Monica A. Millan

Brain and pituitary receptors for corticotropin releasing factor: Localization and differential regulation after adrenalectomy

Specific receptors for corticotropin releasing factor (CRF) were identified in two functionally distinct systems within the brain, the cortex and the limbic system. Autoradiographic mapping of the CRF receptors in the brain revealed high binding density throughout the neocortex and cerebellar cortex, subiculum, lateral septum, olfactory tract, bed nucleus of the stria terminalis, interpeduncular nucleus and superior colliculus. Moderate to low binding was found in the hippocampus, nucleus accumbens, claustrum, nucleus periventricularis thalamus, mammillary bodies, subthalamic nucleus, periaqueductal grey, locus coeruleus and nucleus of the spinal trigeminal tract. As in the anterior pituitary gland, CRF receptors in the brain were shown to be coupled to adenylate cyclase. However, in contrast to the marked decrease in CRF receptors observed after adrenalectomy in the anterior pituitary gland, CRF receptor concentration in the brain and pars intermedia of the pituitary was unchanged. The presence of CRF receptors in areas involved in the control of hypothalamic and autonomic nervous system functions is consistent with the major role of CRF in the integrated response to stress.

Corticotropin-releasing Factor Receptors: Distribution and Regulation in Brain, Pituitary, and Peripheral Tissues

Since the characterization of corticotropin releasing factor (CRF) in 198 1, evidence has accumulated to indicate that the hypothalamic peptide plays an important role in the regulation of ACTH secretion as well as in mediating visceral and behavioral responses to stress.’.’ The initial event in the action of peptide hormones is their binding to a plasma membrane receptor, and labeled ligands were rapidly developed in order to investigate the mode of interaction of C R F with its cellular binding site. Receptors for C R F were first identified in rat pituitary membranes using radioiodinated Tyr-oCRF, and subsequently studied by radioassays in autoradiographic p r o c e d ~ r e s , ~ ~ ~ and cytochernical techniques using biotinylated or fluorescein-conjugated C R F The use of autoradiography facilitated the identification and characterization of C R F receptors in the central and peripheral nervous system, which has contributed to our understanding of the physiological actions of CRF. The most common ligand used for C R F receptor studies is the radiolabeled ovine C R F derivative, Tyr-oCRF, but similar receptor properties have been described using iodinated [NLe”, Tyr3’]oCRFS. Analogues of rat/human C R F have given tracers with reduced biological action and lower binding activity due to peptide damage during the iodination procedure. Since ovine and human C R F bind to the C R F receptor in different species with equal affinities, oCRF can be used for studies in rat and primates. The purpose of this review is to discuss current knowledge of the C R F receptor, including its binding properties, regulation, and distribution in the pituitary, and nervous system.

Receptor-mediated actions of corticotropin-releasing factor in pituitary gland and nervous system.

High-affinity corticotropin-releasing factor (CRF) receptors which mediate the actions of the hypothalamic peptide on adrenocorticotropic hormone (ACTH) release have been identified in the rat anterior pituitary gland. Occupancy of the pituitary receptor by CRF agonists stimulates ACTH release via activation of adenylate cyclase and cyclic adenosine monophosphate dependent protein kinase. In the regulation of ACTH secretion, the effects of CRF on the corticotroph are integrated with the stimulatory actions of cyclic adenosine monophosphate-independent stimuli such as angiotensin II, vasopressin and norepinephrine, and the inhibitory effects of glucocorticoids and somatostatin. In contrast to the major importance of the inhibitory effect of glucocorticoid feedback on ACTH secretion, somatostatin has relatively little effect on CRF-stimulated ACTH release in the normal rat corticotroph. Following adrenalectomy, the progressive elevation of plasma ACTH levels is accompanied by a concomitant decrease in pituitary CRF receptors. The postadrenalectomy loss of CRF receptors, which is prevented by dexamethasone treatment, is caused by a combination of occupancy and processing of the pituitary sites during increased secretion of the hypothalamic peptide. Recently, specific receptors for CRF have been localized in the rat and monkey brain and adrenal medulla, where they are also coupled to adenylate cyclase. Brain CRF receptors are most abundant in the cerebral and cerebellar cortices and in structures related to the limbic system and control of the autonomic nervous system. The actions of CRF on the central and peripheral nervous systems, as well as on the pituitary gland, emphasize the role of CRF as a key hormone in the integrated response to stress.

Amphibian myocardial angiotensin II receptors are distinct from mammalian AT1 and AT2 receptor subtypes.

High‐affinity receptors for angiotensin II were identified on Xenopus laevis cardiac membranes and characterized by binding‐inhibition studies with peptide and non‐peptide AII antagonists. Scatchard analysis of the binding data identified a high‐affinity site with K d1 =1.6 nM and B max1=3.7 pmol/mg protein and a low‐affinity site with K d2=22 nM and B max2 =9.5 pmol/mg protein. Treatment with dithiothreitol reduced the number of binding sites by > 70%. The rank order of potency for AII analogs was (agent, IC50) [Sar1,Ile8]AII, 0.91 nM > AII, 2.0 nM > AI, 5.3 nM > [Sar1, Ala8]AII, 19 nM > CGP42112A, 1.2 μM ⋙ DuP 753≈PD‐123177, > μM. The relative potencies of these compounds differ markedly from their activities on the two known mammalian AII receptor subtypes, AT1 and AT2. These results indicate that amphibian AII receptors are pharmacologically distinct from both the AT1 and AT2 receptors characterized in mammalian tissues.

Developmental changes in brain angiotensin II receptors in the rat

AII binding and distribution were measured in rat brain during development by autoradiographic techniques using radioiodinated [Sar1,Ile8]AII. At all ages, from 2 days to 7 weeks, binding was present in the circumventricular organs, and areas related to pituitary hormone secretion and modulation of sympathetic activity. At early stages of development, AII binding was transiently expressed in a number of motor- and sensory-related areas. These findings support a role for AII in the control of water intake and autonomic activity at all stages of development, and suggest that the peptide may be involved in the maturation of neuronal function during development.

Characterization of angiotensin II receptors in the rat fetus

The presence of AII receptors during early and late embryonic development was studied by binding of 125I[Sar1, Ile8] AII to whole mouse blastocysts and membrane-rich fractions from rat conceptuses, 7 to 21 days in gestation. In early mouse embryos there was no detectable binding under a variety of experimental conditions. However, in late gestation rat fetuses, specific and high affinity binding was observed, with a concentration of sites similar in membranes from whole and eviscerated fetuses. Using less than 100 micrograms of membrane protein, binding was time and temperature dependent, maintaining equilibrium from 30 to 120 min at 23 degrees C and it was enhanced by addition of Mg+2 up to 5 mM, EGTA 2 mM and dithiothreitol up to 2.5 mM. Scatchard analysis of the binding data indicated Kd values ranging between 0.7 and 0.9 nM. Binding was first detectable at day 10 (14.3 +/- 2.3 fmol/mg), increasing to 104 +/- 16, 2,625 +/- 168, 5,993 +/- 152 and 5,902 +/- 92 by days 12, 15, 18, and 21 of gestational age, respectively. Since the functional significance of these binding sites depends on the availability of the agonist ligand, acid extracts from eviscerated 10-day-old fetuses were analyzed for the presence of AII. Measurement of AII by radioimmunoassay revealed immunoreactive AII-like material (845 pg/g of tissue), with an elution pattern identical to that of AII standard in a Sephadex G-50 column. This material was bioactive, as demonstrated by its ability to displace 125I[Sar1, Ile8]AII from adrenal glomerulosa membranes, an effect which was abolished by pretreatment of the extract with AII antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of angiotensin II receptors in the brain of nonhuman primates

Angiotensin II binding sites were demonstrated at discrete nuclei in the brain of three nonhuman primate species by autoradiography, using the agonist ligand, [Sar1]AII. Although there were some differences in location of the binding sites, all three species exhibited a characteristic pattern of distribution in areas related to water intake, vasopressin secretion, and blood pressure regulation through modulation of sympathetic activity. Studies in the cynomolgus monkey with the antagonist ligand, [Sar1,Ile8]AII, which localizes in pathways as well as nuclei, revealed novel regions of binding including the habenular-interpeduncular pathway, ventral bundle, and XII nerve, in addition to the X nerve. These data indicated that AII, as in other species, has a role in the central homeostatic control mechanisms in the primate.