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An orphan receptor discovered in 1993 was called bombesin receptor subtype 3 (BRS-3) because of 47-51% amino acid identity with bombesin (Bn) receptors. Its pharmacology is unknown, because no naturally occurring tissues have sufficient receptors to allow studies. ]Bn-(6 -14) bound to both cell lines with high affinity. Neither Bn nor 14 other naturally occurring Bn peptides bound to hBRS-3 with a K d <1000 nM. Twenty-six synthetic peptides that are high affinity agonists or antagonists at other bombesin receptors had an affinity >1000 nM. Guanosine 5-(␤,␥-imido)triphosphate inhibited binding to both cells due to a change in receptor affinity. These results demonstrate hBRS-3 has a unique pharmacology. It does not interact with high affinity with any known natural agonist or high affinity antagonist of the Bn receptor family, suggesting the natural ligand is either an undiscovered member of the Bn peptide family or an unrelated peptide. The availability of these cell lines and the hBRS-3 ligand should facilitate identification of the natural ligand for BRS-3, its pharmacology, and cell biology. We made two cell lines stably expressing the human BRS-3 (hBRS-3). hBRS-3 was overexpressed in the huRecently, an orphan receptor that is a member of the heptahelical superfamily of receptors was described in both human small cell lung cancer cells (1) and guinea pig uterus (2). Because this orphan receptor had a high degree of homology to mammalian bombesin receptors (i.e. 51-52% for the gastrinreleasing peptide receptor (GRP-R) 1 and 47% for the neuromedin B receptor (NMB-R) (1, 2)), it was named the BRS-3 for bombesin receptor subtype-3 in one study (1). Studies of the distribution of the receptor mRNA show that BRS-3 has a pattern of expression limited to rat secondary spermatocytes (1), guinea pig brain and pregnant uterus (2), and some tumor cell lines (various human small cell and non-small cell lung cancer cell lines (1), the human ductal breast cancer cell line T47D (3), and the human epidermal cancer cell line A431 (3)). However, the natural ligand that interacts with the BRS-3 is unknown, and its pharmacology is largely unknown because of the lack of a radioligand. In addition, little is known about the cellular basis of action of BRS-3 except that it is coupled to phospholipase C when expressed in Xenopus oocytes (1) or when transfected into Balb 3T3 cells (4). The ability to elucidate the pharmacology of the BRS-3 is not only limited by the lack of a radioligand but also by the lack of a cell containing native BRS-3 receptors in sufficient numbers to allow binding studies to identify a possible radioligand.To deal with this latter issue, in the present study we have used two different strategies to produce cell lines stably expressing the human BRS-3 (hBRS-3) receptor whose pharmacology and coupling will probably closely resemble that of the native hBRS-3. Furthermore, we have discovered a unique ligand that is a synthetic analogue of bombesin-(6 -14), which interacts with high affinity with the hBRS-3. With ...

It is well known that smoking during pregnancy is the largest preventable cause of low birth weight, premature delivery, neonatal morbidity, and mortality (1, 2). Perhaps less well appreciated is the recent, strong evidence that smoking during pregnancy directly and adversely effects lung development, as manifested by altered pulmonary function and increased respiratory illness in children born of smoking mothers (3-6). Stoddard and Gray (7) recently estimated that annual costs of increased respiratory illness associated with maternal smoking is in excess of 660 million dollars per year. How smoking produces these effects is not fully understood. While the cause of pulmonary damage caused by maternal smoking is likely to be multifactorial, evidence presented in this paper indicates that much of the effect of maternal smoking on the lung may be mediated by the interaction of nicotine with nicotinic receptors expressed in the developing lung.Multiple studies have shown increased lower respiratory illness in infants born of mothers who smoke (3, 4). Taylor and Wadsworth (3) studied 12,743 children and found significantly increased bronchitis and hospital admissions for lower respiratory illness in children from smoking mothers. Significantly increased hospital admissions were not seen for children whose mothers smoked only after pregnancy, arguing for a prenatal effect. Tager et al. (4) similarly found increased lower respiratory illness with prenatal smoke exposure, but not with postnatal exposure. Correlating the increased respiratory illness with altered pulmonary function, Hanrahan et al. (5) examined pulmonary function of infants shortly after birth (∼4.2 weeks) as a function of smoking during pregnancy (determined both by questionnaire and by urinary cotinine) and found decreased respiratory flow rates. In a follow-up study, Tager et al. (4) showed that the decreased ventilation at functional residual capacity (VFRC) seen with prenatal smoke exposure correlated directly with increased lower respiratory illnesses. Cunningham et al. (6) performed pulmonary function tests on 8,800 nonsmoking school children 8-12 years old and similarly found reduced forced expiratory flow in children whose mothers smoked during pregnancy. Again the correlation was with prenatal, not postnatal, maternal smoking. This confirmed the earlier studies and suggested the deficit continued at least to adolescence.Animal studies on the effects of smoking or nicotine on lung development have been quite limited, and there have been no studies of potential molecular mechanisms. It is well established that maternal smoking during pregnancy is a leading preventable cause of low birth weight and prematurity. Less appreciated is that maternal smoking during pregnancy is also associated with alterations in pulmonary function at birth and greater incidence of respiratory illnesses after birth. To determine if this is the direct result of nicotine interacting with nicotinic cholinergic receptors (nAChRs) during lung development, rhesus monkeys w...

The role of acetylcholine (ACh) as a key neurotransmitter in the central and peripheral nervous system is well established. However, the role of ACh may be broader because ACh may also function as an autocrine or paracrine signaling molecule in a variety of nonneuronal tissues. To begin to establish ACh of nonneuronal origin as a paracrine hormone in lung, we have examined neonatal and adult monkey bronchial epithelium for the components involved in nicotinic cholinergic signaling. Using immunohistochemistry and RT-PCR, we have demonstrated in lung bronchial epithelial cells (BECs) expression of choline acetyltransferase, the vesicular ACh transporter, the choline high-affinity transporter, alpha7, alpha4, and beta2 nicotinic ACh receptor (nAChR) subunits, and the nAChR accessory protein lynx1. Confocal microscopy demonstrates that these factors are expressed in epithelial cells and are clearly distinct from neighboring nerve fibers. Confirmation of RNA identity has been confirmed by partial sequence analysis of PCR products and by cDNA cloning. Primary culture of BECs confirms the synthesis and secretion of ACh and the activity of cholinesterases. Thus, ACh meets all the criteria for an autocrine/paracrine hormone in lung bronchial epithelium. The nonneuronal cholinergic signaling pathway in lung provides a potentially important target for cholinergic drugs. This pathway may also explain some of the effects of nicotine on fetal development and also provides additional mechanisms by which smoking affects lung cancer growth and development.

Epidemiological studies have shown that offspring of women who smoke during pregnancy have abnormal lung function and associated higher incidences of lower respiratory disorders. The recent identification of nicotinic acetylcholine receptors (nAChR) in fetal lung suggests that the direct interaction between nicotine and nAChR in fetal lung may underlie the postnatal pulmonary abnormalities seen in such infants. This hypothesis was tested in monkeys to determine if maternal nicotine exposure would produce changes in lung mechanics in newborn monkeys similar to those observed in human infants whose mothers smoked during pregnancy. Timed pregnant rhesus monkeys were infused with either nicotine (1.5 mg/kg/d, n = 7) or saline (n = 7) using subcutaneous osmotic pumps from Day 26 to 160 of gestation. On Day 160 of pregnancy (term = 165 d), fetuses were delivered by C-section, and the following day were subjected to pulmonary function testing. After testing, animals were sacrificed, and lungs weighed and fixed. Lung weight and fixed lung volume decreased (16% and 14%, respectively) significantly following in utero nicotine exposure. Peak tidal expiratory flow, FEV(0.2), mean mid-expiratory flow, forced expiratory volume at peak expiratory flow (FEV(PEF)), and FEV(PEF)/FVC% were significantly lower in newborns exposed to nicotine during gestation. Absolute and specific pulmonary resistance increased significantly whereas absolute and specific dynamic compliance remained unchanged in prenatally nicotine-treated pups. These changes in pulmonary function are strikingly similar to the changes observed in offspring of human smokers. This suggests that the interaction of nicotine with nAChR in developing lung is responsible for the altered pulmonary mechanics observed in human infants whose mothers smoked during pregnancy.