Performance differences in reaction tasks are reflected in event-related brain potentials (ERPs)

The glycoprotein hormone alpha-subunit gene is expressed and differentially regulated in pituitary gonadotropes and thyrotropes. Previous gene expression studies suggested that cell specificity may be regulated by distinct DNA elements. We have identified an enhancer region between -4.6 and -3.7 kb that is critical for high level expression in both gonadotrope and thyrotrope cells of transgenic mice. Fusion of the enhancer to -341/+43 mouse alpha-subunit promoter results in appropriate pituitary cell specificity and transgene expression levels that are similar to levels observed with the intact -4.6 kb/+43 construct. Deletion of sequences between -341 and -297 resuited in a loss of high level expression and cell specificity, exhibited by ectopic transgene activation in GH-, ACTH-, and PRL-producing pituitary cells as well as in other peripheral tissues. Consistent with these results, transient cell transfection studies demonstrated that the enhancer stimulated activity of a -341/+43 alpha-promoter in both alphaTSH and alphaT3 cells, but it did not enhance alpha-promoter activity significantly in CV-1 cells. Removal of sequences between -341 and -297 allowed the enhancer to function in heterologous cells. Loss of high level expression and cell specificity may be due to loss of sequences required for binding of the LIM homeoproteins or the alpha-basal element 1. These data demonstrate that the enhancer requires participation by both proximal and distal sequences for high level expression and suggests that sequences from -341 to -297 are critical for restricting expression to the anterior pituitary.     

Pharmacological, molecular and functional characterization of vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide receptors in the rat pineal gland

Melatonin secretion from the mammalian pineal gland is strongly stimulated by noradrenaline and also by vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Three types of receptors for VIP and PACAP have been characterized so far: VIP1/PACAP receptors and VIP2/PACAP receptors, which possess similar high affinities for VIP and PACAP, and PACAP1 receptors which exhibit a 100-1000-fold higher affinity for PACAP. The aim of the present study was to characterize the receptor subtype(s) mediating the stimulatory effects of VIP and PACAP on melatonin synthesis in the rat pineal gland. Autoradiographic studies showed that PACAP and VIP were equally potent in displacing binding of radioiodinated PACAP27 from pineal sections. Amplification of pineal complementary DNAs by polymerase chain reaction using specific primers for the different receptor subtypes revealed that all three receptor messenger RNAs are expressed and that VIP1/PACAP receptor messenger RNA was predominant over VIP2/PACAP receptor messenger RNA. In vitro, VIP and PACAP stimulated melatonin synthesis with similar high potency and the effect of the two peptides were not additive. The selective VIP1/PACAP receptor agonists [R16]chicken secretin (1-25) and [K15, R16, L27]VIP(1-7)/growth hormone releasing factor(8-27) were significantly more potent than the selective VIP2/PACAP receptor agonist RO 25-1553 in stimulating melatonin secretion. The stimulatory effects of VIP and PACAP were similarly inhibited by the VIP1/PACAP antagonist [acetyl-His1, D-Phe2, K15, R16, L27]VIP(3-7)/growth hormone releasing factor(8-27). These data strongly suggest that VIP and PACAP exert a stimulatory effect on melatonin synthesis mainly through activation of a pineal VIP1/PACAP receptor subtype.     

Neuroendocrine cell type-specific and inducible expression of the secretogranin II gene: crucial role of cyclic adenosine monophosphate and serum response elements

Secretogranin II, an acidic protein in the chromogranin/secretogranin family, is widely distributed in neuroendocrine secretory granules. What factors govern such widespread, yet selective, expression? The 5' deletions localized neuroendocrine cell type-specific expression to the proximal mouse secretogranin II promoter: such expression was abolished after deletion past the cAMP response element (CRE; [-67 bp]TGACGTCA[-60 bp]), and transfer of the CRE to a neutral promoter conferred 3.4- to 5.3-fold neuroendocrine selectivity. Thus, the CRE is, at least partly, sufficient to confer tissue-specific expression. Substantial (48-59%) loss of cell type-specific expression also occurred upon deletion past the serum response element (SRE; [-302 bp]GATGTCC[-296 bp]), and transfer of the SRE to a neutral promoter also conferred neuroendocrine selectivity. Expression of both the endogenous gene and the transfected secretogranin II promoter was up-regulated after secretagogues, and the degree of trans-activation of the transfected promoter (2.2- to 5.4-fold) paralleled activation of the endogenous gene (1.8- to 3.2-fold). The 5' promoter deletions revealed complete loss of secretagogue responses after deletion past the CRE. Transfer of the CRE to a neutral promoter conferred secretagogue responses (by 2.2- to 18.6-fold). Substantial (59-74%) falls in secretagogue responses also occurred after deletion past the promoter's SRE. Transfer of the SRE to a neutral promoter conferred secretagogue responses (by 2.7- to 8.3-fold). We conclude that the CRE is a crucial determinant of cell type-specific constitutive and secretagogue-inducible expression of the secretogranin II gene and that the SRE also plays a substantial role in both processes.     

Effects of pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal polypeptide on cyclic AMP accumulation in sheep pituitary cells in vitro

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are known to stimulate adenylate cyclase activity in rat pituitary cells but no direct effects have been reported on sheep pituitary cells. In this study we determined whether either peptide could stimulate intracellular cAMP accumulation in dispersed sheep pituitary cells in primary culture. Time course studies with PACAP showed that tachyphylaxis developed rapidly and so a short incubation time (5 min) was used to define the dose-response relationship. PACAP dose-dependently stimulated intracellular cAMP levels with a half-maximum response at 2.9 +/- 0.2 nmol/l (n = 4). In contrast, VIP only caused a small increase in intracellular cAMP levels at the highest dose tested (1 mumol/l). The VIP antagonist [4Cl-D-Phe6,Leu17]VIP had no effect on the cAMP response to either PACAP or VIP while the peptide PACAP(6-38), a putative PACAP antagonist, blocked the cAMP response to PACAP. The desensitisation to PACAP was further investigated by pretreating cells with PACAP for 30 min. After a further 15 min in culture medium alone, these cells showed no cAMP response to subsequent treatment with PACAP but could respond to forskolin. When a longer incubation period of 240 min was used between the first and second treatment with PACAP, a partial return in responsiveness to PACAP was observed. In summary, these results show that PACAP activates adenylate cyclase in sheep pituitary cells but that there is rapid development of tachyphylaxis. Experiments with the antagonists suggest that the response to PACAP is via the PACAP type I receptor. In contrast, physiological doses of VIP do not stimulate cAMP accumulation in sheep pituitary cells.     

Characterization of the equine glycoprotein hormone alpha-subunit gene reveals divergence in the mechanism of pituitary and placental expression

The equine glycoprotein hormone alpha-subunit gene is expressed in both pituitary and placenta, unlike that of all other nonprimate mammals studied, in which expression is limited to pituitary. Previous studies of the 5'-flanking region of the equine alpha-subunit promoter have revealed unique characteristics as well as similarities with the human alpha-subunit promoter, which demonstrates a similar pattern of tissue-specific expression. We have cloned and sequenced the equine alpha-subunit gene and have used tissue culture systems and transgenic mice to characterize its expression. Unlike the human promoter, the cloned equine alpha-subunit promoter failed to direct trophoblast-specific expression in either tissue culture or transgenic mouse models, suggesting an entirely different mechanism for expression. In contrast, the equine alpha-subunit promoter was able to direct gonadotroph expression in both tissue culture and transgenic mouse models. In alphaT3-1 cells, 550 base pair (bp) was sufficient for expression. This expression involves promoter elements identified in other species as playing a role in gonadotroph expression, but mutation of these elements reveals differences in their relative contributions to promoter activity. In mice, 2800 bp of 5'-flanking sequence allowed specific expression in gonadotrophs but not in thyrotrophs or placenta. The pattern of estrogen regulation observed in transgenic mice matched neither the repression that has been observed with human and bovine alpha-subunit promoters in transgenic mice nor the stimulation in mRNA levels reported in mares, suggesting a unique mechanism that is not recapitulated in the transgenic model. Thus the equine alpha-subunit promoter uses a combination of conserved and unique features of gene regulation to direct its pattern of tissue-specific expression.