Chronic nicotine blunts hypoxic sensitivity in perinatal rat adrenal chromaffin cells via upregulation of KATP channels: role of alpha7 nicotinic acetylcholine receptor and hypoxia-inducible factor-2alpha

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 alpha7 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(+) current, membrane depolarization, rise in cytosolic Ca(2+), 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 approximately 1 week, and was prevented by coincubation with selective blockers of alpha7 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 (>90%) of the transcription factor, hypoxia-inducible factor-2alpha (HIF-2alpha), 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.

Figure 1.

K⁺ channel expression in AMCs derived from P0 pups born to saline-treated versus nicotine-treated dams. In A1–A3, mean (±SEM) current density versus voltage plots are shown for saline-treated AMCs exposed to control (C) and either the BK channel blocker IbTx (100 nm; n = 10) (A1), the SK channel blocker Apa (100 nm; n = 10) (A2), or the nonspecific blocker of Ca²⁺ channels cadmium (Cd²⁺) (50 μm; n = 10) (A3), which indirectly blocks Ca²⁺-dependent K⁺ channels. The insets show sample recordings at +30 mV; holding potential was −60 mV. Corresponding data for P0 nicotine-treated AMCs are shown in B1–B3 (n = 10). Note nicotine-treated AMCs expressed similar Ca²⁺-dependent K⁺ currents as saline-treated AMCs. Isolated AMCs from pups exposed to nicotine in utero also showed expression of the K⁺ channel subunits Kv1.2 and Kv1.5, BK (α-subunit), and SK2, as determined by immunofluorescence (C).

Figure 2.

Upregulation of glibenclamide-sensitive K_ATP channels in AMCs derived from P0 pups born to saline-treated versus nicotine-treated dams. Although glibenclamide (glib) had no effect on its own, it potentiated the hypoxia-induced (hox) inhibition of outward K⁺ current (A) and membrane depolarization (C) in P0 saline-treated AMCs. In contrast, P0 nicotine-treated AMCs did not respond to hypoxia alone (B); however, the combination of hypoxia plus glibenclamide resulted in a marked inhibition of outward K⁺ current (B), as well as membrane depolarization accompanied by spike activity (D). The glibenclamide-sensitive difference current density, which provides an estimate of IK_ATP current density, is plotted against voltage for the two conditions in E. Note the significantly larger IK_ATP density in nicotine-treated AMCs suggesting upregulation of K_ATP channels, and the reversal potential of IK_ATP near K⁺ equilibrium potential (E _K = −80 mV) in E. Error bars indicate SEM. In F, Western blot analysis demonstrated a significant upregulation of the Kir 6.2 subunit of the K_ATP channel in P0 nicotine-treated AMCs compared with saline-treated AMCs, with β-actin as control.

Figure 3.

Fura-2 spectrofluorimetric determination of intracellular calcium (Ca_i) levels in AMCs derived from P0 pups born to saline-treated versus nicotine-treated dams. In P0 saline-treated AMCs, significant increases in Ca_i relative to normoxic (Nox) control (ANOVA; *p < 0.001) occurred during exposure to hypoxia (Hox) (PO₂ ∼ 15 mmHg) and the depolarizing stimulus, high extracellular K⁺ (30 mm). Although the K_ATP channel blocker glibenclamide (glib) had no effect on its own, it significantly potentiated the hypoxia-induced rise in Ca_i seen in saline-treated cells as illustrated in the histogram in A (significance compared with hypoxia alone, **p < 0.001, n = 42). Comparative data are shown for nicotine-treated AMCs in B (n = 35). Note lack of effect of hypoxia alone on Ca_i levels in nicotine-treated AMCs, although coapplication of glibenclamide and hypoxia resulted in a significant increase in Ca_i (ANOVA; *p < 0.001). Error bars indicate SEM. Sample recordings are shown in the top traces.

Figure 4.

Role of nAChR subtypes and intracellular signaling pathways in the nicotine-mediated loss of hypoxic sensitivity in neonatal AMCs. Dissociated AMCs from saline-treated P0 pups were grown for ∼1 week in culture under control conditions, or in presence of nicotine base (Nic) (50 μm) with either mecamylamine (Mec) (100 μm; general nAChR blocker), hexamethonium (Hex) (100 μm; blocker of most nAChRs except α7), α-bungarotoxin (α-btx) (100 nm; specific α7 nAChR blocker), or the depolarizing stimulus high K⁺ (30 mm) as indicated. To probe the signaling pathways, neonatal AMCs were cultured with nicotine and one of the following: the PKC blocker GF109203X (GF) (2 μm), CaM kinase inhibitor KN-62 (3 μm), or the PKA inhibitor H-89 (20 μm). Histogram indicates (mean ± SEM) outward current density (in picoamperes per picofarad) at +30 mV during normoxia or hypoxia (A). Note that hypoxic sensitivity, indicated by inhibition of outward current, was lost after chronic nicotine treatment in culture and that this effect was prevented by α7 nAChR antagonists Mec and α-btx only. Also, hypoxia sensitivity was detectable in nicotine-treated AMCs after coincubation with the PKC inhibitor GF, the CaM kinase inhibitor KN-62, but not the PKA inhibitor H-89. Representative traces for each treatment are located above each pair of bins (C, control; H, hypoxia; W, wash). Sample size was n = 7 cells for each treatment, asterisk (*) denotes significantly different from normoxia, p < 0.01. In B, fast application of nicotine (10 μm) induced an inward current at −60 mV (holding potential) that was partially inhibited by the α7 nAChR blocker α-btx (100 nm) applied to the same saline-treated (left traces) or nicotine-treated (right traces) cell; the α-btx-sensitive difference current for each of the two cells is shown in the bottom traces.

Figure 5.

Effects of hypoxia on catecholamine secretion from AMCs after various chronic treatments in culture. Carbon fiber amperometry was used to detect stimulus-evoked release of CATs from AMCs cultured for 7 d in the presence of nicotine (Nic) (50 μm), with or without mecamylamine (Mec) (100 μm), hexamethonium (Hex) (100 μm), and α-bungarotoxin (α-btx) (100 nm), or in the presence of the depolarizing stimulus high K⁺ (30 mm). Hypoxia stimulated quantal CAT release as indicated by an increase in event frequency (events per minute) in control AMCs and AMCs grown chronically in high K⁺. In contrast, hypoxia failed to stimulate CAT secretion from AMCs cultured in the presence of nicotine; however, this blunting effect of nicotine was prevented after coincubation with Mec or α-btx, but not hex. Data were obtained from seven cells for each treatment [the asterisk (*) indicates significant difference, p < 0.01, from normoxic (Nox) control]. Error bars indicate SEM. The top traces show sample recordings of quantal CAT release from AMCs under the conditions indicated.

Figure 6.

Nicotinic AChR-mediated regulation of the expression of the K_ATP channel subunit Kir 6.2 in cultured immortalized chromaffin (MAH) cells. In Western blots, MAH cells cultured for 7 d in the presence of nicotine (Nic) (50 μm) showed an increased expression of the Kir 6.2 subunit, relative to control untreated cultures. This effect of nicotine persisted during coincubation with hexamethonium (Hex) (100 μm), but was blocked during coincubation with the α7 nAChR antagonist α-bungarotoxin (α-btx) (100 nm) or mecamylamine (Mec) (100 μm). Chronic membrane depolarization with 30 mm K⁺ did not result in upregulation of Kir 6.2 (B). Upregulation of Kir 6.2 was also prevented when MAH cells were cultured with nicotine and either the PKC blocker GF or the CaM kinase blocker KN62; the PKA inhibitor H-89 was ineffective. Relative staining intensities (compared with control) are summarized for four Western blots in the corresponding histograms below. *p < 0.005. Error bars indicate SEM.

Figure 7.

Role of HIF-2α in mediating the nicotine-induced loss of hypoxia sensitivity. MAH cells deficient in HIF-2α (>90% knockdown) (shMAH) were cultured with or without nicotine, and hypoxic sensitivity was determined based on inhibition of outward K⁺ current. Control, wild-type MAH cells (wtMAH) were hypoxia-sensitive (A) but became hypoxia-insensitive when cultured with chronic nicotine (B). In contrast, nicotine treatment failed to affect the hypoxic sensitivity of MAH cells deficient in HIF-2α (shMAH) (C); however, in scrambled control MAH cells (scMAH), nicotine exposure still resulted in a loss of hypoxic sensitivity (D). Error bars indicate SEM. In E, the Western blots show that HIF-2α protein was induced in MAH cells by chronic hypoxia (CHox) (2% O₂ for 24 h), but not by chronic nicotine; HIF-2α induction by chronic hypoxia was also present in scMAH cells but was absent in shMAH cells (F). RT-PCR analysis in F shows downregulation of HIF-2α mRNA in shMAH cells, but not in scMAH cells.

Figure 8.

Effect of prenatal nicotine exposure and acute glibenclamide injection on neonatal (postnatal day 1) mortality during a 60 min exposure to hypoxia (10% O₂). Compared with rat pups born to saline-treated dams (control), those born to nicotine-treated dams had a higher percentage of mortality after exposure to hypoxia; both groups received a saline injection (saline inj) just before exposure to hypoxia. In contrast, neonatal nicotine-treated pups that were injected with glibenclamide (glib inj) just before exposure to hypoxia, had a percentage of mortality that was not significantly different from control. N = 40 for each bin. *p < 0.009. Error bars indicate SEM.