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J Appl Physiol 91: 2157-2165, 2001;
8750-7587/01 $5.00
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Vol. 91, Issue 5, 2157-2165, November 2001

Prenatal nicotine affects catecholamine gene expression in newborn rat carotid body and petrosal ganglion

Estelle B. Gauda, Reed Cooper, Patrice K. Akins, and Guimei Wu

Department of Pediatrics, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287-3200


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Nicotine exposure modifies the expression of catecholamine and opioid neurotransmitter systems involved in attenuation of hypoxic chemosensitivity. We used in situ hybridization histochemistry to determine the effect of prenatal and early postnatal nicotine exposure on tyrosine hydroxylase (TH), dopamine beta -hydroxylase (Dbeta H), preproenkephalin (PPE), and D2-dopamine receptor mRNA levels in the rat carotid body and petrosal ganglion during postnatal development. In the carotid body, nicotine increased TH mRNA expression in animals at 0 and 3 postnatal days (both, P < 0.05 vs. control) without affecting TH mRNA levels at 6 and 15 days. At 15 postnatal days, Dbeta H mRNA levels were increased in the carotid body of nicotine-exposed animals. Dopamine D2-receptor mRNA levels in the carotid body increased with postnatal age but were unaffected by nicotine exposure. PPE was not expressed in the carotid body at any of the ages studied in control or treated animals. In the petrosal ganglion, nicotine increased the number of ganglion cells expressing TH mRNA in animals at 3 days (P < 0.01 vs. control). Dbeta H mRNA expression was not induced nor was PPE mRNA expression increased in the petrosal ganglion in treated animals. Prenatal nicotine exposure upregulates mRNAs involved in the synthesis of two inhibitory neuromodulators, dopamine and norepinephrine, in peripheral arterial chemoreceptors, which may contribute to abnormalities in cardiorespiratory control observed in nicotine exposed animals.

peripheral arterial chemoreceptors; tyrosine hydroxylase; dopamine beta -hydroxylase; preproenkephalin; control of breathing; sudden infant death syndrome


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TO BETTER UNDERSTAND the biological mechanisms that help explain the increased association between sudden infant death syndrome and exposure to tobacco smoke (19), several physiology studies have investigated the effect of prenatal nicotine exposure, a major component of tobacco smoke, on ventilatory and cardiovascular control in newborn animals. In newborn rats exposed prenatally to nicotine, abnormalities in ventilation (46), autoresuscitation (11), and cardiovascular responses (32) have been reported. Specifically, prenatal exposure to nicotine blunts the ventilatory responses to short exposures to hypoxia (46) and hyperoxia (2), manipulations that are typically used as a test of peripheral chemoreceptor function in unanesthetized animals and newborn infants.

Nicotine binds to nicotinic cholinergic receptors on catecholamine-containing neurons and affects neurotransmitter expression in the peripheral and central nervous systems (31, 51). Nicotine increases dopamine, norepinephrine, and opioid content and upregulates catecholaminergic synthesizing enzymes and peptide expression (8). The peripheral arterial chemoreceptors express nicotinic receptors (1), contain catecholamines and opioids, and are involved in modulating respiratory and arousal responses to hypoxia (for review, see Ref. 20). In the peripheral arterial chemoreceptors, catecholamines are the most abundant neuromodulators in the carotid body. Dopamine, through binding to dopaminergic D2 receptors, and norepinephrine, through binding to alpha 2-adrenergic receptors on carotid sinus nerve fibers, attenuate hypoxic chemosensitivity (for review, see Ref. 20). Opioids, specifically, Met-enkephalins through binding to delta -opioid receptors, also attenuate hypoxic chemosensitivity (35).

Hypoxic chemosensitivity of peripheral arterial chemoreceptors increases with postnatal age (for review, see Refs. 17 and 10). Associated with maturation of hypoxic chemosensitivity are changes in neurotransmitter profiles and changes in the biochemical and electrical properties of cells in the carotid body (for review, see Ref. 17). Specifically, dopamine content is elevated in the carotid body of newborn rats, and this is associated with blunted chemoreceptor responses (25). Similarly, our laboratory has shown that mRNA levels for tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine synthesis, are significantly elevated in newborn animals, whereas mRNA levels for dopamine D2 receptors are decreased in the same cells in the carotid body (14). However, with increasing maturation, TH mRNA levels decreased, whereas dopamine D2-receptor mRNA levels increased. Less is known about the effect of maturation on the enkephalin content in the carotid body. In addition, our laboratory has previously demonstrated that preproenkephalin (PPE) mRNA is present in petrosal ganglion cells but not in the carotid body of 14-day-old rats (16). Acute nicotine exposure affects catecholaminergic and opioid neurotransmitters in peripheral and central nervous systems. However, how prenatal nicotine exposure affects the expression of these inhibitory neurotransmitter systems in the carotid body and sensory ganglion is not known. Thus the purpose of this study was to elucidate molecular mechanisms involved in alterations in the expression of critical neurotransmitters that may explain alterations in chemosensitivity observed in newborn rats exposed prenatally to nicotine. We determined the effect of prenatal and early postnatal nicotine exposure on TH, PPE, and dopamine D2-receptor gene expression in the carotid body and petrosal ganglion in newborn rat pups during the first 2 wk of postnatal development. We also determined the effect of prenatal nicotine exposure on dopamine beta -hydroxylase (Dbeta H) gene expression in the carotid body and petrosal ganglion in animals at 2 wk postnatal age. Because nicotine exposure upregulates catecholaminergic and opioid traits in central and peripheral nervous systems, we hypothesized that prenatal nicotine exposure would affect neurotransmitters by upregulating mRNAs encoding proteins involved in inhibitory catecholaminergic and opioid systems in peripheral arterial chemoreceptors during early postnatal ages.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Time-dated, pregnant Sprague-Dawley rats were used. On days 2-3 of gestation, before implantation of the embryo in the uterine wall, an osmotic minipump (type 2ML4, Alza, Palo Alto, CA) containing nicotine bitartrate or sterile saline (0.9%) was inserted subcutaneously. The dose of nicotine delivered by the osmotic pump (6 mg · kg-1 · day-1 of free base nicotine) produces plasma nicotine levels that are seen in heavy smokers (3 packs/day) (31, 38). Dams received nicotine or vehicle until parturition on day 22 and then for 1 wk after delivery, i.e., for a total of 4 wk. After spontaneous delivery, the rat pups were cross-fostered to maintain equal litter sizes.

Tissues were taken from Sprague-Dawley rats on postnatal days 0, 3, 6, and 15 (n = 7, each age). All animals were briefly anesthetized with 3% methoxyflurane and decapitated. The bifurcation of the carotid artery with the carotid body and the superior cervical, nodose, petrosal, and jugular ganglia was quickly removed en bloc, placed in embedding media (Fisher), and quickly frozen on dry ice. The right and left tissue blocs were removed from the animals within 5 min of decapitation. The tissues were stored at -70°C until further processing.

In situ hybridization histochemistry. Tissue blocs were cut in 12-µm sections on a cryostat. Sections were thaw-mounted onto gelatin-chrome, alum-subbed slides. Slide-mounted sections were then fixed in 4% paraformaldehyde, acetylated in fresh 0.25% acetic anhydride in 0.1 M triethanolamine, dehydrated in ascending series of alcohols, delipidated in chloroform, and then rehydrated in a descending series of alcohols. Slides were air dried and then stored at -20°C.

Antisense ribonucleotide probes were used for detection of TH, Dbeta H, PPE, and dopamine D2-receptor mRNAs. The antisense probes were constructed from cDNAs for each of these genes by in vitro transcription. The cDNA for the TH, PPE, and dopamine D2 receptors was complementary to base pairs 1,120-1,488 (22), 51-987 (52), and 372-1,174 (3, 37) of the rat genes, respectively. cDNA for Dbeta H contained a 575-base pair fragment corresponding to the nucleotides 205-780 of the rat Dbeta H cDNA described by McMahon et al. (34). The cDNA fragment was obtained by PCR amplification of rat striatum cDNA with sense strand oligonucleotide corresponding to base pairs 205-225 and antisense oligonucleotide strand corresponding to bases 759-780 of the published sequence (34). The PCR products, along with control samples, were electrophoresed through an agarose gel. A single band of ~500 base pairs was excised from the gel and subsequently subcloned into pCR4-TOPO TA cloning vector (Invitrogen, Carlsbad, CA). This cloning vector allows for direct cloning of PCR products into an EcoR I (restriction enzyme) cloning site and contains T3 and T7 RNA polymerase sites for generating sense and antisense ribonucleotide probes for in situ hybridization experiments and direct sequencing. The subcloned cDNA fragment was partially sequenced to determine orientation of the cloned fragment.

To verify specificity of the ribonucleotide probes, coronal brain sections from adult animals corresponding to the following bregma coordinates (bregma: -10.04, -5.80, and 0.20) (41) were used as controls. Control brain slides were hybridized, exposed, and developed simultaneously with the experimental slides. Uniformly, these control slides demonstrated the known patterns of gene expression in striatopallidal neurons for dopamine D2-receptor and (18) PPE mRNAs (44), nigrostriatal neurons for TH (40), and locus coeruleus neurons for Dbeta H (data not shown). All control brain slides showed discrete and specific hybridization signal in the neural cell groups known to express these genes without any background or nonspecific hybridization signal. In addition, the patterns of TH, PPE, Dbeta H, and dopamine D2-receptor mRNA expression differed in the tissue bloc of the carotid body and petrosal and superior cervical ganglia, further demonstrating probe specificity.

Probes were labeled with [35S]UTP via in vitro transcription as outlined by Chesselet et al. (5). Labeled probes of 1.2-1.5 × 106 dpm were added to 100 µl of hybridization buffer [50% formamide, 600 mM NaCl, 300 mM NaCl, 20 mM Tris · HCl, pH 7.5, 1 mM EDTA, 10% dextran sulfate, 1× Denhardt's solution, 100 µg/ml salmon sperm DNA, 250 µg/ml yeast total RNA (type XI), 250 µg/ml yeast tRNA, and 100 mM dithiothreitol], which was applied to slides containing 8-10 sections per slide. Hybridization was performed at 55°C overnight. The slides were then washed in 1× SSC (0.15 M sodium chloride-0.015 M sodium citrate, pH 7.2) at room temperature. After treatment with RNAase A (20 mg/ml), slides were washed at 60°C in 0.2× SSC, rinsed in deionized water, and air dried. Slides were then dipped in Kodak photographic emulsion, dried, and exposed in the dark at -20°C for 4-8 wk. After exposure, the slides were thawed at room temperature, developed with Dektol (Kodak), and counterstained with thionin, and coverslips were applied with Permount.

Data analysis. Comparisons were only made between data obtained from slides that were processed for in situ hybridization, hybridized, exposed, and developed together under the same conditions. Silver grains generated by 35S in the emulsion were analyzed by using a microscope and Macintosh image analysis program [National Institutes of Health (NIH) image, W. Rasband, NIH]. Semiquantitation of silver grains (representing TH, Dbeta H, PPE, and dopamine D2-receptor mRNAs) in the carotid body was done by using a counting algorithm of the image analysis program. After the dark-field image was captured and digitized at ×400, the number of grains was counted in the carotid body. To normalize for the possible different sizes of the sections of the carotid bodies, the number of grains in multiple, contiguous 100-µm-diameter circles was counted, and a mean was obtained for each section of the carotid body. These numbers were combined from two to three sections of the carotid body to obtain an average count per animal. The population of values of average counts per animal was compared between control and treated groups with ANOVA and post hoc analysis via Student's unpaired t-test. Statistical significance was set at P < 0.05. Because of the additional tissue sections that were available from 15-day-old animals, we determined whether prenatal nicotine exposure altered Dbeta H gene expression in the carotid body and petrosal ganglion in this age only.

Semiquantitation of the number of ganglion cells in the petrosal/jugular ganglion (PG/JG) complex expressing TH mRNA was determined by counting the number of positive cells per tissue section with dark-field microscopy at ×400. A mean was obtain from each animal after determining the mean number of TH-positive ganglion cells from five to seven tissue sections per animal. The mean number of TH-positive ganglion cells per PG/JG section between control and nicotine-exposed animals was determined by unpaired t-test in animals at 3 and 15 postnatal days of age.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Prenatal nicotine exposure did not affect litter sizes nor did it affect dam or rat pup mortality. However, rat pups exposed to nicotine were smaller than control pups at each of the postnatal ages studied (Table 1). Prenatal and early postnatal nicotine exposure significantly increased the expression of TH mRNA in the carotid bodies of newborn rats (P = 0.02, ANOVA). TH mRNA levels were greater in animals exposed to nicotine at 0 and 3 postnatal days, as shown in representative photomicrographs from four animals in Fig. 1 and in the histogram of composite data from all animals in Fig. 2. Although TH mRNA levels at days 6 and 15 did not differ between control and treated animals (Fig. 2), nicotine exposure did upregulate Dbeta H mRNA expression in the carotid body of 15-day-old animals (P = 0.03, control vs. nicotine), as shown in the photomicrographs of one control and one prenatally nicotine-exposed animal (Fig. 3, A and B), along with composite data (bar graph) from all animals (Fig. 3, right). Similar to TH mRNA, Dbeta H mRNA was moderately expressed in many cells in the superior cervical ganglion in control animals, as shown in the low- (Fig. 4A) and high-power (Fig. 4B) dark-field photomicrographs from one control animal at day 15. However, prenatal nicotine exposure did not significantly change the level of Dbeta H mRNA levels in the superior cervical ganglion (data not shown). Prenatal nicotine exposure also increased the number of ganglion cells in the PG/JG complex expressing TH mRNA at 3 days (Fig. 5) but not at 15 postnatal days (P = 0.2, control vs. nicotine at 3 days). Dbeta H mRNA was not detected in the PG/JG complex in either control or treated animals at day 15.

                              
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  		Table 1.   Effect of prenatal nicotine exposure on body weight



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  		Fig. 1.   Dark-field photomicrographs of the carotid body showing the expression of tyrosine hydroxylase (TH) mRNAs in control animals at 0 (A) and 3 postnatal days (C) and animals treated prenatally with nicotine at 0 (B) and 3 postnatal days (D). Nicotine exposure increased the expression of TH mRNA in the carotid body in animals at 0 and 3 postnatal days (B and D). Silver grains appear as white dots. Scale bars = 25 µm.



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  		Fig. 2.   Bar graph showing TH mRNA levels in the carotid body during postnatal development in control animals (open bars) and animals treated prenatally with nicotine (solid bars). Values are means ± SE; n = 7 (ANOVA, P = 0.02). Significant difference (Student's unpaired t-test): * P = 0.02 vs. control at day 0; + P = 0.002 vs. control at day 3.



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  		Fig. 3.   Photomicrographs (A and B) and bar graph (right) showing effect of prenatal nicotine exposure on dopamine beta -hydroxylase (Dbeta H) mRNA expression in the carotid body of 15-day-old animals. Representative photomicrographs of a dark-field image of the carotid body (CB) in two 15-day-old animals show the expression of Dbeta H mRNA in a control animal (A) and an animal treated prenatally with nicotine (B). Nicotine exposure increased the expression of Dbeta H mRNA in the carotid body. Arrows are pointing to clusters of silver grains that represent Dbeta H mRNA expression. Scale bars = 25 µm. Right: bar graph showing Dbeta H mRNA levels in the carotid body control animals (open bar) and animals treated prenatally with nicotine (solid bar) at 15 days postnatal age. Values are means ± SE; n = 5. * P = 0.02 vs. control.



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  		Fig. 4.   Representative low- (A) and high-power (B) dark-field photomicrograph showing the specificity and cellular localization of Dbeta H mRNA expression in the superior cervical ganglion (SCG) of a 15-day-old animal. Scale bars = 50 µm.



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  		Fig. 5.   Photomicrographs (A and B) and bar graph (right) showing effect of prenatal nicotine exposure on TH mRNA expression in the petrosal ganglion (PG), jugular ganglion (JG), and SCG of 3-day-old animals. Representative low-power, dark-field photomicrographs are shown of PG, JG, and SCG of 3-day-old animals, showing the increase in PG/JG neurons expressing TH mRNA (arrows) in an animal exposed prenatally to nicotine (B) compared with the control animal (A). Silver grains are clusters of white dots (arrows). Scale bars = 50 µm. Right: bar graph showing the effect of prenatal nicotine exposure on the no. of ganglion cells in the PG/JG complex expressing TH mRNA in 3-day-old animals. Open bar, control animals; solid bar, animals exposed prenatally to nicotine. Values are means ± SE; n = 5 each group. * P < 0.01 vs. control at 3 days.

Dopamine D2-receptor mRNA was expressed in the petrosal ganglion and carotid body in both control and treated animals. The low-power, bright-field photomicrograph of the nissl-stained tissue section (Fig. 6A) depicts the anatomy of the petrosal ganglion, IX cranial nerve, carotid sinus nerve, and carotid body. The high-power, dark-field photomicrograph (Fig. 6B) of the same tissue section shown in Fig. 6A shows clusters of silver grains representing dopamine D2-receptor mRNA within ganglion cells of the petrosal ganglion. As previously described (14) and shown in this study, dopamine D2-receptor mRNA levels in the carotid body increased with postnatal age. However, nicotine exposure did not alter the level of dopamine D2 expression in the carotid body or the petrosal ganglion at any of the four postnatal ages studied (Fig. 7).


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  		Fig. 6.   Photomicrographs [bright-field (A) and dark-field (B)] of nissl-stained tissue section showing the histology of the PG, IX cranial nerve (CN), carotid sinus nerve (CSN), and the CB (A) and the expression of dopamine D2-receptor (D2-R) mRNA in the PG (B) of a 6-day-old animal. Silver grains are clusters of white dots (arrows). Scale bars = 100 µm.



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  		Fig. 7.   Bar graph showing dopamine D2-receptor mRNA levels (grains/area) in the carotid body during postnatal development in control animals (open bars) and animals treated prenatally with nicotine (solid bars). Nicotine exposure did not change the level of dopamine D2-receptor mRNA levels in the carotid body during the first 2 wk of postnatal development. Values are means ± SE; n = 7.

PPE mRNA was not expressed in the carotid body at any of the ages studied in either control or nicotine-exposed animals. Although PPE was not expressed in the carotid body, it was expressed in the superior cervical and petrosal ganglia as previously described (16). Nicotine exposure did not induce PPE gene expression in the carotid body nor did it change PPE mRNA levels in the superior cervical or petrosal ganglia (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental models investigating neurotransmitter expression in the peripheral and central nervous system strongly suggest that alterations in cholinergic neurotransmission affect neurotransmitter phenotypes in immature and mature neurons (30, 31, 39, 51). This study is the first to show that continuous prenatal nicotine exposure during fetal development and early postnatal exposure modifies catecholaminergic gene expression in the peripheral arterial chemoreceptors. Nicotine exposure increases TH mRNA expression in both the carotid body and PG/JG complex in animals within the first 3 days of postnatal life and upregulates Dbeta H mRNA levels in the carotid body of 15-day-old animals. However, prenatal nicotine exposure does not alter dopamine D2-receptor mRNA levels in the carotid body or petrosal ganglion. In addition, we have shown that PPE mRNA is not expressed in the carotid body but is expressed in the superior cervical and petrosal ganglion, and the level of expression is not altered by prenatal exposure to nicotine. Thus alterations in transynaptic neurotransmission affected by prenatal and early postnatal nicotine exposure induce catecholaminergic neurotransmitter expression in peripheral arterial chemoreceptors in newborn rat pups.

We observed an increase in TH mRNA expression in the carotid body of newborn animals at 0 and 3 postnatal days and in the petrosal ganglion in 3-day-old animals exposed to nicotine. However, this effect was no longer seen at 6 and 15 postnatal days. The most plausible explanation for the loss of effect is that the 6- and 15-day-old animals were no longer being exposed to nicotine. The mini-osmotic pump containing nicotine or sterile saline was not removed from the nursing dam, and the pump delivered nicotine or sterile water for ~1 wk postpartum. The 6-day-old animals may have received some nicotine via the milk; however, the relative dose per weight was considerably less than that of 0- and 3-day-old animals.

Cardiorespiratory physiology is altered in rats exposed prenatally to nicotine. Peripheral chemoreceptors are involved in multiple cardiorespiratory functions that include, but are not limited to, modulating respiration in response to changes in temperature, chemical stimuli, and recovery from asphyxial apnea associated with upper airway obstruction (12). Intact peripheral chemoreceptors are not essential for establishment of rhythmic breathing at birth. However, intact peripheral chemoreceptors at birth appear to provide essential trophic influences for maintaining rhythmic breathing during maturation (for review, see Ref. 17). Animals exposed prenatally to nicotine have 1) reduced ventilation during air breathing and in response to hypoxia (46), 2) less reduction in ventilation in response to hyperoxia (test of peripheral chemoreceptor function) (2), 3) reduced ability to autoresuscitate from repeated episodes of hypoxia (11), and 4) increased mortality from severe, continuous hypoxic exposure (45). Alterations in these cardiorespiratory reflexes may involve adverse molecular and cellular changes in peripheral arterial chemoreceptors induced by prenatal nicotine exposure during prenatal development. Bamford and Carroll (2) reported altered peripheral chemoreceptor function (Dejours' test) in awake 3-day-old newborn animals that were exposed prenatally to nicotine. However, these investigators, using a superfused ex vivo preparation, were unable to detect a difference between control and treated animals when measuring activity from the whole carotid sinus nerve in response to a severe hypoxic challenge (5% O2-5% CO2). Yet the preponderance of physiological studies suggest that peripheral arterial chemoreceptor responses could be affected by prenatal exposure to nicotine. One explanation for the observed discrepancy between the whole animal and the isolated carotid body preparations might be secondary to the isolated preparation used. The severe hypoxia stimulus may have saturated the whole nerve response, making it difficult to distinguish differences in the carotid sinus nerve activity in the preparations between control and prenatally treated animals. Our study is the first to demonstrate that changes in gene expression in the peripheral chemoreceptors are induced by prenatal and perinatal exposure to nicotine. Increased expression of inhibitory catecholaminergic traits may in part explain the alterations in cardiorespiratory responses observed in animals treated prenatally with nicotine.

Nicotine exposure affects catecholaminergic traits in the peripheral and central nervous system: comparison to and extension of previous literature. Acute nicotine exposure induces upregulation of catecholaminergic synthesizing enzymes in several dopaminergic and noradrenergic cell groups in the central and peripheral nervous system (43, 47). Furthermore, nicotine significantly increases TH mRNA levels in PC-12 cells (27), an immortalized cell line that is frequently used as a model of chemosensitive type I cells in the carotid body (36). Type I cells in the carotid body contain TH, dopamine, and norepinephrine, and acute nicotine exposure increases dopamine and norepinephrine release from dissociated type I cells (48). In 3-day-old rat pups, acute postnatal nicotine exposure reduced dopamine content and increased TH mRNA expression in the carotid bodies (28). Furthermore, chemoreceptor function was diminished in these 3-day-old rat pups treated acutely with nicotine (28). We have extended the findings of Holgert et al. (28) by showing that chronic prenatal and early postnatal exposure to nicotine is associated with increased TH mRNA expression in the carotid body and petrosal ganglion of newborn rat pups at 0 and 3 postnatal days. Although the effect of nicotine exposure on TH mRNA levels in the carotid body of 15-day-old animals was no longer apparent, we did observe an increase in Dbeta H gene expression in the carotid body in these animals. Because of the limitation of available tissue, we do not know if nicotine exposure also increased Dbeta H mRNA levels in the younger age groups. Similar to the findings of Holgert et al., we report no significant effect of nicotine exposure on dopamine D2-receptor mRNA levels in the carotid body.

Possible mechanisms for nicotine-induced upregulation of TH and Dbeta H mRNAs in peripheral chemoreceptors. Several mechanisms, either indirectly or directly, may account for the upregulation of TH mRNA levels in the carotid body and petrosal ganglion induced by prenatal nicotine. The most plausible indirect mechanism is by inducing hypoxemia in the fetus. It is possible that prenatal nicotine exposure in the dose used in this experiment may have induced vasoconstriction of uterine vessels with subsequent decreased oxygen delivery to the rat pups. The rat pups exposed prenatally to nicotine were smaller than the control animals, an observation that has also been reported by other investigators using the same paradigm of nicotine exposure (2, 45). Litter sizes were matched between nicotine and control animals, although our animals were not pair-fed. We chose the dose of nicotine used in this study because equivalent dosing and exposure paradigms have been used in studies showing abnormalities in hypoxic or hyperoxic chemosensitivity in newborn rat pups (2, 45, 46). Nevertheless, transcription, regulation, and enzymatic function of TH are significantly altered by the partial pressure of oxygen. TH gene expression within 1 h of hypoxic exposure is induced in the carotid body of adult rats (6). The increase in TH mRNA levels appears to be secondary to an increase in the transcription and stabilization of the mRNA transcripts, as suggested by experiments exposing PC-12 cells to hypoxia (7). Although our laboratory has also shown that hypoxia significantly upregulates TH mRNA levels in the carotid body, it did not increase the grains per cell or the number of cells expressing TH mRNA in the PG/JG ganglion complex (15). In contrast, in our present study, prenatal exposure to nicotine did increase the number of ganglion cells expressing TH mRNA.

Alternatively, nicotine may have directly increased TH and Dbeta H mRNA levels in the carotid body and petrosal ganglion by a cascade of intracellular events, thus leading to an increase in gene transcription similar to its effects in PC-12 cells (23) and in the rat adrenal medulla (27). In addition, nicotine may have induced upregulation of TH-positive neurons in the petrosal ganglion via an autocrine or paracrine effect involving the induction of neurotrophins such as brain-derived neurotrophic factor (BDNF). BDNF is released from the carotid body and is essential for the development of full expression of catecholaminergic neurons in the petrosal ganglion (24). Of interest, nicotine has been shown to induce BDNF in the striatum of rodents (33), and BDNF has been implicated as providing a protective effect induced by nicotine on the survival of dopamine-containing nigrostriatal neurons in rodent models of Parkinson's disease (33). Although not addressed by this study, these alternative mechanisms may be operative in the upregulation of TH mRNA and Dbeta H in the carotid body and TH-positive neurons in the petrosal ganglion in animals exposed to nicotine.

Relationship between TH and Dbeta H mRNA levels and changes in dopamine and/or norepinephrine levels. Hypoxia increases dopamine levels, dopamine release, and TH enzyme activity in the carotid body of many mammalian species (for review, see Ref. 20). Furthermore, acute exposure to nicotine increases TH mRNA levels and dopamine release in the carotid body of newborn rats (28). TH is the rate-limiting enzyme for catecholamine synthesis. We do not know if the increase in TH mRNA levels observed in the animals treated prenatally with nicotine represents an increase in dopamine, epinephrine, or norepinephrine levels at any of the ages studied, because levels were not measured. Nicotine did increase Dbeta H mRNA expression in the carotid body of 15-day-old animals.

Nicotine exposure increases norepinephrine levels in the noradrenergic cell groups in the brain (13) and induces Dbeta H gene transcription and Dbeta H enzyme levels. Furthermore, acute nicotine exposure preferentially released norepinephrine vs. dopamine from rat (48) and rabbit (4) carotid bodies. One interpretation of our data is that prenatal and early postnatal exposure to nicotine modifies the ratio of dopamine to norepinephrine protein levels in the rat carotid body, which may be longer lasting than the duration of the exposure. Norepinephrine, through binding to the alpha 2-adrenergic receptors, inhibits output from the carotid body (42). Thus whether the elevation in TH mRNA levels represents an increase in dopamine or norepinephrine levels at the younger age groups or if the elevation of Dbeta H represents an elevation in norepinephrine levels in the older animals is unclear. However, both neurotransmitters may contribute to reduction in peripheral arterial chemoreceptor activity in newborn animals exposed to nicotine.

Prenatal and early postnatal nicotine exposure does not induce PPE mRNA in the carotid body of newborn animals. Enkephalins have been measured in the carotid body, are coreleased with dopamine from the rabbit carotid body (21), and are involved in reduction in hypoxic chemosensitivity. Immunoreactivity for Met-enkephalin has been localized to type I cells in the carotid body of several mammalian species (50). However, in the rat, enkephalin immunoreactivity has only been localized to nerve fibers innervating the carotid body (26). Our previous and present findings show that PPE mRNA was abundantly expressed in the cell bodies in the petrosal ganglion but was not detected in the carotid body, corroborating the immunohistochemical studies in the rat. Similar cellular mechanisms are involved in the nicotine-induced upregulation of PPE gene transcription as they are in TH and Dbeta H gene transcription. PPE mRNA is present in the striatum and adrenal chromaffin cells, and acute nicotine induces the expression of PPE in bovine adrenal chromaffin cells in culture (49) and in the striatum (9, 29) and hippocampus of adult rats (29). However, prenatal exposure to nicotine at the dose used in this study did not induce PPE gene expression in the carotid body nor does it upregulate PPE mRNA in the cell bodies of the petrosal ganglion. Similar to our findings, chronic vs. acute nicotine exposure does not upregulate PPE mRNA expression in the striatum or hippocampus of the adult rat (30).

In summary, prenatal nicotine exposure is associated with blunted peripheral chemoreceptor function (46) and reduced ability to autoresuscitate (11) in newborn animals. We have found that prenatal nicotine exposure upregulates TH mRNA levels in both the carotid body and petrosal ganglion in rat pups at 0 and 3 postnatal days and upregulates Dbeta H mRNA levels in the carotid body of 15-day-old animals. However, dopamine D2-receptor and PPE mRNA levels in the carotid body or petrosal ganglion were unaltered in animals exposed prenatally to nicotine during the first 2 wk of postnatal life. TH and Dbeta H are the rate-limiting enzymes for catecholamine and norepinephrine synthesis, respectively. Dopamine and norepinephrine, through binding to inhibitory dopamine D2- and alpha 2-adrenergic receptors, respectively, on postsynaptic receptors within the carotid body, are associated with reduced hypoxic chemosensitivity. Our data support a possible role for upregulation of catecholamines in peripheral arterial chemoreceptors as one possible cellular mechanism that could explain the reduced physiological responses involving peripheral chemoreceptors in newborn rat pups exposed prenatally to nicotine.


    ACKNOWLEDGEMENTS

The authors thank Dr. Musa Haxhiu for reviewing the manuscript.


    FOOTNOTES

E. B. Gauda is a recipient of National Institute on Drug Abuse Grant R01 DA-13940, which supported this work.

Address for reprint requests and other correspondence: E. B. Gauda, Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, Maryland 21287-3200 (E-mail: egauda{at}welchlink.welch.jhu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 March 2001; accepted in final form 25 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alcayaga, J, Iturriaga R, Varas R, Arroyo J, and Zapata P. Selective activation of carotid nerve fibers by acetylcholine applied to the cat petrosal ganglion in vitro. Brain Res 786: 47-54, 1998[ISI][Medline].

2.   Bamford, OS, and Carroll JL. Dynamic ventilatory responses in rats: normal development and effects of prenatal nicotine exposure. Respir Physiol 117: 29-40, 1999[ISI][Medline].

3.   Bunzow, JR, Van Tol HH, Grandy DK, Albert P, Salon J, Christie M, Machida CA, Neve KA, and Civelli O. Cloning and expression of a rat D2 dopamine receptor cDNA. Nature 336: 783-787, 1988[Medline].

4.   Chen, J, He L, Dinger B, and Fidone S. Stimulus-specific signaling pathways in rabbit carotid body chemoreceptors. Neuroscience 95: 283-291, 2000[ISI][Medline].

5.   Chesselet, MF, MacKenzie L, and Eberle-Wang K. In situ hybridization using 35S and digoxigenin-labeled RNA probes. In: In Situ Hybridization Techniques for the Brain, edited by Henderson Z.. Chichester, UK: Wiley, 1996, p. 79-109.

6.   Czyzyk-Krzeska, MF, Bayliss DA, Lawson EE, and Millhorn DE. Regulation of tyrosine hydroxylase gene expression in the rat carotid body by hypoxia. J Neurochem 58: 1538-1546, 1992[ISI][Medline].

7.   Czyzyk-Krzeska, MF, Furnari BA, Lawson EE, and Millhorn DE. Hypoxia increases rate of transcription and stability of tyrosine hydroxylase mRNA in pheochromocytoma (PC12) cells. J Biol Chem 269: 760-764, 1994[Abstract/Free Full Text].

8.   Dani, JA, and Heinemann S. Molecular and cellular aspects of nicotine abuse. Neuron 16: 905-908, 1996[ISI][Medline].

9.   Dhatt, RK, Gudehithlu KP, Wemlinger TA, Tejwani GA, Neff NH, and Hadjiconstantinou M. Preproenkephalin mRNA and methionine-enkephalin content are increased in mouse striatum after treatment with nicotine. J Neurochem 64: 1878-1883, 1995[ISI][Medline].

10.   Donnelly, DF. Developmental aspects of oxygen sensing by the carotid body. J Appl Physiol 88: 2296-2301, 2000[Abstract/Free Full Text].

11.   Fewell, JE, and Smith FG. Perinatal nicotine exposure impairs ability of newborn rats to autoresuscitate from apnea during hypoxia. J Appl Physiol 85: 2066-2074, 1998[Abstract/Free Full Text].

12.   Fewell, JE, Taylor BJ, Kondo CS, Dascalu V, and Filyk SC. Influence of carotid denervation on the arousal and cardiopulmonary responses to upper airway obstruction in lambs. Pediatr Res 28: 374-378, 1990[ISI][Medline].

13.   Gallardo, KA, and Leslie FM. Nicotine-stimulated release of [3H]norepinephrine from fetal rat locus coeruleus cells in culture. J Neurochem 70: 663-670, 1998[ISI][Medline].

14.   Gauda, EB, Bamford OS, and Gerfen CR. Developmental expression of tyrosine hydroxylase, D2-dopamine receptor and substance P genes in the carotid body of the rat. Neuroscience 75: 969-977, 1996[ISI][Medline].

15.   Gauda, EB, Bamford OS, and Northington FJ. Lack of induction of substance P gene expression by hypoxia and absence of neurokinin 1-receptor mRNAs in the rat carotid body. J Auton Nerv Syst 74: 100-108, 1998[ISI][Medline].

16.   Gauda, EB, and Gerfen CR. Expression and localization of enkephalin, substance P, and substance P receptor genes in the rat carotid body. In: Frontiers in Arterial Chemoreception, edited by Zapata P, Eyzaguirre C, and Torrance R.. New York: Plenum, 1996, p. 313-318.

17.   Gauda, EB, and Lawson EE. Developmental influences on carotid body responses to hypoxia. Respir Physiol 121: 199-208, 2000[ISI][Medline].

18.   Gerfen, CR, Engber TM, Mahan LC, Susel Z, Thomas TN, Monsma FJ, Jr, and Sibley DR. D1 and D2 dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons. Science 250: 1429-1432, 1996.

19.   Golding, J. Sudden infant death syndrome and parental smoking---a literature review. Paediatr Perinat Epidemiol 11: 67-77, 1997[ISI][Medline].

20.   Gonzalez, C, Dinger BG, and Fidone SJ. Mechanisms of carotid chemoreception. In: Regulation of Breathing, edited by Dempsey JA, and Pack AI.. New York: Dekker, 1994, p. 391-470.

21.   Gonzalez-Guerrero, PR, Rigual R, and Gonzalez C. Opioid peptides in the rabbit carotid body: identification and evidence for co-utilization and interactions with dopamine. J Neurochem 60: 1762-1768, 1993[ISI][Medline].

22.   Grima, B, Lamouroux A, Blanot F, Biguet NF, and Mallet J. Complete coding sequence of rat tyrosine hydroxylase mRNA. Proc Natl Acad Sci USA 82: 617-621, 1985[Abstract/Free Full Text].

23.   Gueorguiev, VD, Zeman RJ, Hiremagalur B, Menezes A, and Sabban EL. Differing temporal roles of Ca2+ and cAMP in nicotine-elicited elevation of tyrosine hydroxylase mRNA. Am J Physiol Cell Physiol 276: C54-C65, 1999[Abstract/Free Full Text].

24.   Hertzberg, T, Fan G, Finley JC, Erickson JT, and Katz DM. BDNF supports mammalian chemoafferent neurons in vitro and following peripheral target removal in vivo. Dev Biol 166: 801-811, 1994[ISI][Medline].

25.   Hertzberg, T, Shellstrom S, Holgert H, Lagercrantz H, and Pequignot JM. Development of the arterial chemoreflex and turnover of carotid body catecholamines in the newborn rat. J Physiol (Lond) 425: 211-225, 1990[Abstract/Free Full Text].

26.   Heym, C, and Kummer W. Immunohistochemical distribution and colocalization of regulatory peptides in the carotid body. J Electron Microsc (Tokyo) 12: 331-342, 1989.

27.   Hiremagalur, B, Nankova B, Nitahara J, Zeman R, and Sabban EL. Nicotine increases expression of tyrosine hydroxylase gene. Involvement of protein kinase A-mediated pathway. J Biol Chem 268: 23704-23711, 1993[Abstract/Free Full Text].

28.   Holgert, H, Hokfelt T, Hertzberg T, and Lagercrantz H. Functional and developmental studies of the peripheral arterial chemoreceptors in rat: effects of nicotine and possible relation to sudden infant death syndrome. Proc Natl Acad Sci USA 92: 7575-7579, 1995[Abstract/Free Full Text].

29.   Houdi, AA, Dasgupta R, and Kindy MS. Effect of nicotine use and withdrawal on brain preproenkephalin A mRNA. Brain Res 799: 257-263, 1998[ISI][Medline].

30.   Lauder, JM. Neurotransmitters as growth regulatory signals: role of receptors and second messengers. Trends Neurosci 16: 233-240, 1993[ISI][Medline].

31.   Lichtensteiger, W, Ribary U, Schlumpf M, Odermatt B, and Widmer HR. Prenatal adverse effects of nicotine on the developing brain. Prog Brain Res 73: 137-157, 1988[ISI][Medline].

32.   Lin, W, Seidler FJ, McCook EC, and Slotkin TA. Overexpression of alpha 2-adrenergic receptors in fetal rat heart: receptors in search of a function. J Dev Physiol 17: 183-187, 1992[ISI][Medline].

33.   Maggio, R, Riva M, Vaglini F, Fornai F, Molteni R, Armogida M, Racagni G, and Corsini GU. Nicotine prevents experimental parkinsonism in rodents and induces striatal increase of neurotrophic factors. J Neurochem 71: 2439-2446, 1998[ISI][Medline].

34.   McMahon, A, Geertman R, and Sabban EL. Rat dopamine beta-hydroxylase: molecular cloning and characterization of the cDNA and regulation of the mRNA by reserpine. J Neurosci Res 25: 395-404, 1990[ISI][Medline].

35.   McQueen, DS, and Ribeiro JA. Inhibitory actions of methionine-enkephalin and morphine on the cat carotid chemoreceptors. Br J Pharmacol 71: 297-305, 1980[ISI][Medline].

36.   Millhorn, DE, Conforti L, Beitner-Johnson D, Zhu W, Raymond R, Filisko T, Kobayashi S, Peng M, and Genter MB. Regulation of ionic conductances and gene expression by hypoxia in an oxygen sensitive cell line. Adv Exp Med Biol 410: 135-142, 1996[Medline].

37.   Monsma, FJ, Jr, Mahan LC, McVittie LD, Gerfen CR, and Sibley DR. Multiple D2 dopamine receptors produced by alternative RNA splicing (Abstract). Nature 342: 926, 1989[Medline].

38.   Murrin, LC, Ferrer JR, Zeng WY, and Haley NJ. Nicotine administration to rats: methodological considerations. Life Sci 40: 1699-1708, 1987[ISI][Medline].

39.   Oliff, HS, and Gallardo KA. The effect of nicotine on developing brain catecholamine systems. Front Biosci 4: D883-D897, 1999[Medline].

40.   Pasinetti, GM, Morgan DG, Johnson SA, Lerner SP, Myers MA, Poirier J, and Finch CE. Combined in situ hybridization and immunocytochemistry in the assay of pharmacological effects on tyrosine hydroxylase mRNA concentration. Pharm Res 21: 299-311, 1989.

41.   Paxinos, G, and Watson C. The Brain in Sterotaxic Coordinates. Orlando, FL: Academic, 1986.

42.   Prabhakar, NR, and Kou YR. Inhibitory sympathetic action on the carotid body responses to sustained hypoxia. Respir Physiol 95: 67-79, 1994[ISI][Medline].

43.   Role, LW, and Berg DK. Nicotinic receptors in the development and modulation of CNS synapses. Neuron 16: 1077-1085, 1996[ISI][Medline].

44.   Simpson, JN, and McGinty JF. Forskolin induces preproenkephalin and preprodynorphin mRNA in rat striatum as demonstrated by in situ hybridization histochemistry. Synapse 19: 151-159, 1995[ISI][Medline].

45.   Slotkin, TA, Saleh JL, McCook EC, and Seidler FJ. Impaired cardiac function during postnatal hypoxia in rats exposed to nicotine prenatally: implication for perinatal morbidity and mortality, and for sudden infant death syndrome. Teratology 55: 177-184, 1997[ISI][Medline].

46.   St. John, WM, and Leiter JC. Maternal nicotine depresses eupneic ventilation of neonatal rats. Neurosci Lett 267: 206-208, 1999[ISI][Medline].

47.   Summers, KL, and Giacobini E. Effects of local and repeated systemic administration of (-)nicotine on extracellular levels of acetylcholine, norepinephrine, dopamine, and serotonin in rat cortex. Neurochem Res 20: 753-759, 1995[ISI][Medline].

48.   Vicario, I, Rigual R, Obeso A, and Gonzalez C. Characterization of the synthesis and release of catecholamine in the rat carotid body in vitro. Am J Physiol Cell Physiol 278: C490-C499, 2000[Abstract/Free Full Text].

49.   Wang, X, Bacher B, and Hollt V. Nicotine-induced gene expression of proenkephalin in bovine chromaffin cells. Clin Investig 72: 925-929, 1994[ISI][Medline].

50.   Wharton, J, Polak JM, Pearse AGE, McGregor GP, Bryant MG, Bloom SR, Emson PC, Bisgard GE, and Will JA. Enkephalin-, VIP-, and substance P-like immunoreactivity in the carotid body. Nature 284: 269-271, 1980[Medline].

51.   Yamada, S, Kagawa Y, Isogai M, Takayanagi N, and Hayashi E. Ontogenesis of nicotinic acetylcholine receptors and presynaptic cholinergic neurons in mammalian brain. Life Sci 38: 637-644, 1986[ISI][Medline].

52.   Yoshikawa, K, Williams C, and Sabol SL. Rat brain preproenkephalin mRNA. cDNA cloning, primary structure, and distribution in the central nervous system. J Biol Chem 259: 14301-14308, 1984[Abstract/Free Full Text].

J APPL PHYSIOL 91(5):2157-2165
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