No abstract

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.

Hypothyroxinemia affects 35–50% of neonates born prematurely (12% of births) and increases their risk of suffering neurodevelopmental alterations. We have developed an animal model to study the role of maternal thyroid hormones (THs) at the end of gestation on offspring's cerebral maturation. Pregnant rats were surgically thyroidectomized at embryonic day (E) 16 and infused with calcitonin and parathormone (late maternal hypothyroidism [LMH] rats). After birth, pups were nursed by normal rats. Pups born to LMH dams, thyroxine treated from E17 to postnatal day (P) 0, were also studied. In developing LMH pups, the cortical lamination was abnormal. At P40, heterotopic neurons were found in the subcortical white matter and in the hippocampal stratum oriens and alveus. The Zn-positive area of the stratum oriens of hippocampal CA3 was decreased by 41.5% showing altered mossy fibers’ organization. LMH pups showed delayed learning in parallel to decreased phosphorylated cAMP response element-binding protein (pCREB) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) expression in the hippocampus. Thyroxine treatment of LMH dams reverted abnormalities. In conclusion, maternal THs are still essential for normal offspring's neurodevelopment even after onset of fetal thyroid function. Our data suggest that thyroxine treatment of premature neonates should be attempted to compensate for the interruption of the maternal supply.