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Notes: Abstract: Continuous exposure of cells to agonists develops a process that determines the extent to which the cells eventually respond to further stimuli. Here we used CATH.a cells (a catecholaminergic neuron-like cell line), which express pituitary adenylate cyclase-activating polypeptide (PACAP) receptors linked to both adenylyl cyclase and phospholipase C-β pathways, to investigate the influence of prolonged hormonal treatment on dual signaling and gene transcription. Prolonged incubation of cells with PACAP failed to down-regulate the density and affinity of membrane binding sites and caused opposite changes in messenger systems: PACAP-stimulated cyclic AMP accumulation was attenuated in a time- and dose-dependent fashion (t1/2 = 6.7 h and IC50 = 0.1 nM), whereas phosphoinositide turnover was overstimulated. Both effects were insensitive to pertussis toxin, whereas the drop in cyclic AMP concentration was also unchanged in the presence of 3-isobutyl-1-methylxanthine, indicating that neither Gi-like proteins nor cyclic nucleotide phosphodiesterases play a critical role in these processes. Blockade of protein synthesis with cycloheximide, as well as inhibition by H89 of cyclic AMP-dependent protein kinase (but not by bisindolylmaleimide of protein kinase C) antagonized the influences exerted by PACAP on adenylyl cyclase activity and inositol phosphate formation. Transcription of the chimeric GAL4-CREB construct, transiently transfected into CATH.a cells, was stimulated by PACAP, and this effect was potentiated as a result of chronic PACAP treatment. The results of the present investigation provide new insight into the possible differential regulation and cross-talks of transduction signals of receptors linked to multiplex signaling. They demonstrate that prolonged exposure of CATH.a cells to PACAP results in the desensitization of the cyclic AMP pathway and superinduction of the inositol phosphate signal, through protein neosynthesis and cyclic AMP-dependent protein kinase activation. At the same time, they show that desensitization of cyclic AMP signaling not only fails to hamper, but actually amplifies PACAP-stimulated CREB-regulated transcription.

Notes: Abstract: We used a catecholaminergic neuron-like cell line (CATH.a cells) as a model system to investigate the likelihood that pituitary adenylate cyclase-activating polypeptide (PACAP) may participate in the regulation of specific gene expression in catecholaminergic neurons. Analysis by reverse transcriptase-PCR amplification revealed the presence in these cells of type I PACAP receptors, with a short isoform, together with a heavier so-called Hop splice variant. PACAP38 and PACAP27 enhanced, in a dose-dependent manner, both cyclic AMP formation and phosphoinositide breakdown, with EC50 values of, respectively, 0.6 × 10−10 and 2 × 10−9M. These peptides, in addition, also elevated [Ca2+]i by mobilizing intracellular calcium pools. Vasoactive intestinal peptide (VIP) was ∼1,000-fold less potent in stimulating cyclic AMP (with EC50 = 2 × 10−7M) and failed to change the turnover of phosphoinositides and to alter [Ca2+]i. Both forms of PACAP, as well as forskolin, stimulated transcriptional induction of tyrosine hydroxylase (TH) and c-fos promoters fused to a chloramphenicol acetyltransferase (CAT) reporter gene in transiently transfected cells (p 〈 0.01 vs. controls). Induction of CAT activity linked to both TH and c-fos promoters was obliterated upon coexpression of a dominant inhibitory mutant (Mt-RAB) of cyclic AMP-dependent protein kinase. We conclude that CATH.a cells do express functional PACAP type I receptors, the activation of which impinges on TH and c-fos transcription according to a process that is primarily dependent on the cyclic AMP-PKA pathway.

Notes: The leporidae (rabbit and hare)pituitary intermediate lobe(IL)differs from that of other mammals by its neuroendocrine regulation. In particular, it is not submitted to the classic dopaminergic inhibitory control, which has been considered as a repressive factor for the expressionof glucocorticoid receptors (GR) in the mammalian IL. Hence, the present experiments aimed at examining the rabbit IL for the possible existence of GR. Using both immunocytochemistry and binding studies with (3H)-dexamethasone, we localized GR in the nuclei of IL cells and showed the presence of saturable and high-affinity type II receptor sites, with Kd∼3.9nM. Also, exposure of cultured IL cells to 10 nM dexamethasone (DEX) resulted in the blockade of melanocyte-stimulating hormone(αMSH) secretion stimulated by oxytocin (OT). Importantly, the inhibitory effect was reversed by a 100-fold excess of the glucocorticoid antagonist RU 38486. This is the first study which clearly demonstrates in a mammalian IL, namely the rabbit, the presence of functional GR, involved in the negative regulation of the melanotrophic activity of this gland.

Notes: The locus cœruleus is innervated by proopiomelanocortin (POMC)-derived peptide immunoreactive fibres. The biological effects of α melanocyte-stimulating hormone (αMSH) and β-endorphin on second messengers (cAMP, inositol phosphates) and gene transcription were studied in the locus cœruleus-derived cell line CATH.a. RT-PCR analysis revealed the presence of four MSH receptor subtypes (1, 3, 4 and 5). Activation of these receptors by diacetyl αMSH stimulated cAMP accumulation in a dose-dependent manner (EC50: 4 × 10–9m). Diacetyl αMSH stimulated transcription from reporter genes driven by the c-fos or tyrosine hydroxylase promoter. This effect was abolished when protein kinase A was inactivated with a dominant inhibitory mutant. RT-PCR analyses revealed the presence of δ-, but not μ-and κ-opioid receptor. Pharmacological analysis showed that β-endorphin (EC50: 2.5 × 10–8m), but not N-acetyl β-endorphin, antagonized the biological effect of diacetyl αMSH on cAMP production and gene transcription. Since N-acetylation regulates the biological activity of αMSH and β-endorphin in an opposite manner, we propose a model where the rate of secretion dictated by the bioelectric activity of the presynaptic neuron modulates POMC-derived peptide maturation and the resulting biological signal sensed by the postsynaptic plate.