Corticotropin-releasing Factor Binding Sites Research Articles

Corticotropin-releasing factor receptors in the rat central nervous system: characterization and regional distribution

A stable, iodine-125-labeled analog of rat/human corticotropin-releasing factor (CRF) was used to define the characteristics of CRF receptors in a crude mitochondrial/synaptosomal membrane preparation of rat olfactory bulb, and to study the distribution of CRF binding sites in discrete regions of the rat CNS. The binding of 125I-Tyro rat/human CRF (125I-rCRF) was time- and temperature-dependent, was sensitive to the pH, ionic strength, and cationic composition of the incubation buffer, and was linear over a broad range of membrane protein concentrations. 125I-rCRF binding to olfactory bulb membrane was saturable, reversible, and, on Scatchard analysis, revealed a high-affinity component with an apparent equilibrium dissociation constant (Kd) of 0.2 nM and a low-affinity binding site with Kd of approximately 20 nM. Data from pharmacological studies indicated that the ability of a variety of CRF fragments and analogs to inhibit 125I-rCRF to olfactory bulb membranes correlates well with their reported relative potencies in stimulating pituitary adrenocorticotropic hormone secretion in vitro. Consistent with a coupling of CRF receptors to adenylate cyclase, the binding of 125I-rCRF was decreased by guanine nucleotides and increased by magnesium ions. A heterogeneous distribution of 125I-rCRF binding sites was found in the rat CNS, with highest densities present in olfactory bulb, cerebellum, cerebral cortex and striatum, and progressively lower but significant levels of binding were detected in cervical spinal cord, hypothalamus, medulla, midbrain, thalamus, pons, and hippocampus. These data, using a rat CRF ligand homologous to the endogenous peptide, are consistent with those from previous studies demonstrating the presence of specific binding sites for ovine CRF in rat brain, and provide further support for the suggestion that endogenous CRF may function as a neurotransmitter in the CNS.

Increased corticotropin-releasing factor receptors in rat cerebral cortex following chronic atropine treatment

Rats were treated chronically with atropine (14 days, 20 mg/kg/day, s.c.) and corticotropin-releasing factor (CRF) receptors and CRF-mediated adenylate cyclase activity were measured in discrete brain regions. Chronic atropine treatment produced significant increases in muscarinic cholinergic receptors in the frontoparietal cortex (30% increase) and hippocampus (20% increase). No significant changes in the concentration of [ 125I]Tyr o-rat CRF binding sites were observed in olfactory bulb, cerebellum, striatum and hippocampus. In contrast, there was a significant and selective increase (35%) in CRF receptors in the frontoparietal cortex of atropine-treated rats. However, no significant corresponding changes in the V max or EC 50 of CRF-stimulated adenylate cyclase activity accompanied the upregulation of CRF receptors in the cerebral cortex. These results demonstrate that (1) CRF receptors in rat brain are subject to receptor regulation, (2) the upregulation of CRF receptors occurs as a consequence of chronic muscarinic cholinergic receptor blockade, and (3) this interaction between acetylcholine and CRF may be limited to the cerebral cortex.

Demonstration of corticotropin-releasing factor binding sites on human and rat erythrocyte membranes and their modulation by chronic ethanol treatment in rats

In a previous study we reported the presence of specific corticotropin-releasing factor (CRF) binding sites in peripheral tissues of the rat (Endocrinology, 116 , 2152, 1985). Using 125I-labeled rat or human CRF, specific CRF binding sites were identified on rat and human erythrocytes, but not on lymphocytes or platelets. Furthermore, identical CRF binding was observed in the presence of intact erythrocytes or lysed erythrocyte membranes. Maximal binding of 125I-CRF occurred within 25 min at 4°C and was saturable. Scatchard analysis of CRF binding to erythrocyte membranes revealed the existence of a single class of binding site. Chronic exposure of rats to ethanol vapor, known to lower specific CRF binding to pituitary tissue by 35%, also decreased 125I-rat CRF binding to erythrocyte membranes by approximately 45%, which was due to a decrease in the number of CRF binding sites. The parallel decrease of CRF binding to rat-erythrocyte and pituitary membranes following chronic ethanol treatment suggests that CRF binding to erythrocyte and pituitary membranes is modulated in a similar direction, which further suggests that the determination of CRF binding to erythrocytes may provide an important clinical tool to indirectly assess CRF-receptor levels in the pituitary gland and thereby enhance our understanding of ethanol-induced disorders of the hypothalamic-pituitary-adrenal axis in patients.

Demonstration that corticotropin-releasing factor binding to rat peripheral tissues is modulated by glucocorticoid treatment in vivo and in vitro

In a recent study we reported the presence of specific binding sites for corticotropin-releasing factor (CRF) in peripheral tissues of the rat (Endocrinology, 116 , 2151, 1985). The objective of this study was to determine if CRF binding to peripheral tissues was modified following adrenalectomy and glucocorticoid replacement therapy. Adult male rats were adrenalectomized and CRF binding to liver, spleen and testicular membranes was determined at 5,7 or 14 days following adrenalectomy. An additional group of adrenalectomized rats received subcutaneous injections of dexamethasone (75 μg/day) for 14 days. Adrenalectomy of rats for 14 days increased CRF binding to liver, kidney, testis, spleen and ventral prostate by approximately 65%–125% above sham-control values. CRF binding to membrane preparations obtained from the pancreas of sham-operated rats was undetectable; however, adrenalectomy produced detectable CRF binding in this tissue. Adrenalectomy produced a time-related increase in CRF binding to ventral prostate, spleen and liver tissue. Administration of dexamethasone to adrenalectomized animals prevented increased CRF binding to peripheral tissues observed following adrenalectomy alone. In vitro dexamethasone treatment of prostatic or hepatic homogenates from adrenalecomized rats resulted in a dose-related decrease in CRF binding activity. However, similar in vitro treatment of prostatic or hepatic homogenate with progesterone exhibited no significant effects on CRF binding. Our results suggest that glucocorticoids may be a regulator of peripheral CRF receptors.

Corticotropin-Releasing Factor Receptors in Human Pituitary Gland: Autoradiographic Localization

Corticotropin-releasing factor (CRF) receptors were localized in the human pituitary gland by an in vitro labeling light microscopic autoradiographic technique using an 125I-labeled analog of ovine CRF substituted with norleucine and tyrosine at amino acid residues 21 and 32, respectively. Specific binding sites for CRF were observed in the anterior lobe while no specific binding sites for CRF were present in the posterior pituitary lobe. The CRF binding sites in the anterior lobe occurred in clusters which predominated in the anteromedial portion of the lobe. This pattern of localization of CRF binding sites resembles previously reported distributions of corticotrophs in anterior pituitary. These data support the physiological role of endogenous CRF in regulating hormone secretion from the anterior lobe of the human pituitary.

Corticotropin-releasing factor receptors in rat pituitary gland: autoradiographic localization

Corticotropin-releasing factor (CRF) receptors were localized in rat pituitary gland by an in vitro labeling light microscopic autoradiographic technique using the radioiodinate ovine CRF analog, [Nle 21, 125I-Tyr 32]-CRF. Our data confirms the existence of CRF receptors in anterior pituitary and demonstrates the presence of specific CRF binding sites in the intermediate lobe of rat pituitary. The distribution of CRF binding sites closely resembles the clustering of corticotrophs in anterior lobe and the uniform distribution of opiomelanocortin-producing cells in the intermediate pituitary. These results suggest that endogenous CRF plays a physiological role in regulating secretion of pro-opiomelanocortin-derived peptides from both anterior and intermediate lobes of rat pituitary.

Properties and regulation of high-affinity pituitary receptors for corticotropin-releasing factor

Specific receptors for corticotropin-releasing factor (CRF) were identified in the rat anterior pituitary gland by binding studies with 125I-Tyr-CRF. Binding of the labeled CRF analog to pituitary particles was rapid and temperature-dependent, and reached steady state within 45 min at 22°C. The CRF binding sites were saturable and of high affinity, with dissociation constant (Kd) of 0.76 × 10 −9 M. Pituitary binding of 125I-Tyr-CRF was inhibited by CRF, Tyr-CRF and the active 15–41 fragment of CRF, but not by the inactive 21–41 CRF fragment and unrelated peptides. The binding-inhibition potencies of the CRF peptides were similar to their activities as stimuli of adrenocorticotropic hormone (ACTH) release. The high-affinity CRF sites were markedly reduced in adrenalectomized rats, and this change was reversed by dexamethasone treatment. These data indicate that the high-affinity CRF sites demonstrated in the anterior pituitary are the functional receptors which mediate the stimulatory action of the peptide on ACTH release, and that CRF receptors are down-regulated during increased secretion of the hypothalamic hormone.