Neuronal nicotinic acetylcholine receptors from bovine adrenomedullary chromaffin cells play a primary role in triggering catecholamine secretion. In the present study, their constituent subunits were characterized. In addition to the cx3 subunit, which we have previously cloned, the presence of a5 and /94 but not of /32 subunits was detected by reverse transcription-PCR analysis of mRNA from adrenal medulla. In situ hybridization indicated that cr3, cr5, and~34subunits are coexpressed in all chromaffin cells. The primary structure of a5 and /34 subunits was determined and functional receptors were obtained upon coinjection of subunit cRNAs into Xenopus oocytes. In contrast to other /34-containing nicotinic receptors, the ones formed by the bovine /94 subunit are insensitive to the agonist cytisine. Finally, we characterized the intergenic region of cr3 and cr5 subunits, which together with the /34 subunit, form a gene cluster in rats and chickens. RNase assays and the existence of overlapping cDNAs indicate that, in the bovine genome, the cr3 and cr5 genes overlap at their 3' ends. This fact is probably due to inefficient transcription termination, as a result of weak polyadenylation signals.

Adrenomedullary chromaffin cells express at least two subtypes of acetylcholine nicotinic receptors, which differ in their sensitivity to the snake toxin ␣-bungarotoxin. One subtype is involved in the activation step of the catecholamine secretion process and is not blocked by the toxin. The other is ␣-bungarotoxin-sensitive, and its functional role has not yet been defined. The ␣7 subunit is a component of this subtype. Autoradiography of bovine adrenal gland slices with ␣-bungarotoxin indicates that these receptors are restricted to medullary areas adjacent to the adrenal cortex and colocalize with the enzyme phenylethanolamine N-methyl transferase (PNMT), which confers the adrenergic phenotype to chromaffin cells. Transcripts corresponding to the ␣7 subunit also are localized exclusively to adrenergic cells. To identify possible transcriptional regulatory elements of the ␣7 subunit gene involved in the restricted expression of nicotinic receptors, we isolated and characterized its 5Ј flanking region, revealing putative binding sites for the immediate early gene transcription factor Egr-1, which is known to activate PNMT expression. In reporter gene transfection experiments, Egr-1 increased ␣7 promoter activity by up to sevenfold. Activation was abolished when the most promoter-proximal of the Egr-1 sites was mutated, whereas modification of a close upstream site produced a partial decrease of the Egr-1 response. Because Egr-1 was found to be expressed exclusively in adrenergic cells, we suggest that this transcription factor may be part of a common mechanism involved in the induction of the adrenergic phenotype and the differential expression of ␣-bungarotoxin-sensitive nicotinic receptors in the adrenal gland.

These results indicate that the magnitude of immunoproliferative responses are unrelated to maternal mite allergen exposure and cannot be used as evidence for in utero sensitization to inhalant allergens. The immunoproliferative responses at 1 year seem to shift away from the genetically influenced responses at birth towards responses to specific stimulants which correlate with environmental exposure to those specific stimulants. These data support the concept of sensitization to inhalant allergens occurring in early life, but not in utero.

We investigated the effect of in utero and postnatal environmental tobacco smoke (ETS) exposure on respiratory symptoms and atopy in the first 3 years of life in children at high risk of allergic disease (both parents atopic). Three hundred and sixty-nine children were followed from birth and reviewed at ages 1 and 3 years (respiratory questionnaire, skin testing). Parental smoking questionnaires were administered, and plasma cotinine in cord and peripheral blood (at age 1 year) was measured (capillary column gas-liquid chromatography). Wheezing starting in the first year of life was significantly more common in children of smoking mothers (54.2% vs. 39.5%, P = 0.017), but not wheezing starting after age 1 year (10.8% vs. 10.9%, smoking and nonsmoking mothers, P = 0.99). Detectable cord cotinine was not associated with wheeze. More frequent wheeze in infancy was significantly more common in those with detectable 1-year cotinine (e.g., wheeze without colds, 17.8% vs. 5.6%, P = 0.02; wheeze most days, 6.5% vs. 0%, P = 0.04). ETS exposure was not associated with atopy. In the multivariate regression analysis, maternal smoking during pregnancy and/or in the first year of life remained associated with wheeze in the first year of life (odds ratio, 1.88; 95% confidence interval, 1.14-3.12; P = 0.01). ETS exposure in "high-risk" infants increases the risk of wheezing starting in the first year of life, but not after age 1 year. However, ETS exposure has little or no effect on the development of atopy. Measurement of plasma cotinine was no more useful than tobacco exposure assessment by questionnaire in our cohort.