We reported previously that acute stress and intracerebroventricular (i.c.v.) injection of corticotropin-releasing factor (CRF) increased neuronal activation and CRF type-1 receptor (CRFR-1) mRNA expression in the CRF-producing neurons of the parvocellular paraventricular nucleus (PVN) of the hypothalamus. In this study, to determine whether CRF can act directly on hypothalamic CRF neurons, thereby increasing CRFR-1 expression, microinjection of CRF into PVN neurons in vivo and primary cultures of dispersed rat fetal hypothalami in vitro were performed. Microinjection of 0.1 microg of CRF into the PVN significantly increased c-fos and CRFR-1 mRNA expression in the CRF-producing parvocellular PVN, 30 min or 180 min after injection, respectively. This effect was blocked by a CRF antagonist, alpha-helical CRF. CRF, when injected into the lateral ventricle at the same dose, increased neither CRFR-1 nor c-fos mRNA levels in the PVN. Primary culture of hypothalamic neurons revealed that CRFR-1 like immunoreactivity was located in CRF-containing neurons, and that the CRFR-1 mRNA level was significantly increased 4 h after incubation with 10(-8) M CRF. These results demonstrate that CRF directly affects hypothalamic neurons to increase CRFR-1 mRNA expression, providing evidence of a direct role for CRF in the regulation of CRFR-1 expression of hypothalamic neurons.
In rats, acute stress substantially increases corticotropin-releasing factor (CRF) type 1 receptor (CRFR-1) mRNA expression in the paraventricular nucleus (PVN) and osmotic stimulation induces both CRF and CRFR-1 mRNA in magnocellular PVN and supraoptic nucleus (SON). However, these phenomena have not been analysed in other species. We compared CRF and CRFR-1 expression in rat and mouse hypothalamus. Male C57BL/6 mice and Wistar rats were exposed to acute restraint stress for 3 h, or to hypertonic saline ingestion for 7 days. Restraint stress increased CRF and c-fos mRNA expression in both rat and mouse PVN. CRFR-1 mRNA was barely detectable in controls, whereas restraint stress substantially increased CRFR-1 mRNA in rat PVN, but not in mouse. Hypertonic saline ingestion induced CRF mRNA in magnocellular PVN and SON of the rat, but did not alter CRF mRNA levels in mouse hypothalamus. CRFR-1 mRNA was also induced in magnocellular PVN and SON of the rat in response to osmotic stimulation, but not in mouse. Immunohistochemistry demonstrated that CRFR-1-like immunoreactivity (ir) was distributed within parvocellular and magnocellular PVN of mouse and rat. CRFR-1-ir in rat PVN was increased by acute stress and osmotic stimulation. By contrast, these treatments did not alter CRFR-1-ir in mouse PVN. Combined immunohistochemistry and in situ hybridization revealed that CRFR-1-ir was most frequently colocalized to CRF in mouse PVN, whereas only a small percentage of oxytocin and vasopressin-producing cells coexpressed CRFR-1-ir. These results indicate that (i) by contrast to rats, neither acute stress nor osmotic stimulation induces CRFR-1 mRNA expression in the mouse PVN; (ii) osmotic stimulation does not alter CRF mRNA expression in parvocellular and magnocellular neurones of mouse PVN; and (iii) acute stress increases c-fos and CRF mRNA to a similar degree in mouse and rat PVN. Thus, differences may exist between mouse and rat in the regulation of CRF and CRFR-1 gene expression in hypothalamus following stress and osmotic stimulation.
Corticotropin-releasing factor (CRF) is a major secretagogue of adrenocorticotopic hormone from the anterior pituitary and a key activator of the hypothalamic-pituitary-adrenal axis. We previously reported that CRF down-regulates expression of the CRF type-1 receptor (CRF-R1) mRNA in cultured rat anterior pituitary cells. The present study was conducted to clarify the signal transduction systems involved in CRF-induced down-regulation of CRF-R1 gene expression in the anterior pituitary. Northern blot analysis revealed that, under serum-free conditions, 10 nM CRF decreased CRF-R1 mRNA levels in cultured rat anterior pituitary cells as we reported previously. Treatment with 5 mM 8-Br-cAMP reduced CRF-R1 mRNA levels within 2 h. The mRNA level fell to 37+/-3% of the basal level at 2 h and remained low for 16 h after treatment. This CRF-induced reduction of CRF-R1 mRNA expression was inhibited completely by pretreatment with protein kinase A (PKA) inhibitor (1 microM H-89). Further examination revealed that after pretreatment with 10 microM of antisense oligodeoxynucleotide for cyclic AMP-response element binding protein (CREB), the CRF-induced inhibition of CRF-R1 mRNA was partially decreased to 79+/-4% of the control level 2 h after administration of CRF. These findings indicate that CRF may down-regulate CRF-R1 mRNA expression via a cAMP-PKA-mediated mechanism in rat anterior pituitary cells, and that CREB may mediate at least a portion of this inhibitory effect.
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