The brain corticotropin-releasing factor (CRF) receptor is of lower apparent molecular weight than the CRF receptor in anterior pituitary. Evidence from chemical cross-linking studies

The ligand binding subunits of the corticotropin-releasing factor (CRF) receptors in brain and anterior pituitary of a number of species have been identified by chemical affinity cross-linking using the homobifunctional cross-linking agent disuccinimidyl suberate and 125I-Tyr0-oCRF (ovine CRF). In homogenates of rat, monkey, and human cerebral cortex, 125I-Tyr0-oCRF was covalently incorporated into a protein of Mr = 58,000. Under identical conditions in the anterior pituitary of rat, monkey, cow, and pig, 125I-Tyr0-oCRF was incorporated into a protein of apparent Mr = 75,000. The specificity of the labeling was typical of the CRF binding site since both the cerebral cortex- and pituitary-labeled proteins exhibited the appropriate pharmacological rank order profile characteristic of the CRF receptor (Nle21,Tyr32-oCRF approximately equal to rat/human CRF approximately equal to ovine CRF approximately equal to alpha-helical CRF(6-41) greater than alpha-helical oCRF(9-41) greater than or equal to oCRF(7-41) greater than rat/human CRF(1-20) approximately equal to vasoactive intestinal peptide). In addition to the major labeled proteins, 125I-Tyr0-oCRF was incorporated into higher molecular weight peptides which may represent precursors and into lower molecular weight components which may represent fragments of the major labeled proteins or altered forms of the CRF binding subunit. In summary, these data indicate a heterogeneity between brain and pituitary CRF receptors with the ligand binding subunit of the brain CRF receptor residing on a Mr = 58,000 protein, while in the anterior pituitary, the identical binding subunit resides on a protein of apparent Mr = 75,000.     

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 degrees C. The CRF binding sites were saturable and of high affinity, with dissociation constant (Kd) of 0.76 X 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.     

Diurnal variation of corticotropin-releasing factor binding sites in the rat brain and pituitary

Summary 1. Corticotropin-releasing factor (CRF) is thought to be involved in the regulation of the diurnal activity of the hypothalamus-pituitary-adrenal (HPA) axis and to act as a neurotransmitter in the brain. To date it is unknown whether the binding sites of the central CRF system are subject to diurnal variations. 2. We measured the number of CRF binding sites over the course of a complete 24-hr light-dark cycle in the pituitary, amygdala, bed nucleus of the stria terminalis (BNST), cingulate cortex, visceral cortex, paraventricular nucleus of the hypothalamus, hippocampus, and locus ceruleus of rats byin vitro receptor autoradiography with iodinated ovine CRF. A 24-hr time course was also established for plasma CRF and corticosterone. 3. The diurnal pattern of plasma CRF does not correlate with the pattern of plasma corticosterone. Within the brain, CRF binding in the basolateral nucleus of the amygdala showed a U-shaped curve with maximum levels in the morning and a wide hallow between 1500 and 0100. A biphasic profile with a small depression in the afternoon and a more pronounced depression in the second half of the activity period is characteristic for the other brain areas and the pituitary. The profile for the pituitary correlates with those for the BNST and the area of the locus ceruleus. Furthermore, the diurnal pattern of CRF binding sites in the BNST correlates with that of the hippocampus, and the daytime pattern of the visceral cortex is similar to that of both the hippocampus and the BNST. 4. Since the CRF-binding profiles in the brain and the pituitary clearly differ from the profiles of both plasma CRF and corticosterone, one may assume that the diurnal pattern of central CRF binding sites is not directly coupled to the activity of the HPA axis.     

Differential loss in function of angiotensin II receptor subtypes during tissue storage

In vitro receptor autoradiography was performed on rat brain and kidney sections stored frozen at -20 degrees C for extended time periods (17, 40, 64, 121, 183, 251, and 333 days). The results indicate that prolonged tissue storage has a differential effect upon 125I sar1ile8 angiotensin II binding to AT1 and AT2 receptor sites. Binding at AT1 receptor rich tissues studied (renal medulla, renal cortex, anterior pituitary, ventral hippocampus, spinal trigeminal nucleus, and nucleus of the solitary tract) shows a first order exponential decay pattern. The logarithmic linear regression slope (log(e) specific binding versus time), is significantly different from zero (p<0.05) in all AT1 rich tissues except for nucleus of the solitary tract (p=0.086). There is no detected loss of 125I sar1ile8 angiotensin II binding at the AT2 prominent regions in the superior colliculus, medial geniculate nucleus, and the inferior olivary nucleus. The half lives of AT1 receptors are highly variable, ranging from 36 days in the anterior pituitary to 442 days in the nucleus of the solitary tract, and this might be related to variable stability of AT1A and AT1B receptors. These observations should be taken into account when assessing and comparing AT1 and AT2 receptor subtype densities.     

Corticotropin-releasing factor (CRF) receptors in intermediate lobe of the pituitary: biochemical characterization and autoradiographic localization

CRF receptors were characterized using radioligand binding and chemical affinity cross-linking techniques and localized using autoradiographic techniques in porcine, bovine and rat pituitaries. The binding of 125I-[Tyr0]-ovine CRF (125I-oCRF) to porcine anterior and neurointermediate lobe membranes was saturable and of high affinity with comparable KD values (200-600 pM) and receptor densities (100-200 fmoles/mg protein). The pharmacological rank order of potencies for various analogs and fragments of CRF in inhibiting 125I-oCRF binding in neurointermediate lobe was characteristic of the well-established CRF receptor in anterior pituitary. Furthermore, the binding of 125I-oCRF to both anterior and neurointermediate lobes of the pituitary was guanine nucleotide-sensitive. Affinity cross-linking studies revealed that the molecular weight of the CRF binding protein in rat intermediate lobe was identical to that in rat anterior lobe (Mr = 75,000). While the CRF binding protein in the anterior lobes of porcine and bovine pituitaries had identical molecular weights to CRF receptors in rat pituitary (Mr = 75,000), the molecular weight of the CRF binding protein in porcine and bovine intermediate lobe was slightly higher (Mr = 78,000). Pituitary autoradiograms from the three species showed specific binding sites for 125I-oCRF in anterior and intermediate lobes, with none being apparent in the posterior pituitary. The identification of CRF receptors in the intermediate lobe with comparable characteristics to those previously identified in the anterior pituitary substantiate further the physiological role of CRF in regulating intermediate lobe hormone secretion.     

Molecular characterization of D2 dopamine-like receptors from brain and from the pituitary gland.

D2 dopamine-like receptors have been purified from five bovine brain regions (caudate nucleus, putamen, olfactory tubercle, frontal cortex, cerebellum) and the anterior and neurointermediate lobes of the pituitary gland using a combined ligand-affinity and lectin-affinity chromatography procedure. In all the brain regions except cerebellum and in the neurointermediate lobe of the pituitary gland the purified species appeared as a M(r) 95,000 doublet on SDS-PAGE. In the anterior lobe of the pituitary an additional M(r) 142,000-145,000 species was seen. The M(r) 95,000 species had a low affinity for the lectin wheat germ agglutinin (WGA) whereas the M(r) 142,000-145,000 species had a higher affinity for WGA and additionally showed some affinity for concanavalin A. It is concluded that both the M(r) 95,000 and 142,000-145,000 species are D2 dopamine-like receptors and that the differences between the species are mainly at the oligosaccharide level. Some evidence was also obtained for heterogeneity at the protein level which may correspond to the D2(short) and D2(long) isoforms of these receptors.     

Physicochemical characterization of corticotrophin releasing factor receptor in rat pituitary and brain

The physicochemical characteristics of solubilized crosslinked CRF receptor-ligand complexes were studied in the anterior and intermediate pituitary lobes and brain of the rat. In all tissues studied, there was a major labeled band with a molecular weight of 72 +2- 3.5 kDa, (n = 15), 71 +/- 1.3 (n = 6), 73 +2- 2.5 (n = 7) and 75 +2- 3.5 (n = 7) kDa in the anterior and intermediate lobes of the pituitary, amygdala and cerebral cortex, respectively. The density of this band was inhibited by CRF analogs, but not by unrelated peptides. This is consistent with the comparable binding properties of CRF in membrane preparations of these tissues. These results suggest that differences in receptor regulation and the reported ability of CRF to stimulate cAMP is due to differences in ligand receptor processing and coupling to membrane transduction systems, rather than to major differences in the binding protein.     

Species Differences in the Brain Regional Distribution of Receptor Binding for Thyrotropin-Releasing Hormone

Abstract: A survey of the regional distribution of binding of 1 nM [³H](3-MeHis²)thyrotropin-releasing hormone ([³H]MeTRH) to TRH receptors in the brains of eight mammalian species revealed major species differences in both the absolute and relative values of TRH receptor binding in different brain regions. Several brain regions exhibited binding equal to or exceeding that in the anterior pituitary gland of the same species, including the amygdaia in the guinea pig and rat, the hypothalamus in the guinea pig, the nucleus accumbens in the rabbit, and all these and other regions in the cat and dog, for which pituitary binding was exceptionally low. Species could be divided into two groups according to which brain region appeared highest in binding: rabbits, sheep, and cattle had highest binding in the nucleus accumbens/septal area, whereas guinea pigs, rats, dogs, cats, and pigs had highest binding in the amygdala/temporal cortex area. The nucleus accumbens consistently exceeded the caudate-putamen in receptor binding. For most brain regions, rabbits, rodents, and sheep tended to be higher than carnivores, cattle, or pigs. Further regions that exhibited appreciable binding in most species included the olfactory bulb and tubercle, hippocampus, and various cortical and brain stem areas. In fact, essentially all brain regions appeared to have detectable levels of TRH receptors in at least some species, but no rat peripheral tissues have yet shown detectable receptor binding. The species differences appeared to reflect largely if not entirely differences in receptor density, although this was not tested in every species.     

Insulin-like growth factor (IGF)-I binding to a cell membrane associated IGF binding protein-3 acid-labile subunit complex in human anterior pituitary gland

The binding characteristics of [(125) I]insulin-like growth factor (IGF)-I were studied in human brain and pituitary gland. Competition binding studies with DES(1-3)IGF-I and R(3) -IGF-I, which display high affinity for the IGF-I receptor and low affinity for IGF binding proteins (IGFBPs), were performed to distinguish [(125) I]IGF-I binding to IGF-I receptors and IGFBPs. Specific [(125) I]IGF-I binding in brain regions and the posterior pituitary was completely displaced by DES(1-3)IGF-I and R(3) -IGF-I, indicating binding to IGF-I receptors. In contrast, [(125) I]IGF-I binding in the anterior pituitary was not displaced by DES(1-3)IGF-I and R(3) -IGF-I, suggesting binding to an IGF-binding site that is different from the IGF-I receptor. Binding affinity of IGF-I to this site was about 10-fold lower than for the IGF-I receptor. Using western immunoblotting we were also unable to detect IGF-I receptors in human anterior pituitary. Instead, western immunoblotting and immunoprecipitation experiments showed a 150-kDa IGFBP-3-acid labile subunit (ALS) complex in the anterior pituitary and not in the posterior pituitary and other brain regions. RT-PCR experiments showed the expression of ALS mRNA in human anterior pituitary indicating that the anterior pituitary synthesizes ALS. In the brain regions and posterior pituitary, IGFBP-3 was easily washed away during pre-incubation procedures as used in the [(125) I]IGF-I binding experiments. In contrast, the IGFBP-3 complex in the anterior pituitary could not be removed by these washing procedures. Our results indicate that the human anterior pituitary contains a not previously described tightly cell membrane-bound 150-kDa IGFBP-3-ALS complex that is absent in brain and posterior pituitary.     

Autoradiographic distribution of I-galanin binding sites in the rat central nervous system

Galanin (GAL) binding sites in coronal sections of the rat brain were demonstrated using autoradiographic methods. Scatchard analysis of ¹²⁵I-GAL binding to slide-mounted tissue sections revealed saturable binding to a single class of receptors with a Kd of approximately 0.2 nM. ¹²⁵I-GAL binding sites were demonstrated throughout the rat central nervous system. Dense binding was observed in the following areas: prefrontal cortex, the anterior nuclei of the olfactory bulb, several nuclei of the amygdaloid complex, the dorsal septal area, dorsal bed nucleus of the stria terminalis, the ventral pallidum, the internal medullary laminae of the thalamus, medial pretectal nucleus, nucleus of the medial optic tract, borderline area of the caudal spinal trigeminal nucleus adjacent to the spinal trigeminal tract, the substantia gelatinosa and the superficial layers of the dorsal spinal cord. Moderate binding was observed in the piriform, periamygdaloid, entorhinal, insular cortex and the subiculum, the nucleus accumbens, medial forebrain bundle, anterior hypothalamic, ventromedial, dorsal premamillary, lateral and periventricular thalamic nuclei, the subzona incerta, Forel's field H₁ and H₂, periventricular gray matter, medial and superficial gray strata of the superior colliculus, dorsal parts of the central gray, peripeduncular area, the interpeduncular nucleus, substantia nigra zona compacta, ventral tegmental area, the dorsal and ventral parabrachial and parvocellular reticular nuclei. The preponderance of GAL-binding in somatosensory as well as in limbic areas suggests a possible involvement of GAL in a variety of brain functions.     

Thyrotropin-Releasing Hormone Receptor Binding Sites: Autoradiographic Distribution in the Rat and Guinea Pig Brain

Thyrotropin-releasing hormone (TRH) binding sites were labeled in vitro in mounted brain tissue sections from rat and guinea pig brains with [3H]methyl TRH and localized autoradiographically using 3H-sensitive film. Regional densities of TRH binding sites were measured by computer-assisted microdensitometry. The distribution of sites in both species was highly heterogeneous. In both guinea pig and rat brains, the highest densities of binding sites were seen in the amygdaloid nuclei and the perirhinal cortex. In contrast, in other brain areas, a clear difference between the distribution of sites in rat and guinea pig was found. The temporal cortex, pontine nuclei, and interpeduncular nucleus, which contained high densities of binding in the guinea pig, were scarcely labeled in the rat. The accessory olfactory bulb and the septohippocampal area presented in the rat higher concentrations of binding sites than in the guinea pig. Other brain areas showing intermediate to low densities in both species were accumbens nucleus, bed nucleus of the stria terminalis, dentate gyrus, facial and hypoglossal nuclei, and gelatinosus subnucleus of the trigeminal nerve, among others. The anterior pituitary also presented low to intermediate concentrations of receptors. The distribution of TRH sites here described does not completely correlate with that of endogenous TRH, but is in good agreement with previous biochemical data. The results are discussed in correlation to the physiological effects that appear to be mediated by TRH.     

Selective effect of castration on the anterior pituitary VIP receptor of male rats

We investigated the effect of surgical castration of male rats on the binding of [Tyr(¹²⁵I)¹⁰]VIP to receptors on the anterior pituitary gland, superior mesenteric artery, brain, liver, and prostate gland. In anterior pituitary membranes the maximum number of VIP binding sites was increased whereas binding affinity was decreased 24 hours following castration. In particular, the high affinity equilibrium dissociation constant (K_D) increased from 0.13±0.02 nM (mean±SEM) to 0.67±0.07 nM and the maximum number of high affinity binding sites (B_max) increased from 71±9 to 470±112 fmol/mg protein. No significant change was observed in the other tissues. Anesthesia or sham operation did not alter the anterior pituitary VIP receptor binding parameters. The changes in the VIP receptor 24 hours after castration were prevented by prior injection of testosterone. These findings demonstrate tissue-selective alterations to the anterior pituitary VIP receptor by castration that are likely mediated by withdrawal of testosterone.     

Exposure to an Antisense Oligonucleotide Decreases Corticotropin-Releasing Factor Receptor Binding in Rat Pituitary Cultures

Abstract: Corticotropin-releasing factor (CRF) appears to integrate the endocrine, autonomic, immunologic, and behavioral responses of mammals to stress. To investigate further the role of CRF in the CNS, we have begun investigating the usefulness of "antisense knockdown" strategies directed against the CRF receptor using rat anterior pituitary gland primary cell cultures. The 15-mer antisense (5' CTG-CGG-GCG-CCG-TCC 3') and "scrambled" control (5' CGT-CCG-CGC-GCT-GCG 3') oligonucleotides were synthesized based on the rat CRF receptor sequence just downstream of the initiation codon. In each of four separate experiments, exposure to 10 µmol/L of antisense oligonucleotide for 40–67 h resulted in significant (17–36%) decreases in ¹²⁵I-ovine CRF binding to pituitary cells as compared with either control (no oligonucleotide) or 10 µmol/L of "scrambled" oligonucleotide. Moreover, compared with scrambled oligonucleotide, exposure to 10 µmol/L of antisense oligonucleotide, which produced a 22% decrease in CRF receptor binding, also resulted in a significant attenuation of the adrenocorticotrophic hormone response following a 30-min challenge with 100 pmol/L of CRF. Thus, CRF receptor antisense oligonucleotides apparently reduce functional expression of CRF receptors. This technique may be useful in studying the kinetics of CRF receptor production and the physiological functions of CRF receptors within the CNS.     

Lectin target cells in human central nervous system and the pituitary gland

Summary Peanut lectin (PNL), Concanavalin A (Con A) and Ulex europaeus lectin I (Ulex) were chosen to map their binding sites in different regions of formalin fixed and paraffin embedded human central nervous system tissue and pituitary gland tissues. An extended PaP method was used for PNL and Ulex, whereas a direct peroxidase technique was employed for Con A. In astrocytes, the cytoplasm as well as the delicate processes were stained by PNL and Con A; the most conspicious binding of PNL was seen in the ependymal cells and on the surface of plexus epithelial cells; in the anterior part of the pituitary gland a selective population was PNL positive. Intracytoplasmic Con A acceptors could be demonstrated in neurons, in ependymal cells, and in plexus epithelial cells. Intracytoplasmic Con A receptors were finely granular in astrocytes, oligodendrocytes, and in some cells in the pituitary gland. Ulex binding was restricted to the vascular endothelial cells and a selective population of cells in the pituitary gland. Our results suggest that lectins may be good tools for the evaluation of their respective target cells in the central nervous system and in the pituitary gland.     

Lectin target cells in human central nervous system and the pituitary gland

Peanut lectin (PNL), Concanavalin A (Con A) and Ulex europaeus lectin I (Ulex) were chosen to map their binding sites in different regions of formalin fixed and paraffin embedded human central nervous system tissue and pituitary gland tissues. An extended PaP method was used for PNL and Ulex, whereas a direct peroxidase technique was employed for Con A. In astrocytes, the cytoplasm as well as the delicate processes were stained by PNL and Con A; the most conspicuous binding of PNL was seen in the ependymal cells and on the surface of plexus epithelial cells; in the anterior part of the pituitary gland a selective population was PNL positive. Intracytoplasmic Con A acceptors could be demonstrated in neurons, in ependymal cells, and in plexus epithelial cells. Intracytoplasmic Con A receptors were finely granular in astrocytes, oligodendrocytes, and in some cells in the pituitary gland. Ulex binding was restricted to the vascular endothelial cells and a selective population of cells in the pituitary gland. Our results suggest that lectins may be good tools for the evaluation of their respective target cells in the central nervous system and in the pituitary gland.     

Autoradiographic distribution of 125I calcitonin gene-related peptide binding sites in the rat central nervous system

Using autoradiographic method and 125I-Tyro rat CGRP as a ligand, receptor binding sites were demonstrated in the rat central nervous system. Saturation studies and Scatchard analysis of CGRP-binding to slide mounted tissue sections containing primarily cerebellum showed a single class of receptors with a dissociation constant of 0.96 nM and a Bmax of 76.4 fmol/mg protein. 125I-Tyro rat CGRP binding sites were demonstrated throughout the rat central nervous system. Dense binding was observed in the telencephalon (medial prefrontal, insular and outer layers of the temporal cortex, nucleus accumbens, fundus striatum, central and inferior lateral amygdaloid nuclei, most caudal caudate putamen, organum vasculosum laminae terminalis, subfornical organ), the diencephalon (anterior hypothalamic, suprachiasmatic, arcuate, paraventricular, dorsomedial, periventricular, reuniens, rhomboid, lateral thalamic pretectalis and habenula nuclei, zona incerta), in the mesencephalon (superficial layers of the superior colliculus, central nucleus of the geniculate body, inferior colliculus, nucleus of the fifth nerve, locus coeruleus, nucleus of the mesencephalic tract, the dorsal tegmental nucleus, superior olive), in the molecular layer of the cerebellum, in the medulla oblongata (inferior olive, nucleus tractus solitarii, nucleus commissuralis, nuclei of the tenth and twelfth nerves, the prepositus hypoglossal and the gracilis nuclei, dorsomedial part of the spinal trigeminal tract), in the dorsal gray matter of the spinal cord (laminae I-VI) and the confines of the central canal. Moderate receptor densities were found in the septal area, the "head" of the anterior caudate nucleus, medial amygdaloid and bed nucleus of the stria terminalis, the pyramidal layers of the hippocampus and dentate gyri, medial preoptic area, ventromedial nucleus, lateral hypothalamic and ventrolateral thalamic area, central gray, reticular part of the substantia nigra, parvocellular reticular nucleus. Purkinje cell layer of the cerebellum, nucleus of the spinal trigeminal tract and gracile fasciculus of the spinal cord. The discrete distribution of CGRP-like binding sites in a variety of sensory systems of the brain and spinal cord as well as in thalamic and hypothalamic areas suggests a widespread involvement of CGRP in a variety of brain functions.     

Characterization of tachykinin NK2 receptor in the anterior pituitary gland

Tachykinins are a family of bioactive peptides that interact with three subtypes of receptors: NK1, NK2 and NK3. Substance P has greater affinity for NK1, and neurokinin A (NKA) for NK2 receptor subtype. Although only NK1 receptor has been characterized in the anterior pituitary gland, some evidence suggests the existence of NK2 receptors in this gland. Therefore, we investigated the presence of NK2 receptors in the anterior pituitary gland of male rats by radioligand binding studies using labeled SR48968, a non peptidic specific antagonist. [3H]SR48968 specific binding to cultured anterior pituitary cells was time-dependent and saturable, but with a lower affinity than previously reported values for cells expressing NK2 receptors. Unlabeled NKA inhibited only partially [(3)H]SR48968 specific binding to whole anterior pituitary cells. Since SR48968 is a non polar molecule, we performed experiments to discriminate surface from intracellular binding sites. SR48968 exhibited both surface and intracellular specific binding. Analysis of the surface-bound ligand indicated that [3H]SR48968 binds to one class of receptor with high affinity. Neurokinin A completely displaced [3H]SR48968 surface specific binding fitting to a two-site/two-state model with high and low affinity. Additionally, immunocytochemical studies showed that the NK2 receptor is expressed at least in a subset of lactotropes. These results demonstrate the presence of NK2 receptors in the anterior pituitary gland and suggest that NKA actions in this gland are mediated, at least in part, by the NK2 receptor subtype.     

Atrial natriuretic peptide binding sites in the brain and pituitary gland of the toad, Bufo marinus: localisation and receptor characterisation

The distribution and nature of 125I-atrial natriuretic peptide binding sites have been examined in the brain and pituitary gland of the toad, Bufo marinus, using tissue section autoradiography, affinity cross-linking and electrophoresis, guanylyl cyclase assays and molecular analysis of natriuretic peptide receptor C (NPR-C) and NPR-GC mRNA expression. The highest density of 125I-atrial natriuretic peptide binding sites occurred in the dorsal pallium, the habenular region, the torus semicircularis, the choroid plexus, and the pituitary gland. Less dense binding was observed in the medial pallium, the thalamic region, the hypothalamus, the optic tectum, and the interpeduncular nucleus. The natriuretic peptide receptor-C specific ligand, C-ANF, displaced the binding in all brain regions; however, some residual binding was observed in the habenular region, the hypothalamus, the choroid plexus, and the pituitary gland. In isolated brain membranes, 1 microM rat atrial natriuretic peptide increased cyclic guanosine monophosphate levels to 90% above basal. Affinity cross-linking followed by reducing electrophoresis showed that 125I-atrial natriuretic peptide bound to proteins of 65 kDa and 135 kDa respectively. Furthermore, molecular analysis demonstrated that natriuretic peptide receptor-C and guanylyl cyclase messenger ribonucleic acid are expressed in the brain. In combination with the autoradiography, the data indicated that atrial natriuretic peptide acting via specific receptors could be important in natriuretic peptide regulation of the brain.     

Endocytosis of the vasopressin receptor by anterior pituitary cells is increased by corticotropin-releasing factor (CRF)

Endocytosis of certain receptors such as the transferrin receptor and the EGF-receptor appears to be influenced by second messengers. If second messengers are involved in modulation of endocytosis, not only endocytosis of the stimulated receptor itself but also of receptors for other ligands on the cell surface may be influenced by receptor occupancy. Corticotropin-releasing factor (CRF) and vasopressin act synergistically on secretion of ACTH from the anterior pituitary. The results presented here demonstrate that CRF increases retrieval of the vasopressin receptor in anterior pituitary cells in primary culture without influencing the surface binding of vasopressin. This is not a function of an increased membrane turnover since endocytosis of the transferrin receptor is not influenced by CRF.