ90% knockdown) MAH cell line (shMAH) allowed us to investigate the role of HIF-2␣ in the opioid signaling cascade. Because protein kinases [e.g., protein kinase C (PKC) and calmodulin kinase (CaMK)] were implicated in the upregulation of Kir6.2 in nicotine-treated chromaffin cells (5), we also investigated their potential involvement in opioid receptor signaling in MAH cells using pharmacological inhibitors. Finally, to validate the predictions of this in vitro model, we used a physiologically relevant in vivo model where pregnant dams received daily injections of morphine just before and throughout gestation.…”