PERSPECTIVE
Prenatal Nicotine Exposure and Abnormal Lung Function

Richard A. Pierce and Nguyet M. Nguyen

Department of Internal Medicine, Pulmonary Division, Washington University School of Medicine, St. Louis, Missouri

	Nicotine and the Neonatal Lung

It has long been recognized that maternal smoking during pregnancy increases the incidence of premature delivery, spontaneous abortions, low birth weight, and neonatal morbidity and mortality (1). There is also increased occurrence of lower respiratory illnesses with associated altered pulmonary mechanics on pulmonary function testing of children whose mothers smoked during pregnancy, suggesting that prenatal smoking affects lung development in utero (4). Nicotine, which crosses the placental barrier, may be the primary mediator of many of these effects of maternal smoking during pregnancy. In experimental models, maternal nicotine exposure alone elicits many of the same effects on neonates as maternal direct or sidestream tobacco smoke exposure.

In animal models, maternal nicotine exposure causes a variety of effects on neonatal lung, summarized in Table 1. These effects include structural changes such as decreased elastin staining of lung parenchyma, increased mean linear intercept, decreased radial alveolar counts, and increased alveolar volume, indicative of emphysema-like changes in the neonatal lung (12). In this issue, Sekhon and colleagues present the third of a series of studies focusing on the effects of maternal nicotine exposure on lung structural development in a primate model. Their finding that maternal nicotine exposure alters fibrillar collagen expression in developing primate lungs helps explain how maternal nicotine exposure could lead to impaired pulmonary function in neonates. Such recent advances follow the discovery that receptors for nicotine are found on many different cell types in the lung and other tissues (10, 16).

	Nicotinic Acetylcholine Receptors in the Lung

Nicotine exerts its effects through a family of nicotinic acetylcholine receptors that are composed of five homologous subunits arranged around a central ion channel. Ligand binding to the receptor opens the central ion channel, allowing the flow of cations, usually calcium, potassium, or sodium. There are currently nine  and four  known subunits, which form nicotinic receptors of  and  heteromers or  homomers. The majority of early studies focused on nicotinic acetylcholine receptors in the brain and the effects of nicotine on neural activity (17). More recently, members of this receptor family have been found to be expressed in a wide variety of cell types and to mediate an array of downstream effects (18). Table 2 summarizes the nicotinic receptor subunits and their locations in the lung. Human and rodent bronchial epithelial cells express 3, 5, 7, 2, and 4 subunits, 4 subunits are seen in alveolar epithelial cells, and 7 subunits are seen in the submucosal glands (19). However, the expression of nicotinic receptors on these cell types may not readily explain nicotine-mediated changes in the structural development of the lung.

	Linking Nicotine to Collagen Content

Sekhon and colleagues developed a primate model of maternal nicotine exposure to determine whether nicotine exposure altered fetal lung nicotinic receptor expression and mediated downstream structural effects (10). In the developing rhesus monkey lung, antibody specific for 7 showed immunoreactivity in airway and arterial smooth muscle cells and in fibroblasts surrounding walls of airways and vessels. These cells synthesize the abundant extracellular matrix that determines the structure of conducting airways and blood vessels. Prenatal exposure to nicotine led to an increased intensity of immunoreactivity of 7 in cartilaginous airway wall cells, blood vessel walls, and airway epithelial cells, but no change in staining of other nicotinic receptor subnunits. Additionally, there was a significant increase in Masson's trichrome staining of collagen surrounding large airways and vessels after nicotine treatment. This finding suggested that changes in airway wall thickness or stiffness could be the basis for altered lung mechanics in neonates exposed to nicotine in utero. Within the distal lung, significant increases in mean linear intercept and volume density of alveolar airspace, and decrease in internal surface area of the lung, were seen with maternal nicotine exposure, all consistent with failure of alveolar development. Maternal nicotine administration during pregnancy in monkeys also resulted in increased numbers of neuroendocrine cells, size of neuroepithelial bodies, and number of alveolar type II epithelial cells, a result that is consistent with an earlier study in rat lungs (9).

	Altered Lung Function

A follow up study by Sekhon and colleagues of newborn rhesus monkeys showed that prenatal nicotine exposure alters pulmonary function (20). Significant decreases in lung weight and lung volume were seen. During tidal breathing, nicotine exposed newborns had significantly lower FEV_0.2 and peak tidal expiratory flow. The mid-expiratory flow (FEF_25%-75%) was significantly decreased, and all other forced expiratory flow rates were also decreased, but not to a significant level. Pulmonary resistance was significantly increased and static and dynamic lung compliance was decreased, but not significantly. All these changes were consistent with those seen in children of human smokers, supporting the hypothesis that the interaction between nicotine and the nicotine receptor may be responsible for the pulmonary changes.

	Different Models of Nicotine Effects on Lung Extracellular Matrix

In this issue, Sekhon and colleagues report that abnormal lung mechanics after maternal nicotine exposure are associated with increased type I and III collagen mRNAs and protein expression in airway and alveolar walls and increased airway wall area (11). Elastin mRNA expression was increased, but elastin protein level showed a modest decrease that was not statistically significant. The basis for altered elastin content is still unexplained. The increased type I and type II collagen expression and staining in airway walls with nicotine exposure lend support to a model in which nicotine upregulates the 7 nicotinic receptor in the cells that synthesize the connective tissue surrounding large airways. Signaling through a nicotinic receptor containing the 7 subunit then leads to increased collagens type I and III deposition, which increases the airway wall area. Increased airway wall thickness may be the basis for abnormal pulmonary function tests, including decreased FEV_0.2 and increased pulmonary resistance. Similar upregulation of type I and type III collagens has been reported in chick chorioallantoic membranes exposed to sidestream cigarette smoke, while cultured cardiac fibroblasts exposed to nicotine show a decrease in collagen type I mRNA expression (21, 22). These varying data indicate that the effects of nicotine on fibrillar collagen content may be cell type-specific.

Maritz and coworkers, who have studied the effects of prenatal and neonatal nicotine exposure on alveolar development in rats, have proposed a distinct model for the effects of prenatal nicotine exposure on lung development. They report arrested or incomplete alveolar development, decreased elastin content, and decreased lung copper content with prenatal and neonatal nicotine exposure (12, 13, 23). This supports a model in which nicotine exposure decreased lung copper content, inhibiting the copper-requiring enzyme lysyl oxidase, which is required for crosslinking elastin and collagen in the extracellular matrix. Previous studies have shown that inhibiting lysyl oxidase activity by copper starvation (24) or use of -aminoproprionitrile, an inhibitor of lysyl oxidase (25), irreversibly suppresses alveolar development in the neonatal rat. Are these models mutually exclusive or could they coexist?

	Unanswered Questions

A number of important questions remain concerning the connection between nicotine exposure and altered lung development and mechanics in these distinct models of prenatal nicotine exposure. Sekhon and coworkers have focused on altered airway morphology, collagen content, and lung mechanics in prenatal monkeys; Maritz and colleagues focused on elastin content and arrested alveolar development in neonatal rats. Is it likely that different species respond differently to prenatal nicotine exposure? In rats, the alveolar stage of development occurs postnatally, whereas in primates and most large mammals, alveolar development is well-established by birth. Nicotine exposure may elicit different responses during different stages of lung development. For example, although nicotine clearly has adverse effects on the latter stages of prenatal lung development, nicotine exposure actually enhances branching morphogenesis of isolated lung buds in a nicotine receptor-dependent mechanism (26).

In their different approaches to studying the effects of nicotine on lung development, Sekhon and coworkers report increased collagen staining and gene expression in the cells surrounding conducting airways, while Maritz and coworkers have reported that prenatal nicotine exposure decreases available lung copper and likely suppresses lysyl oxidase, the enzyme that crosslinks collagen and elastin in the extracellular matrix. Is increased collagen accumulation in the airway extracellular matrix plausible if lysyl oxidase activity is limiting in that lung compartment? Localizing collagen, elastin, and lysyl oxidase expression, and determining the content of crosslinked collagen and elastin in specific lung compartments, would begin to answer such fundamental questions in these distinct models. For example, it may be possible to infer the ratio of crosslinked elastin to the soluble tropoelastin precursor by staining with antibodies specific for desmosine, the elastin crosslink, versus staining for the tropoelastin precursor. Similarly, histochemical staining for collagen could be compared with staining for the immature collagen propeptide. Technological advances such as laser-capture-microdissection could be applied to different lung compartments and perhaps combined with assays for hydroxyproline to quantify collagen content and desmosine to determine elastin content.

Genetic tools could also be applied to a better understanding of the pathways linking nicotine exposure to impaired lung structural development that leads to altered lung mechanics. Mice without functional 7 nicotinic acetylcholine receptors are viable, anatomically normal (27), and have no detectable abnormalities of high-affinity nicotine binding sites in the brain, but lack high affinity -bungarotoxin binding sites. Further studies showed that these mice have altered baroreflex, indicating that the 7 nicotinic acetylcholine receptor participates in the autonomic reflex that maintains blood pressure (28). A threonine-to-leucine substitution at position 247 of the channel domain of the chick 7 receptor increases agonist affinity and creates a gain of function allele. Homozygous mutations for this allele confer neonatal lethality in mice due to neuronal apoptosis (29), but mice bearing one gain of function allele and one wild type allele appear normal (30). Such lines of genetically altered mice could be exposed to nicotine prenatally and tested for changes in lung morphology, copper content, collagen and elastin biosynthesis, and lung mechanics.

	Role of the Extracellular Matrix in the Alveolar Stage of Lung Development

These newly discovered connections between maternal nicotine exposure and altered extracellular matrix synthesis or deposition are an important first step in bridging the gap between nicotine exposure in utero and altered lung structure and function in newborns and young children. Furthermore, they join a growing body of data emphasizing the importance of the extracellular matrix in the alveolar stage of lung development. Altering the expression or deposition of elastin during the latter stages of lung development by various methods, including blocking lysyl oxidase activity (24, 25), or altering elastin expression through blocking retinoid signaling (31), or "knocking out" the elastin gene (32), or eliminating alveolar myofibroblasts through genetic manipulation of the PDGF-A signaling pathway (33, 34) all lead to impaired alveolar development. Other components of elastic fiber assembly machinery in the lung are also critical for normal alveolar development. A genetic duplication in the fibrillin-1 gene, a primary component of microfibrils that comprise a scaffold for elastic fibers, leads to arrested alveolar development in the tight-skin (Tsk) mouse (35). Recently, Ramirez and coworkers have found arrested alveolar development in mice heterozygous for a null allele of the fibrillin-1 gene (personal communication). These new data showing connections between prenatal nicotine exposure, collagen and elastin biosynthesis, and lung structure and mechanics strengthen the premise that the alveolar stage of lung development is dependent on elaboration and maturation of a complex connective tissue architecture.

	Footnotes

Address correspondence to: Richard A. Pierce, Ph.D., Washington University School of Medicine, Barnes-Jewish Hospital, North, 216 S. Kingshighway Blvd., St. Louis, MO 63110. E-mail: rpierce{at}im.wustl.edu

Acknowledgments: This work was supported by NIH-HL-UO1 64049 (RAP) and an ALA Research Training Grant (NMN).

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