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* Department of Biology, and
Department of Obstetrics and Gynecology, McMaster University, Hamilton, Ontario, Canada
1Correspondence: Department of Biology, McMaster University, 1280 Main St. West, Hamilton, Ontario, Canada L8S 4K1. E-mail: nursec{at}mcmaster.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 wk in the presence of a subsaturating concentration of nicotine base (50 µM), and this effect was abolished by the nicotinic acetylcholine receptor (nAChR) blocker mecamylamine (100 µM). Taken together, these data suggest that the adverse effects of maternal smoking on sympathoadrenal function in the offspring are due in part to a loss or suppression of acute hypoxic sensitivity in adrenal chromaffin cells, triggered by the direct action of nicotine on endogenous nicotinic acetylcholine receptors.—Buttigieg, J., Brown, S., Zhang, M., Lowe, M., Holloway, A. C., Nurse, C. A. Chronic nicotine in utero selectively suppresses hypoxic sensitivity in neonatal rat adrenal chromaffin cells.
Key Words: catecholamine • nAChR • SIDS • fetus • asphyxia
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Despite the numerous chemicals present in cigarette smoke, many of the deleterious side effects on the fetus and newborn are thought to arise from the presence of nicotine. Nicotine passes readily from the maternal to fetal circulation and binds to nicotinic acetylcholine receptors (nAChRs) on target cells that regulate various autonomic functions including respiration, cardiac function, blood pressure, and arousal responses (2)
.
The adrenal medulla forms part of the autonomic nervous system, and catecholamine (CA) release from this organ in response to hypoxic stress is critical for neonatal survival and adaptation to extrauterine life (3
, 4)
. For example, hypoxia-induced CA release from adrenomedullary chromaffin cells (AMCs) is necessary for the maintenance of cardiac conduction and for the preparation of the lungs for air breathing via stimulation of fluid reabsorption and surfactant secretion (4)
. More recent studies (5
, 6)
suggest that neonatal AMCs are also CO2/pH sensors, and during perinatal asphyxia the combined effects of hypoxia, increased CO2 (hypercapnia), and increased acidity appear to elicit a robust CA secretory response. Among the deficits associated with fetal nicotine exposure are impaired CA secretion and increased neonatal mortality after a sustained (75 min) hypoxic challenge (5% O2) (7)
. The mechanisms underlying this deficient adrenal CA secretory response are not completely understood but appear central to the loss of hypoxia tolerance and increased neonatal mortality (2
, 7)
.
In the present study, we considered the possibility that nicotine exposure in the perinatal period leads to a suppression of the direct, acute hypoxia-sensing mechanism in adrenal chromaffin cells via binding and activation of endogenous nAChRs. This mechanism is present in neonatal chromaffin cells and is nonneurogenic, i.e., occurs before maturation of the preganglionic splanchnic innervation (3
, 4)
. Several studies suggest that acute hypoxia sensing in neonatal chromaffin cells is mediated by inhibition of a variety of K+ channels, leading to, or facilitating, membrane depolarization, voltage-gated Ca2+ entry, and CA secretion (6
, 8
9
10
11
12
13
14
15)
. Interestingly, this direct hypoxia-sensing mechanism, as well as CO2 sensing, in rodent chromaffin cells is lost or suppressed postnatally along a time course that roughly parallels maturation of the splanchnic innervation, which is itself cholinergic (4
5
6
, 9
, 11
, 12)
.
To assess chromaffin cell responses to hypoxia or hypercapnia, we used several complementary approaches. In particular, whole-cell recording was used to measure membrane currents and membrane potential, Fura-2 spectrofluorimetry was used to measure cytosolic calcium, whereas carbon fiber amperometry was used to measure secretion of CA. We show that exposure of pregnant rats to chronic nicotine bitartrate (1 mg/kg/day) leads to a dramatic suppression of acute hypoxic sensitivity (relative to saline controls) in adrenal chromaffin cells isolated from newborn offspring. Interestingly, CO2 sensitivity remained largely intact, suggesting a selective effect of in utero nicotine on hypoxic sensitivity. Moreover, we show that this suppression of hypoxia sensing can be reproduced in vitro by exposing isolated neonatal chromaffin cells to chronic nicotine for 7 days in culture.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Dams were allowed to deliver naturally and pups were collected soon after birth [postnatal day 0 (P0)]. Before the adrenal glands were removed, P0 pups were first rendered unconscious by a blow to the head and then immediately killed by decapitation. Isolated adrenal glands were kept in sterile medium, where most of the outer cortex was removed before enzymatic digestion of the medullary tissue.
Cell culture
Primary cultures enriched in adrenal chromaffin cells were prepared from P0 rat pups by combined enzymatic and mechanical dissociation as described in detail elsewhere (9
, 11
, 12)
. After being preplated for 2 h to remove most of the cortical cells, the nonadherent chromaffin cells were plated on modified culture wells coated with Matrigel (Collaborative Research, Bedford. MA, USA). Cells were grown at 37°C in a humidified atmosphere of 95% air-5% CO2 for 18–36 h before use. The growth medium consisted of F-12 nutrient medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and other additives as described previously (12)
. In some experiments, nicotine base (50 µM) and/or the nicotinic receptor blocker mecamylamine (100 µM) was added to the culture medium.
Electrophysiology
Voltage-clamp data were obtained using the nystatin perforated-patch technique as described previously (10
, 11
, 13)
. The pipette solution contained (mM) the following: 110 K gluconate, 25 KCl, 5 NaCl, 2 CaCl2, 10 HEPES at pH 7.2, and nystatin (300–450 µg/ml). Some experiments were carried out at room temperature (20–22°C) in HEPES-buffered extracellular medium that contained (mM) the following: 135 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 10 glucose, and 10 HEPES at pH 7.4, with or without 0.5 µM tetrodotoxin. Other experiments were carried at 35°C in bicarbonate/5% CO2-buffered solution (24 mM NaHCO3 substituted for equimolar NaCl) as described previously (17)
. Hypoxic solutions (PO2=5–15 mmHg) were generated by bubbling N2 gas and were applied to the cells by gravity flow or by a rapid perfusion system (12
, 17)
. Hypercapnic solutions (10% CO2; pH=7.4) were prepared by increasing bicarbonate concentration to 48 mM and bubbling 10% CO2 as described previously (17)
. In voltage-clamp experiments, cells were held at –60 mV and step depolarized to the indicated test potential (between –100 and +80 mV in 10 mV increments) for 100 ms at a frequency of 0.1 Hz. In some cases, cells were held at –60 mV and the voltage was ramped from –80 to +50 mV over 500 ms at a frequency of 0.1 Hz. In the majority of experiments, cells were studied within 24 h of isolation. In cases where cultures were treated chronically with nicotine and/or mecamylamine (see above), recordings were carried out 1 wk after cell isolation. All electrophysiological data are expressed as mean ± SE and compared using the paired or independent Student’s t tests (Microcal Origin version 7.0).
Intracellular Ca2+ measurements
Intracellular Ca2+ (Ca2+i) was monitored using the fluorescent Ca2+ indicator Fura-2 AM. Cells were loaded with 5 µM Fura-2 AM for 30 min at 37°C then rinsed (3 times) before use (30 min later). Ratiometric Ca2+ imaging was performed using a Nikon Eclipse TE2000-U inverted microscope (Nikon, Mississauga, ON, Canada) equipped with a Lambda DG-4 ultra high-speed wavelength changer (Sutter Instrument Co., Novato, CA, USA; exposure time 100 ms), a Hamamatsu OCRCA-ET digital CCD camera (Hamamatsu, Sewickley, PA, USA), and a Nikon S-Fluor x40 oil-immersion objective lens with a numerical aperture of 1.3. Dual images (340 and 380 nm excitation and 510 nm emission) were collected, and pseudocolor ratiometric data were obtained using Simple PCI software version 5.3 (Hamamatsu). The imaging system was standardized with a two-point calibration, using Ca2+ free solution and Ca2+ solution (39 µM) from Molecular Probes (Eugene, OR, USA; F-6774). The parameters used for the two point calibration included the dissociation constant of Fura-2 (Kd=224 nM), the ratio values for the (–) and (+) concentration standards (Rmin=0.026 and Rmax=4.4), and the β value of 5.6. Ca2+i concentration (in nM) was calculated according to the equation described by Grynkiewicz et al. (18)
. All experiments were performed at 34°C, and cells were continuously perfused with bicarbonate/CO2-buffered extracellular medium (see above). The switch between control and test solutions was aided with a double-barrel fast perfusion system (17)
.
Carbon fiber amperometry
CA secretion from chromaffin cells was monitored using carbon fiber amperometry after the culture dish was placed on the stage of a Zeiss Axioskop 2 upright microscope equipped with a x40 water immersion objective (Carl Zeiss, Toronto, ON, Canada). The culture was perfused under gravity with extracellular solution containing (mM) the following: 135 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 10 glucose, and 10 HEPES at pH 7.4 and 37°C. In some experiments, high K+ (30 mM) solutions were used after equimolar substitution for NaCl. Hypoxic solution (PO2=15–20 mmHg) was obtained by continuously bubbling with N2. CA secretion was monitored from single cells that were usually part of a cell cluster with ProCFE low noise carbon fiber electrodes (5 µm diameter tip; Dagan Corporation, Minneapolis, MN, USA) connected to a CV 23BU headstage and an Axopatch 200B amplifier set at 800 mV. Data acquisition and analysis were performed using Clampfit 9.2 (Axon Instruments, Foster City, CA, USA); currents were filtered at 100 Hz, digitized at 250 Hz, and stored on a personal computer. Individual secretory events were quantified by measuring the charge, calculated by integrating the area under each amperometric spike. Total secretion during stimulus application was plotted as the cumulative charge [femtocoulombs (fC)]. Events <3 pA were excluded from the analysis, and spike frequency was calculated as the number of spike events per min. Samples were compared using Student’s t test, and level of significance was set at P < 0.05. Unless otherwise noted, the data are expressed as mean ± SE.
Western blot
Protein was extracted from enriched chromaffin cell cultures in lysis buffer containing (mM) the following: 10 HEPES pH 7.6, 10 KCl, 0.1 EDTA pH 8, 0.1 EGTA pH 8, and 1 DTT). Thirty micrograms of protein was loaded onto an 8% SDS-PAGE and run at 120 V for 2 h. Protein was transferred from the gel onto a PVDF membrane (Millipore, Bedford, MA, USA) and incubated in either sheep polyclonal antibody against carbonic anhydrase II (Biogenesis, Oxford, UK) or mouse monoclonal antibody against β-actin at 4°C overnight. The membrane was washed with PBS, incubated for 1 h at room temperature with a horseradish peroxidase (HRP) -linked secondary antibody, and rewashed in PBS. The blot was visualized using Immobilon Western Chemiluminescent HRP substrate (Millipore) and autoradiography.
Quantitative reverse transcription-polymerase chain reaction
RNA from enriched chromaffin cell cultures was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). RNA was quantified in an Eppendorf Biophotometer, and 500 ng was treated with DNase I (Invitrogen,). Reverse transcription (RT) was carried out on 100 ng of DNase-treated RNA using Superscript III (Invitrogen) and random primers (100 ng). Quantitative polymerase chain reaction (QPCR) was carried out and analyzed using a Stratagene MX3000P machine and the Absolute QPCR SYBR Green Mix (ABgene, Epson, UK). Analysis was based on the 
CT method, and CT values were in the linear phase of the PCR reaction. Gene-specific primers were designed using GeneFisher Software (19)
and synthesized by the central facility of the Institute for Molecular Biology and Biotechnology (MOBIX; McMaster University, Hamilton, ON, Canada). The following intron spanning primers were used and listed as gene amplified, sequence (forward, reverse), and annealing temperature: lamin A/C: 5'-GCAGTACAAGAAGGAGCTA-3' and 5'-CAGCAATTCCTGGTACTCA-3', 55°C; carbonic anhydrase I or CAI: 5'-AACCAGCGAAGCCAAAC-3' and 5'-TGTGGTGGA CGGTGGTTG-3', 55°C; and carbonic anhydrase II or CAII: 5'-CCGACAGTCCCCTGTGGA-3' and 5'-GCGGAGTGGTCAGAGAGCCA-3', 55°C. Verification of the PCR products was done using the QIAquick Gel Extraction Kit (Qiagen). The DNA sample was then sequenced (at MOBIX). The sequencing results were analyzed by BLAST and matched to the Rattus norvegicus lamin A/C (GenBank accession number BC062018.1 and X99257.1), CAI (XM_226922.4), and CAII (NM_019291.1).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The maternal steady-state levels of serum cotinine (the major metabolite of nicotine) resulting from this exposure of 135.9 ± 7.86 ng/ml are within the range of cotinine concentrations reported in pregnant smokers during both early pregnancy and late pregnancy (21)
.
Electrophysiological comparison of the sensitivity of nicotine- and saline-treated P0 adrenal chromaffin cells to hypoxia and hypercapnia
In neonatal AMCs, hypoxia-induced inhibition of outward current and membrane depolarization represent common hallmarks of O2 sensitivity and are mediated by closure of different K+ channel subtypes (8
, 12
13
14)
. These properties enhance voltage-dependent Ca2+ entry and CA secretion during hypoxia in part by prolonging action potential repolarization (6
, 9
, 11
, 12
, 15)
. In HEPES-buffered extracellular solution, hypoxia caused the expected inhibition of outward K+ current at positive potentials during voltage-clamp whole-cell recordings in the majority (n=28/32) of control P0 saline-treated AMCs (Fig. 1
A, C). Also, hypoxia induced a depolarizing receptor potential in the majority (n=8/10) of saline-treated P0 AMCs, and in a few cases it triggered action potentials (e.g., Fig. 1E
). In contrast, both features were conspicuously absent or blunted in nicotine-treated P0 AMCs, where hypoxia had a negligible effect on whole-cell outward current (Fig. 1B, D
; 31/37 cells nonresponsive) and membrane potential (Fig. 1F
; 13/16 cells nonresponsive). Data sets comparing mean ± SE whole-cell outward K+ current density at +30 mV and membrane potential for all sampled cells (n) under the different treatments are summarized in Table 1
. These data suggest that chronic nicotine exposure in utero leads to a premature loss or blunting of hypoxic sensitivity in neonatal AMCs, thereby compromising the ability of the newborn to survive hypoxic stress (3
, 22)
.
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Neonatal AMCs are also CO2 sensors, and their sensitivity to elevated CO2 (hypercapnia) leading to CA secretion is mediated by an intracellular pH-dependent inhibition of outward K+ current and membrane depolarization (5)
. In contrast to hypoxia, the depolarization in this case appears to be due to activation of a resting membrane conductance (5)
. To test whether or not nicotine exposure in utero also affects hypercapnic sensitivity of neonatal AMCs, we examined the effects of high CO2 (10%; pH=7.4) in bicarbonate-buffered medium. Interestingly, high CO2 induced inhibition of outward K+ current in most P0 AMCs from both saline –treated (n=17/19) and nicotine-treated (n=28/31) pups (Fig. 2
A–D). Indeed, we could demonstrate both CO2 and hypoxic sensitivity in the same saline-treated cells (e.g., Fig. 2A
) and retention of CO2 but loss of hypoxic sensitivity in the same nicotine-treated cells (e.g., Fig. 2B
). Current density (pA/pF) plots comparing hypercapnic and hypoxic responses in the same groups of saline-treated (n=17) and nicotine-treated (n=15) cells are summarized in Fig. 2C, D
, respectively, for voltage steps to +30 mV. The percent inhibition of outward K+ current induced by hypercapnia was similar (45%) for both saline- and nicotine-treated cells (Fig. 2A
–D). In addition, during current clamp recordings in bicarbonate-buffered medium, depolarizing receptor potentials were induced in the majority of saline-treated cells by hypercapnia (n=12/13) and hypoxia (n=8/11). Robust chemoexcitation due to separate application of these stimuli to the same saline-treated cell is shown in Fig. 2
E1, E2. In contrast, although hypercapnia induced a depolarizing receptor potential in the majority (n=14/18) of nicotine-treated cells, hypoxia produced no detectable response in 87% of nicotine-treated cells (13/15 cells nonresponsive). An example of a robust hypercapnic, but absent, hypoxic response in the same nicotine-treated cell is shown in Fig. 2
F1, F2. Data sets comparing mean ± SE whole-cell outward K+ current density at +30 mV and membrane potential for all sampled cells (n) under the different treatments are summarized in Table 1
. Taken together, these data suggest that CO2 sensing remains relatively intact in neonatal AMCs after chronic nicotine exposure in utero, whereas hypoxia sensing is markedly impaired.
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Expression of CO2 markers in saline- vs. nicotine-treated P0 adrenal chromaffin cells
Neonatal rat AMCs express CAI and CAII intracellularly, and this appears to be the basis of CO2 sensitivity (5)
. We used RT-QPCR to compare the expression patterns of CAI and CAII in saline- vs. nicotine-treated P0 AMCs. As illustrated in Fig. 3
A, in utero nicotine exposure had no significant effect on normalized CAI and CAII mRNA expression in P0 AMCs relative to saline controls. In control experiments (no RT), no product was observed (data not shown). Moreover, Western blot analysis showed that CAII protein expression was similar in both saline- and nicotine-treated AMCs (Fig. 3B
). Thus, in concert with our electrophysiological studies showing preservation of CO2 sensitivity (see above), the expression of CO2 molecular markers in neonatal AMCs remains relatively unaffected by prenatal nicotine exposure.
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Effects of chemostimuli on Ca2+i levels in control saline- vs. nicotine-treated P0 adrenal chromaffin cells
Elevation in Ca2+i, arising principally from voltage-gated Ca2+ entry, represents a key step leading to CA secretion from neonatal AMCs after exposure to hypoxic and hypercapnic stimuli (5
6
7
, 10
11
12
, 15)
. We used Fura-2 spectrofluorimetry to monitor Ca2+i in P0 AMCs after exposure to various stimuli. Only cells that responded to a depolarizing stimulus, i.e., high (30 mM) extracellular K+, were included in the analysis (Fig. 4
). Hypoxia caused a significant rise in cytosolic Ca2+i in the majority (106/127; 83%) of control, saline-treated P0 AMCs as illustrated in Fig. 4A
and summarized in Fig. 4C
and Table 1
. A small fraction (17%) of these cells did not respond to hypoxia. A slightly larger proportion (114/127; 90%) of saline-treated cells responded to hypercapnia (10% CO2; pH=7.4) with a substantial rise in Ca2+i (Fig. 4A, C
). By contrast, the majority of nicotine-treated P0 AMCs (e.g., Fig. 4B
) failed to respond to hypoxia (only 32/153 responsive; 21%) but remained responsive to hypercapnia (127/153 responsive; 83%). In addition, nicotine (10 µM) evoked substantial elevations of Ca2+i in 90% (115/127) of saline-treated P0 cells compared to 84% (129/153) for nicotine-treated P0 cells (e.g., Fig. 4A, B
). Mean (±SE) data for these experiments are summarized in Fig. 4C, D
and Table 1
. Thus, nAChR remained functional in neonatal AMCs even after chronic nicotine exposure in utero.
|
Secretory responses in control saline- vs. nicotine-treated P0 adrenal chromaffin cells during acute hypoxia
Using carbon fiber amperometry, we monitored CA secretion from P0 chromaffin cells to contrast the effects of chronic nicotine exposure on hypoxia-evoked secretion. As illustrated in Fig. 5
A, C, acute hypoxia stimulated quantal CA secretion from saline-treated P0 AMCs, as did exposure to the depolarizing stimulus high extracellular K+ (30 mM). In marked contrast, however, hypoxia failed to induce secretion of CA from nicotine-treated P0 AMCs although high K+ was effective (Fig. 5B, D
; see also Table 1
), suggesting the secretory machinery was intact. Similar results were obtained when release of ATP, which is costored with CA in chromaffin granules, was monitored using the luciferin-luciferase assay (data not shown; see ref. 10
). These data indicate that CA secretion from P0 AMCs during acute hypoxia is markedly impaired after fetal nicotine exposure.
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Effects of chronic nicotine in vitro on hypoxic sensitivity of isolated neonatal adrenal chromaffin cells
The hypoxia-sensing mechanism in neonatal AMCs is gradually lost or suppressed postnatally in parallel with nicotinic cholinergic innervation via the splanchnic nerve and returns after denervation (3
, 9
, 12)
. This, together with our findings reported above, raises the possibility that direct activation of nicotinic cholinergic receptors on AMCs may normally contribute to the loss or suppression of hypoxic sensitivity. We tested this possibility in vitro by growing cultures of neonatal AMCs in the presence or absence of chronic nicotine base (50 µM), with and without the nicotinic blocker mecamylamine. This dose of nicotine (50 µM) is subsaturating for nAChR present on neonatal AMCs and close to the EC50 of 25.5 ± 4.5 µM, as determined in voltage-clamp experiments at –60 mV (Fig. 6
A). Hypoxic sensitivity, as determined by inhibition of outward K+ current at more positive potentials under voltage clamp, was retained after saline-treated P0 AMCs were cultured in control medium for 7 days (Fig. 6B
). In contrast, however, hypoxic sensitivity was lost when such cells were cultured for 7 days in medium supplemented with 50 µM nicotine base (Fig. 6C
). This blunting effect of nicotine was prevented by the continuous presence of the nAChR blocker mecamylamine (100 µM; Fig. 6D
), suggesting it depended on activation of nAChR. In other control experiments, AMCs grown in media supplemented with 100 µM mecamylamine alone retained their hypoxic sensitivity, indicating that mecamylamine on its own had no effect (data not shown). A similar conclusion was drawn in parallel experiments where P0 AMCs were isolated from nicotine-treated pups and used at the initial plating (Fig. 6E
–G). Notably, these nicotine-treated P0 cells developed hypoxic sensitivity when grown in nicotine-free media for 7 days (Fig. 6E
), whereas they retained their hypoxic insensitivity when grown in nicotine-supplemented medium over the same period (Fig. 6F
). Likewise, nicotine-treated AMCs acquired hypoxic sensitivity when grown in the presence of both nicotine base and mecamylamine for 7 days in vitro (Fig. 6G
) or in the presence of mecamylamine alone (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 7
, 22)
. This CA release is required for redistribution of blood flow to the brain and heart, for maintenance of cardiac contractility during O2 deprivation, and for transformation of the lung into an air breathing organ (7
, 23)
. Our main conclusion was based on the use of several independent assays that were previously established for monitoring hypoxic sensitivity in adrenal chromaffin cells. These included monitoring of hypoxia-induced inhibition of outward K+ current and hypoxia-induced membrane depolarization by whole-cell recording, elevation of cytosolic calcium by Fura-2 spectrofluorimetry, as well as secretion of CA by carbon fiber amperometry. It is noteworthy that the newborn may experience prolonged and intermittent periods of asphyxia during which blood levels of O2 fall, whereas both CO2 and acidity increase (5
, 23)
. These three stimuli appear to act directly on perinatal chromaffin cells (6
, 8
, 9
, 12
, 15)
, and their combined effects cause a markedly enhanced CA release.
Although we demonstrated that fetal nicotine bitartrate exposure markedly impaired the ability of neonatal chromaffin cells to sense a fall in O2 (hypoxia), it had negligible effects on their ability to sense increased CO2 (hypercapnia). Whether doses of nicotine bitartrate higher than the ones used here (1 mg/kg/day) might lead to impaired hypercapnic sensitivity remains to be determined. Nevertheless, the retention of CO2 sensitivity in the present study may have partly compensated for the adverse effects associated with loss of hypoxic sensitivity, since elevations of CO2 during apneic episodes also contribute to CA secretion (5)
. In previous studies, higher maternal doses of nicotine bitartrate (6 mg/kg/day) did not alter perinatal survival but did result in a marked increase in hypoxia-induced mortality (7)
and frequency of apneas between bouts of intermittent hypoxia (18)
. The fact that lower doses (2 mg/kg/day) of nicotine bitartrate did not cause any increase in hypoxia-induced mortality (7)
could well be due to the preservation of other mechanisms, e.g., CO2 sensitivity, that contribute to CA secretion. Similarly, the dose of nicotine bitartrate (1 mg/kg/day), which did not alter CO2 sensitivity in this study, also did not result in increased neonatal death.
Our results favor the idea that the action of nicotine and the resulting suppression of hypoxic sensitivity were mediated via direct activation of nAChRs known to be present on fetal adrenal chromaffin cells (19)
. Indeed, we found that when neonatal chromaffin cells were cultured in the presence of chronic nicotine base at subsaturating doses (50 µM) for 1 wk, they lost hypoxic sensitivity, in contrast to control cultures grown without nicotine base. Moreover, this loss was prevented when mecamylamine, a known blocker of nAChR, was present concomitantly with nicotine. These data suggest that the action of nicotine was mediated via direct stimulation of nAChR on chromaffin cells, although the signaling pathway leading to the loss of hypoxia sensing remains to be determined. The molecular identity of the O2 sensor in these cells is still unknown, although it may be a component of the mitochondrial electron transport chain (9
, 10)
. Thus, chronic nicotine may cause alterations of the O2 sensor itself or other downstream components that ultimately lead to K+ channel regulation (10)
. In this regard, both fetal and neonatal adrenal chromaffin cells express a KATP current that is actually activated by hypoxia (8
, 13
, 24)
. This mechanism appears to be a protective one that tends to hyperpolarize the cell during hypoxia, thereby opposing or limiting the depolarization due to hypoxic inhibition of other O2-sensitive K+ channels (e.g., large conductance KCa channels). Moreover, in the late fetal stages there is a normal developmental switch such that KATP channel expression decreases while KCa channel expression increases in adrenal chromaffin cells (24)
. Thus, a plausible mechanism by which fetal nicotine exposure might lead to an apparent loss of hypoxic sensitivity is via delaying this developmental switch or simply up-regulating KATP channel expression. Our preliminary data suggest that this interesting possibility may indeed occur, although further experiments are required to validate this point as well as to determine whether other mechanisms may also contribute.
Despite the loss of hypoxic sensitivity in neonatal chromaffin cells after fetal nicotine exposure, sensitivity to elevated CO2 (hypercapnia) was retained. One or both carbonic anhydrase isoforms CAI and CAII appear to be the molecular substrate(s) for CO2 sensitivity in these cells and mediate their effects via acidification of intracellular pH leading to regulation of membrane ion channels (5)
. In the present study, the expression of both isoforms was unaffected by prenatal nicotine, in agreement with functional evidence for retention of CO2 sensitivity. Thus, the regulation of hypoxic and CO2 sensitivity in chromaffin cells appears to involve separate pathways, of which hypoxia sensing seems most sensitive to nicotinic receptor stimulation. Interestingly, these findings also provide an explanation for the developmental loss or suppression of hypoxic sensitivity in adrenal chromaffin cells that occurs normally during early postnatal life (4
, 6
, 9
, 12
, 15)
. This loss occurs in parallel with preganglionic cholinergic innervation of chromaffin cells via the splanchnic nerve and can be reversed by denervation (4)
. Thus, nAChR activation via preganglionic ACh release might contribute to the normal developmental mechanisms that suppress hypoxic sensitivity. Alternatively, the action of nicotine in utero might be viewed as a precocious activation of these nAChR leading to a premature suppression of hypoxic sensitivity. The mechanisms by which activation of these nAChR leads to suppression of hypoxic sensitivity need further investigation and are clearly of interest in light of the adverse effects of maternal smoking on the sympathoadrenal system of the offspring. The possibility of an alteration in function of nAChR containing β2-subunits also requires further consideration in light of the finding that mutant mice lacking the β2 subunit display similar deficits as nicotine-exposed wild-type mice at birth (18)
.
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ACKNOWLEDGMENTS |
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Received for publication June 26, 2007. Accepted for publication November 1, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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