Characterization of corticotropin-releasing factor receptor-mediated adenylate cyclase activity in the rat central nervous system

We report here that corticotropin-releasing factor (CRF) stimulates adenylate cyclase activity in the rat central nervous system (CNS). In frontoparietal cortex homogenates, the stimulation by CRF was dependent on time, temperature, tissue protein concentration, and guanine nucleotides. The rank order of potency for CRF analogs and fragments in stimulating adenylate cyclase activity [(Nle21,38) rat CRF greater than rat CRF approximately equal to acetyl ovine CRF (4-41) approximately equal to alpha helical ovine CRF greater than ovine CRF much greater than ovine CRF (1-39) approximately equal to ovine CRF (7-41)] was consistent with their affinities for CRF receptors in the brain and their relative potencies in stimulating pituitary adrenocorticotropic hormone secretion in vitro. The putative CRF receptor antagonist, alpha helical ovine CRF (9-41), did not stimulate adenosine 3',5'-cyclic monophosphate (cAMP) production but was able to attenuate the stimulation by various concentrations of rat CRF. The regional distribution of 125I-Tyr(o)-ovine CRF binding (olfactory bulb greater than frontoparietal cortex approximately equal to cerebellum greater than hypothalamus greater than striatum greater than or equal to midbrain greater than hippocampus greater than or equal to spinal cord) did not correspond with the regional degree of CRF receptor-mediated stimulation of adenylate cyclase (frontoparietal cortex greater than olfactory bulb greater than or equal to cerebellum greater than midbrain greater than or equal to hippocampus greater than striatum greater than or equal to hypothalamus greater than spinal cord). In addition, marked differences were observed in the ability of forskolin to potentiate CRF-stimulated cAMP production in the various brain areas examined. In summary, these data demonstrate that at least one of the second-messenger systems mediating the effects of CRF in the CNS involves stimulation of cAMP production and provides further support for a neurotransmitter role for this neuropeptide in the brain. Significant differences in the regulation of CRF-stimulated cAMP production and the disparity between CRF receptor number and receptor-mediated adenylate cyclase activity in discrete regions of the rat CNS suggest that some populations of CRF receptors in the brain may be functionally coupled to alternative signal transduction mechanisms.     

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 EC₅₀ 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.     

Postnatal development of regional binding of corticotropin-releasing factor and adenylate cyclase activity in the rat brain.

1. Corticotropin-releasing factor (CRF) plays a major role in the endocrine, autonomic and behavioral responses to stress. The distribution of CRF and CRF receptors in hypothalamic and extra-hypothalamic brain regions is consistent with its stress-related functions. 2. In most brain regions, CRF acts primarily, if not exclusively, through activation of the adenylate cyclase systems. 3. While previous studies have demonstrated the prenatal presence of CRF receptors, in the early postnatal period the abundance of CRF receptors relative to the magnitude of CRF-stimulated cAMP production suggests that CRF receptors are not fully linked to adenylate cyclase. 4. Because of our interest in the possible involvement of CRF signal transduction in the development of the neonatal stress response, we have examined postnatal development of CRF receptors in relation to adenylate cyclase activity in the rat. 5. CRF binding decreased significantly in the hippocampus and striatum from postnatal days 7-21. Basal adenylate cyclase activity peaked in the second-third week of postnatal life in each brain region. Preliminary studies suggest that early stress can alter the maturation of second messenger systems in the frontal cortex.     

The role of adenylate cyclase in relaxation of brain arteries: studies with forskolin

The natural product, forskolin, which stimulates adenylate cyclase by a direct, non-receptor-mediated mechanism, was studied for its effect on the tension of isolated brain arteries and adenylate cyclase activity of cerebral arteries. Helical strips of bovine and porcine basilar arteries and bovine middle cerebral arteries, which had been precontracted with prostaglandin F2 alpha (PGF2 alpha) or KCl, relaxed potently to administration of forskolin with ED50 values, ranging from 22 to 69 nM. Incubation of forskolin with a broken cell preparation of bovine cerebral arteries resulted in an efficacious stimulation of adenylate cyclase, approximating 5 times basal activity at a forskolin concentration of 1 microM. The metal salts nickel chloride and manganese chloride decreased the potency of vasorelaxation by vasoactive intestinal peptide (VIP), which stimulates adenylate cyclase via the VIP receptor. In contrast, nickel chloride had little effect on vasorelaxation by forskolin. The endogenous nucleoside, adenosine, which acts via the adenosine receptor and adenylate cyclase, relaxed bovine basilar and middle cerebral arteries with ED50 values ranging from 0.26 to 0.94 microM. The data presented support a role for adenylate cyclase in mediating vasodilation of brain blood vessels.     

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.     

Characterization of corticotropin-releasing factor receptors in dissociated brain cell cultures

Although corticotropin-releasing factor (CRF) receptors have been identified throughout the brain, relatively little is known about the regulation of CRF receptors. Recent investigations aimed at developing an in vitro model for studying the regulation of CRF receptors demonstrated CRF binding in brain cell cultures. To test the hypothesis that dissociated brain cell cultures contain CRF receptors and may provide a model for studying their regulation, studies characterizing binding of labeled CRF were performed. Dissociated cells derived from hypothalamus and extrahypothalamic forebrain (predominantly cortex) of day 17 fetal rats were maintained in chemically defined medium. We used a stable 125I-labeled analog of ovine CRF, 125I-Tyro-ovine CRF (125I-oCRF), to identify and characterize CRF receptors. Although specific binding of 125I-oCRF was demonstrated in both hypothalamic and extrahypothalamic cell cultures, the concentration of CRF receptors was much greater (3-5 fold) in extrahypothalamic cells. Binding of 125I-oCRF in extrahypothalamic cells was saturable and was composed of high affinity (Kd = 0.51 nM) and low affinity (Kd = 17.25 nM) sites. Pharmacological displacement of labeled CRF from cells with a variety of CRF fragments and analogs was similar to that in studies of pituitary and brain homogenates. Extrahypothalamic cells studied at several times between 4 and 13 days in culture revealed an increase in the number of CRF receptors; the concentration of CRF receptors at 13 days was 3.5 times that observed at 4 days. Studies directed toward determining whether CRF receptor concentration could be modulated by CRF, adrenocorticotropic hormone, atropine or a CRF antagonist showed a change (36% decrease) only in response to chronic exposure with CRF. Conclusions: (1) dissociated fetal rat brain cell cultures derived from extrahypothalamic forebrain and hypothalamus contain CRF receptors; (2) CRF receptors in brain cells exhibit a differential distribution and characteristics similar to those previously reported in brain and pituitary; (3) dissociated fetal rat brain cell cultures may provide a relatively simplified in vitro model for studying the regulation of CRF receptors; and (4) CRF down-regulates its own receptor in extrahypothalamic forebrain cells.     

The effect of capsaicin on the adenylate cyclase activity of rat brain.

The effect of capsaicin on the adenylate cyclase activity in different regions of the rat brain (preoptic area of the hypothalamus, cerebral cortex and cerebellum) was investigated. Capsaicin added in vitro (10(-7)-10(-5) M) increased the adenylate cyclase activity of different brain regions. Following systemic capsaicin desensitization adenylate cyclase activity was significantly increased in the preoptic area. The enhanced adenylate cyclase activity in the preoptic area was inhibited by the vitro addition of capsaicin or 5-HT, whereas desensitization did not affect the in vitro activating effect of capsaicin in other brain regions (cerebral cortex, cerebellum). It is assumed that the pharmacological effect of capsaicin in the preoptic area is mediated through the activation of adenylate cyclase. Since capsaicin induces irreversible impairment of the function of warmsensitive hypothalamic neurons it is assumed that adenylate cyclase is involved in maintaining normal thermoregulatory functions.     

Colorimetric assay for rapid screening of corticotropin releasing factor receptor ligands

The corticotropin releasing factor (CRF) receptor is known to be coupled to G_s and transduces its signal through stimulation of cyclic AMP (cAMP) production. Here we describe the characterization of several stable CRF receptor-expressing LVIP2.0Zc cell lines that also contain an exogenous cAMP-responsive β-galactosidase reporter gene construct. The CRF receptor activity was assayed by measuring the induction of β-galactosidase in response to CRF. Rat/human and bovine CRF stimulated β-galactosidase activity in a dose-dependent manner with EC₅₀ values of ∼0.1 nM; the biologically weak deamidated analog of bovine CRF was ∼500-fold less potent. The CRF receptor antagonist, [d-Phe¹²,Nle^21,38,Ala³²r/hCRF(12–41) produced a dose-dependent inhibition of CRF-stimulated β-galactosidase activity, further demonstrating the pharmacological specificity of the interaction. The magnitude of the maximal response to CRF varied among individual cell lines. This variation was independent of the level of CRF receptor expression, but reflected differences in the intrinsic activity of adenylate cyclase. In contrast to most cAMP assay systems, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine decreased the CRF-induced β-galactosidase activity when used in the context of the assay regimen described here. Since the assay can be easily performed in a high-throughput 96-well plate format, these cell lines provide an efficient way for the identification of CRF receptor agonists and antagonists.     

The ontogeny of brain receptors for corticotropin-releasing factor and the development of their functional association with adenylate cyclase

This study reports the ontogeny of corticotropin-releasing factor (CRF) receptor binding sites in rat brain, using both membrane binding assays and in vitro receptor autoradiography. CRF binding sites are evident by prenatal day 17, increase to 312% of their adult density by postnatal day 8, then decrease to reach adult values by day 21. Not only the density, but the distribution of CRF binding undergoes major modifications in development. CRF binding sites are most numerous in striatum prenatally, but postnatally, binding is more dense in the cortex, reaching the adult laminar distribution by postnatal day 14. Brain CRF receptors are linked to adenylate cyclase early in postnatal life. This contrasts with the later appearance of most of the guanine nucleotide stimulatory protein and catalytic subunit sites in the rat brain and suggests that CRF receptors may become functional earlier than several other brain receptors that are linked to adenylate cyclase.     

Homologous desensitization of human corticotropin-releasing factor, receptor in stable transfected mouse fibroblast cells

Previous radioligand binding and second messenger studies have shown that corticotropin-releasing factor (CRF) modulates its receptor following both in vivo and in vitro treatment. In the present study, we determined the sequence of events leading to CRF-induced downregulation and desensitization of cloned CRF receptors in murine fibroblast cells (Ltk⁻) stably transfected with CRF, DNA (from human pituitary). Treatment of cells with rat/human CRF produced a dose- and time-dependent decrease in [¹²⁵I]Tyr^o-ovine CRF ([¹²⁵I]oCRF) binding and a concomitant decrease in CRF-stimulated adenylate cyclase activity. Significant decreases in [¹²⁵I]oCRF binding and agonist-stimulated cAMP production were evident minutes after CRF treatment with maximal (60–80%) reductions seen following 1 h of CRF treatment. Scatchard analysis revealed that the decrease in [¹²⁵I]oCRF binding was due to the downregulation of the receptor with no significant alteration seen in the affinity of the ligand. Since the transfected cell line is engineered using an artificial promoter, we did not detect any significant changes in CRF₁ receptor mRNA levels following CRF treatment for up to 24 h.     

Adenylate cyclase in striatal cholinergic interneurons regulates acetylcholine release

Fractional [³H]ACH efflux from dissociated rat striata tested whether tonic inhibition prevents stimulation of acetylcholine (ACH) release by adenylate cyclase. Forskolin stimulated release from the dissociated cells (threshold at 300 nM; EC₅₀ ≥ 1 μM). Release was also stimulated by 3-isobutyl-l-methylxanthine and was additive with forskolin. The 1,9-dideoxy forskolin analog that lacks cyclase-stimulating activity was ineffective. Thus, stimulation of adenylate cyclase within striatal cholinergic interneurons increases ACH secretion but is tonically inhibited by endogenous striatal transmitters. Disinhibition of the excitatory cyclase by denervation of striatal cholinergic interneurons in situ could contribute to supersensitivity without receptor upregulation.     

Corticotropin-releasing factor (CRF)--a review

Corticotropin-releasing factor (CRF), a 41 amino acid polypeptide, has been isolated from ovine hypothalamic extracts, sequenced, and synthesized. It has a high potency for stimulating the secretion of corticotropin-like and beta-endorphin-like immunoactive substances in vitro and in vivo in laboratory animals and humans. The high concentration of CRF-like immunoactivity in hypophyseal portal plasma supports the hypothesis that CRF is the physiological hypothalamic factor. Human and rat CRF (rCRF) also have been purified and synthesized. They have an 83% sequence homology with ovine CRF (oCRF). oCRF-like activity has been found in human hypothalamus, pituitary stalk, posterior pituitary, thalamus, cerebral cortex, cerebellum, pons, medulla oblongata, spinal cord and in the adrenal, lung, liver, stomach, duodenum and pancreas. oCRF-like activity also has been found in the human placenta and in tissues producing ectopic ACTH. The action of CRF can be potentiated by vasopressin, oxytocin, epinephrine, norepinephrine, VIP, and angiotensin II. Intracerebroventricular administration of CRF in the rat produces prolonged elevations of plasma epinephrine, norepinephrine, glucose and glucagon; elevates mean arterial pressure and heart rate; increases motor activity and exploration in familiar surroundings and oxygen consumption; and decreases feeding and sexual behavior. Testing with CRF has enabled the separation of patients with hypothalamic and pituitary adrenal insufficiency. The CRF stimulation test has been useful in distinguishing pituitary from ectopic causes of Cushing's disease. The distribution of CRF within and beyond the hypothalamus provides an anatomical context for the observation that CRF can simultaneously activate and coordinate metabolic, circulatory and behavioral responses that are adaptative in 'stressful' situations. CRF not only stimulates the pituitary-adrenal axis in man, but it also influences several aspects of CNS function which may be of relevance to psychiatric illnesses.     

Agaridoxin: A fungal catecholamine which acts as an alpha 1 agonist of mammalian hypothalamic adenylate cyclase

Agaridoxin, a catecholamine isolated from mushrooms, and 4 synthetic analogues cause activation of adenylate cyclase in the presence of guanylyl imidodiphosphate (Gpp(NH)p) in membrane particles prepared from rat hypothalamus. These compounds also activate adenylate cyclase preparations from rat kidney, liver and cerebral cortex. In the presence of tyrosinase, these compounds are readily oxidized to quinones which lack agonist activity. Studies with selective adrenergic blockers suggest that agaridoxin acts at an alpha 1-type receptor. Agaridoxin-mediated adenylate cyclase stimulation is most effectively antagonized by WB-4101 and phenoxybenzamine, while propranolol and yohimbine are without inhibitory effect. Agaridoxin and the alpha 1 agonist methoxamine inhibited the binding of [3H]WB-4101 in rat hypothalamic and cerebral cortical membranes. The values of Ki for both compounds are lower than that of norepinephrine. The agaridoxin analogue, 4-aminocatechol hydrochloride, is a more effective and potent adenylate cyclase activator than agaridoxin or methoxamine.     

Postnatal development of 5-HT1 receptors: [3H]5-HT binding sites and 5-HT induced adenylate cyclase activations in rat brain cortex

The postnatal development of the 5-HT1 receptor system was studied in young rat brain cortex from birth to adulthood (14 successive ages). The high-affinity binding of [3H]5-HT was low at birth but developed markedly between the 8th and the 15th day postnatally. The basal adenylate cyclase activity produced 50 pmoles cAMP/mg protein/min at birth and increased from the 8th to the 15th day. 5-HT could stimulate the adenylate cyclase activity in adult rat brain cortex with two different affinity constants: Km = 1 nM and Km = 0.5 microM; these low- and high-affinity constants presumably correspond to 5-HT1A and 5-HT1non-A.non-B.non-C (5-HT1D) respectively. These two activities developed parallelly from the 14-15th to the 28th day. The 8-hydroxy-2-(di-n-propylamino-tetralin) (8-OH-DPAT)-induced activity described a curve similar to the one that corresponded to 10 microM 5-HT. These results establish that 5-HT1A and 5-HT1non-A.non-B.non-C receptors mainly develop during the synaptogenesis.     

Corticotropin-releasing factor receptors in the rabbit brain visualized by in vitro autoradiography

Corticotropin-releasing factor (CRF) binding sites were visualized in the rabbit brain by in vitro autoradiography using the radioligand 125I-[Tyr0]ovine CRF. The radioligand binding to sections of rabbit cingulate cortex were competed for by ovine and rat CRF with inhibitory constants (Ki) of 26 and 37 nM, respectively, whereas sauvagine and alpha-helical CRF9-41 were approximately 10-fold less potent. In the rabbit brain, the highest densities of binding sites for CRF are found in the pineal gland and the choroid plexus. The cerebral cortex is labelled throughout, with the highest concentration of binding sites in the piriform and primary olfactory divisions. In the cerebellar cortex, the granular layer is more intensely labelled than the molecular layer. The distribution of CRF binding sites in the hippocampus follows a laminar pattern; the molecular layer of the dentate gyrus is intensely labelled, the oriens, radiatum and lacunosum moleculare layers of Ammon's horn contain moderate densities of binding and no binding is observed in the granular layer of the dentate gyrus and the pyramidal cell layer. The ventral subnucleus of the lateral septum, the zonal and superficial layers of the superior colliculus contain high densities of receptors. A moderate concentration of binding sites is observed in the caudate nucleus, putamen, bed nucleus of the stria terminalis, paraventricular, anterodorsal and anteroventral thalamic nuclei and the medial nucleus of the mammillary body.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of PGE2 inhibition of corticotropin releasing factor-mediated ACTH release

The role of prostaglandin E2 (PGE2) on the mechanism of corticotropin releasing factor (CRF) induced adrenocorticotropin (ACTH) release was studied in primary rat pituitary cell culture. The continuous incubation of pituitary cells with PGE2 inhibited CRF-stimulated ACTH with an ED50 of 1.2 X 10(-9) M PGE2. PGE2, however, did not alter the spontaneous release of ACTH. PGE (10(-8) M) significantly decreased 10(-10) M, 10(-9) M, and 10(-8) M CRF-mediated ACTH release by 42%, 47%, and 31% of total CRF stimulated ACTH release. Time course experiments demonstrated a PGE2-induced inhibition by 20 min of CRF incubation which continued for 3 h. After a 2-h incubation with PGE2, the wash-out of PGE2 from the culture medium just prior to the addition of CRF eliminated the inhibitory activity of PGE2. PGE2 decreased the amount of CRF-stimulated ACTH from cells incubated with cycloheximide (P less than 0.01). The inhibitory activity of PGE2 (10(-8) M) on CRF-stimulated ACTH was unaltered by the addition of 3 mM or 7 mM CaCl2 to the standard culture media (1.6 mM CaCl2). The inhibition of CRF-induced ACTH release by maximal inhibitory concentrations of PGE2 (10(-7) M) and cortisol (5 X 10(-7) M) were not additive. In conclusion, PGE2 may play an important role in modulating pituitary ACTH release. Its inhibitory activity occurs by 20 min of CRF incubation, is in part independent of protein synthesis, requires the continued presence of PGE2, is not reversed with CaCl2, and is not additive with the inhibitory activity of cortisol.     

Guanosine-5'-O-thiodiphosphate functions as a partial agonist for the receptor-independent stimulation of neural adenylate cyclase

GTP-binding proteins (G proteins) have been implicated as mediators of several aspects of neuronal signal transduction including ion channels, phosphatidyl inositol turnover and the stimulation or inhibition of adenylate cyclase. Several investigators have employed the stable guanosine diphosphate (GDP) analog, guanosine 5'-O-thiodiphosphate (GDP beta S) to block putative G protein-mediated processes. Although GDP beta S is assumed to block G protein function, some investigators have reported partial activation of G protein-mediated processes by this compound. In this study we demonstrate that GDP beta S functions as a partial agonist for the adenylate cyclase system. In rat cerebral cortex membranes, GDP beta S activates adenylate cyclase with an EC50 similar to the hydrolysis resistant GTP analog, guanylylimidodiphosphate (GppNHp), but to a far lower extent. Further, GDP beta S antagonizes the activation of adenylate cyclase by high doses of GppNHp or GTP gamma S (another stable GTP analog) but potentiates adenylate cyclase activation by low doses of these nucleotides. High doses of GDP beta S provoke, only partially, exchange of nucleotides among G proteins, as measured by the transfer of the photoaffinity GTP analog, azidoanilido-GTP, between the inhibitory and stimulatory GTP-binding proteins. In the presence of the beta-adrenergic agonist, isoproterenol, GDP beta S fails to support stimulation of C6 glioma membrane adenylate cyclase and inhibits GppNHp- or GTP gamma S-mediated stimulation of that enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

The resolution of dopamine and beta 1- and beta 2-adrenergic-sensitive adenylate cyclase activities in homogenates of cat cerebellum, hippocampus and cerebral cortex.

The stimulation of adenylate cyclase by dopamine and various β-adrenergic agonists has been investigated in homogenates from 3 areas of cat brain: the cerebral cortex, cerebellum and hippocampus. The purpose of the study was to determine whether the β-adrenergic receptors coupled to adenylate cyclase could be classified as either β₁ and β₂ subtypes in the different regions studied.

The stimulation of adenylate cyclase by the β-adrenergic agonist, (−)isoproterenol (5 × 10⁻⁶M), was completely blocked by the specific β-adrenergic antagonist, (−)alprenolol (10⁻⁵ M), but not by the dopaminergic antagonist, fluphenazine (10⁻⁵ M), whereas the stimulation of adenylate cyclase by (−)epinephrine (10⁻⁴ M) was blocked to varying extents by these two drugs in each of the 3 regions studied. The (−)epinephrine effect was always blocked in the combined presence of (−)alprenolol and fluphenazine. The adenylate cyclase stimulation by (−)epinephrine which is not blocked by (−)alprenolol was due to interaction of (−)epinephrine with a dopaminergic-sensitive adenylate cyclase which has been characterized in cerebral cortex, hippocampus and cerebellum.

Regional differences in the affinity of β-adrenergic-sensitive adenylate cyclase for various agonists were investigated in the presence of fluphenazine (10⁻⁵ M). In the cerebellum the potency order was (±)protokylol> (±)hydroxybenzylisoproterenol> (±)isoproterenol> (−)epinephrine> (±)salbutamol> (−)norepinephrine, indicating the presence of a β₂-adrenergic receptor. In the cerebral cortex the potency order was (−)isoproterenol> (±)protokylol> (±)hydroxybenzylisoproterenol> (−)epinephrine= (−)norepinephrine((±)salbutamol being inactive). A similar pattern was found in the hippocampus indicating the presence of a β₁-adrenergic receptor in these two regions. (±)Salbutamol was a partial agonist in the cerebellum and a competitive antagonist in the cerebral cortex.

The ratio of the antagonist potencies of (±)practolol and (±)butoxamine preferential β₁- and β₂-adrenergic antagonists respectively, to block the stimulation of adenylate cyclase was 25 in the cerebellum, compared to 0.5 in the cerebral cortex and 1.6 in the hippocampus. These results confirm the presence of a β₂ subtype of receptor coupled to adenylate cyclase in the former and β₁ subtypes in the latter two regions. The comparison between the affinities of a series of β-adrenergic agonists and antagonists for the β-adrenergic receptors coupled with an adenylate cyclase in cerebral cortex and cerebellum with their affinities for well characterized β₂-adrenergic receptors in lung and β₁-adrenergic receptor in heart substantiated this conclusion.     

Corticotropin-releasing factor receptors: Physiology, pharmacology, biochemistry and role in central nervous system and immune disorders

Corticotropin-releasing factor (CRF) plays a major role in coordinating the endocrine, autonomic, behavioral and immune responses to stress through actions in the brain and the periphery. CRF receptors identified in brain, pituitary and spleen have comparable kinetic and pharmacological characteristics, guanine nucleotide sensitivity and adenylate cyclase-stimulating activity. Differences were observed in the molecular mass of the CRF receptor complex between the brain (58,000 Da) and the pituitary and spleen (75,000 Da), which appeared to be due to differential glycosylation of the receptor proteins. The recently cloned CRF receptor in the pituitary and the brain (designated as CRF,) encodes a 415 amino acid protein comprising seven putative membrane-spanning domains and is structurally related to the calcitonin/vasoactive intestinal peptide/growth hormone-releasing hormone subfamily of G-protein-coupled receptors. A second member of the CRF receptor family encoding a 411 amino acid rat brain protein with ≈70% homology to CRF, has recently been identified designated as CRF₂); there exists an additional splice variant of the CRF₂ receptor with a different N-terminal domain encoding a protein of 431 amino acids. In autoradiographic studies, CRF receptors were localized in highest densities in the anterior and intermediate lobes of the pituitary gland, olfactory bulb, cerebral cortex, amygdala, cerebellum and the macrophage-enriched zones and red pulp regions of the spleen. CRF can modulate the number of CRF receptors in a reciprocal manner. For example, stress and adrenalectomy increase hypothalamic CRF secretion which, in turn, down-regulates CRF receptors in the anterior pituitary. CRF receptors in the brain and pituitary are also altered as a consequence of the development and aging processes. In addition to a physiological role for CRF in integrating the responses of the brain, endocrine and immune systems to physiological, psychological and immunological stimuli, recent clinical data implicate CRF in the etiology and pathophysiology of various endocrine, psychiatric, neurologic and inflammatory illnesses. Hypersecretion of CRF in the brain may contribute to the symptomatology seen in neuropsychiatric disorders, such as depression, anxiety-related disorders and anorexia nervosa. Furthermore, overproduction of CRF at peripheral inflammatory sites, such as synovial joints may contribute to autoimmune diseases such as rheumatoid arthritis. In contrast, deficits in brain CRF are apparent in neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease and Huntington's disease, as they relate to dysfunction of CRF neurons in the brain areas affected in the particular disorder. Strategies directed at developing CRF-related agents may hold promise for novel therapies for the treatment of these various disorders.