RESUMO
Neuropeptide W (NPW) was isolated as an endogenous ligand for NPBWR1, an orphan G protein-coupled receptor localized in the rat brain, including the paraventricular nucleus. It has been reported that central administration of NPW stimulates corticosterone secretion in rats. We hypothesized that NPW activates the hypothalamic-pituitary-adrenal (HPA) axis via corticotrophin-releasing factor (CRF) and/or arginine vasopressin (AVP). NPW at 1 pM to 10 nM did not affect basal or ACTH-induced corticosterone release from dispersed rat adrenocortical cells, or basal and CRF-induced ACTH release from dispersed rat anterior pituitary cells. In conscious and unrestrained male rats, intravenous administration of 2.5 and 25 nmol NPW did not affect plasma ACTH levels. However, intracerebroventricular (icv) administration of 2.5 and 5.0 nmol NPW increased plasma ACTH levels in a dose-dependent manner at 15 min after stimulation (5.0 vs. 2.5 nmol NPW vs. vehicle: 1802 ± 349 vs. 1170 ± 204 vs. 151 ± 28 pg/mL, respectively, mean ± SEM). Pretreatment with astressin, a CRF receptor antagonist, inhibited the increase in plasma ACTH levels induced by icv administration of 2.5 nmol NPW at 15 min (453 ± 176 vs. 1532 ± 343 pg/mL, p<0.05) and at 30 min (564 ± 147 vs. 1214 ± 139 pg/mL, p<0.05) versus pretreatment with vehicle alone. However, pretreatment with [1-(ß-mercapto-ß, ß-cyclopentamethylenepropionic acid), 2-(Ο-methyl)tyrosine]-arg-vasopressin, a V1a/V1b receptor antagonist, did not affect icv NPW-induced ACTH release at any time (p>0.05). In conclusion, we suggest that central NPW activates the HPA axis by activating hypothalamic CRF but not AVP.
Assuntos
Hormônio Adrenocorticotrópico/metabolismo , Arginina Vasopressina/fisiologia , Hormônio Liberador da Corticotropina/fisiologia , Neuropeptídeos/farmacologia , Hormônio Adrenocorticotrópico/sangue , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos , Células Cultivadas , Corticosterona/metabolismo , Avaliação Pré-Clínica de Medicamentos , Antagonistas de Hormônios/administração & dosagem , Antagonistas de Hormônios/farmacologia , Injeções Intravenosas , Masculino , Neuropeptídeos/administração & dosagem , Adeno-Hipófise/citologia , Adeno-Hipófise/efeitos dos fármacos , Adeno-Hipófise/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Regulação para Cima/efeitos dos fármacosRESUMO
CONTEXT: Methods for preoperative diagnosis of prohormone convertase 2 (PC2)-positive ACTH-producing pituitary adenomas (APPAs) have not been established. Also, their characteristics are not evident. OBJECTIVE: This study was designed to understand the meaning of plasma alphaMSH levels and the role of cell proliferation-signaling molecules in PC2-positive APPAs. PATIENTS AND MAIN OUTCOME MEASURES: Nineteen human APPAs (four males and 15 females) were examined for the expression of PC2, phosphorylated ERK1/2, phosphorylated Akt1/2/3 (p-Akt) and receptor tyrosine kinases. alphaMSH was measured in extracted plasma from 17 APPA patients and 30 healthy volunteers. RESULTS: Nine adenomas (47.4%) were immunopositive for PC2 and were large and invasive in nature. In all normal controls and eight PC2-negative cases, plasma alphaMSH was undetectable, whereas in four PC2-positive cases, it was detected at abnormally higher levels. Eight adenomas (42.1%) were immunopositive for both PC2 and p-Akt, and seven others (36.8%) were immunonegative for both, suggesting significant coexpression of PC2 and p-Akt in tumors. Quantitative RT-PCR revealed that PC2 expression is associated with phosphorylation of Akt but not with its gene expression. Most APPAs expressed receptor tyrosine kinases, but membrane-bound receptors could not be identified. CONCLUSIONS: Our study suggests that PC2 expression and Akt phosphorylation are related at the molecular level, resulting in a change in cell cycle and an increase in pituitary adenoma size. An elevation of plasma alphaMSH could conjecture the activation of the phosphatidylinositol 3/Akt cascade in PC2-positive APPAs and may become a valuable clinical marker of tumor growth in Cushing's disease.
Assuntos
Adenoma Hipofisário Secretor de ACT/metabolismo , Adenoma/metabolismo , Pró-Proteína Convertase 2/metabolismo , alfa-MSH/sangue , Adenoma Hipofisário Secretor de ACT/genética , Adenoma/genética , Adolescente , Adulto , Idoso , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Pró-Proteína Convertase 2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Radioimunoensaio , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cytoplasmic calcium ([Ca(2+)](i)) provided through voltage-dependent Ca(2+) channels (VDCC) plays an important role in adrenocorticotropin (ACTH)-induced steroidogenesis in adrenocortical cells. To identify alternative mechanisms for [Ca(2+)](i) supply, we investigated the 2-aminoethoxydiphenyl borate (2APB)-sensitive pathway as one of the possible signaling pathways involved in [Ca(2+)](i) supply for ACTH-induced steroidogenesis. In monolayers of cultured rat adrenal fasciculate and reticularis cells, ACTH at 10(-11) M stimulated corticosterone synthesis without increasing intracellular cAMP, and corticosterone synthesis was decreased by 10 microM 2APB by 51.8% (6.71 +/- 0.97 vs. 3.23 +/- 0.05 ng/mL/4 hours; p<0.05). Furthermore, 2APB significantly decreased the 10(-11) M ACTH-stimulated [Ca(2+)](i). ACTH increased the intracellular inositol-1,4,5-trisphosphate (IP3) content with a peak at 10(-13) M ACTH, which illustrates the possibility that ACTH activates IP3/diacylglycerol- dependent protein kinase C signal transduction. However, the difference in ACTH concentrations between that responsible for the IP3 increase and steroidogenesis without elevated cAMP, suggest a hypothesis that IP3 is not required for steroidogenesis, but does involve an unknown messenger, which stimulates the release of Ca(2+) from the ER or the subsequent store-operated Ca(2+) entry (SOCE). The pregnenolone concentration in the culture medium was increased by ACTH, which was significantly suppressed by 2APB, showing that the 2APB-sensitive Ca(2+) supply affects cholesterol transport into the mitochondrial membrane via steroidogenic acute regulatory protein. Therefore, the SOCE may contribute to ACTH-induced steroidogenesis in the mitochondrial region. In conclusion, the [Ca(2+)](i) used for steroidogenesis may be derived from a 2APB-sensitive pathway and via VDCCs, particularly at physiological concentrations of ACTH. We suggest that ACTH receptors activate steroidogenesis via inositol triphosphate, or an unknown downstream messenger, which could be inhibited by 2APB.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Compostos de Boro/farmacologia , Cálcio/farmacologia , Corticosterona/biossíntese , Animais , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Células Cultivadas , AMP Cíclico/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Tapsigargina/farmacologiaRESUMO
In this study, we investigated the role of store-operated Ca2+ channels (SOCC) on ACTH release using microperifusion system. The SOCC blockers, SKF96365 and MRS1845, did not affect the ACTH response to single AVP stimulation. After the depletion of intracellular Ca2+ stores by treating with ionomycin, SOCC blockers reduced the initial spike phase of ACTH response to AVP, which is mediated by inositol 1,4,5-trisphosphate-induced intracellular Ca2+ release from the endoplasmic reticulum (ER). The sustained plateau phase of ACTH response, which is mediated by protein kinase C leading Ca2+ influx via L-type voltage-dependent Ca2+ channels, was not affected. Addition of L-type voltage-dependent Ca2+ channel blocker nimodipine with the SOCC blockers reduced both the initial spike and sustained phases of ACTH response to AVP. Even after ER Ca2+ depletion, the SOCC blockers did not affect the ACTH response to CRH, which is mediated by cAMP-dependent protein kinase A. Transient receptor potential (TRP) C channel is the strongest candidate for SOCC, and RT-PCR revealed that all types of TRPC homologue mRNA were expressed in rat anterior pituitary cells. In conclusion, the SOCC mediates the initial spike phase of ACTH response to AVP, possibly via ER Ca2+ store refilling to induce maximum response.