Can Spirulina maxima reduce the mutagenic potential of sibutramine?

The worldwide obesity pandemic requires the use of anti-obesity drugs. Sibutramine is an anti-obesity drug that has been used worldwide but is indiscriminately consumed in Brazil. Several studies have demonstrated that sibutramine promotes weight loss and weight maintenance, but several side effects have been associated with its systematic consumption. For this reason, sibutramine was withdrawn from the European and American markets, but still remains legal for use in Brazil. Studies have shown that a 5-10% reduction in body weight results in outstanding health benefits for obese patients. However, in order to promote significant weight loss, it is necessary to use sibutramine for at least 2 years. This long-term exposure has carcinogenic potential, as sibutramine causes DNA damage. Thus, this study evaluated the in vivo mutagenic potential of sibutramine alone (5, 7, 10, 15, and 20 mg/kg) and in association with Spirulina maxima (150 and 300 mg/kg), a cyanobacterium with antioxidant potential, using the polychromatic erythrocyte micronucleus test. Our results reinforced the mutagenic potential of sibutramine alone, which showed a time-dependent action. Combinatory treatments with S. maxima were not able to reduce the genotoxicity of sibutramine. These results were confirmed in vitro with the cytokinesis-blocked micronucleus test. In conclusion, our data showed that new alternative anti-obesity treatments are needed since the consumption of sibutramine can increase the risk of cancer in overweight patients.

Mutagenic potential of Cordia ecalyculata alone and in association with Spirulina maxima for their evaluation as candidate anti-obesity drugs.

Obesity is one of the most important nutritional disorders, and can be currently considered as an epidemic. Although there are few weight reduction drugs available on the market, some new drug candidates have been proposed, including Cordia ecalyculata, a Brazilian plant with anorectic properties, and Spirulina maxima, a cyanobacterium with antioxidant and anti-genotoxic activity. In this study, we evaluated the mutagenic potential of C. ecalyculata at doses of 150, 300, and 500 mg/kg alone and in association with S. maxima at doses of 75, 150, and 250 mg/kg, respectively, through an in vivo micronucleus test, using mice of both sexes, and an in vitro micronucleus test and comet assay, using human peripheral blood. For all tests, cyclophosphamide was used as a positive control. The results showed that treatment of 300 mg/kg C. ecalyculata and the combination treatment of 500 mg/kg C. ecalyculata with 250 mg/kg S. maxima resulted in anorectic effects. The mutagenic tests did not reveal any clastogenic or genotoxic activity for any treatment, indicating that these candidates could be marketed as weight-reduction drugs. Moreover, the drugs contain chemo-preventive substances that can protect against tumorigenesis, which has been associated with obesity.

DNA extraction from hair shafts of wild Brazilian felids and canids.

Wild felids and canids are usually the main predators in the food chains where they dwell and are almost invisible to behavior and ecology researchers. Due to their grooming behavior, they tend to swallow shed hair, which shows up in the feces. DNA found in hair shafts can be used in molecular studies that can unravel, for instance, genetic variability, reproductive mode and family structure, and in some species, it is even possible to estimate migration and dispersion rates in given populations. First, however, DNA must be extracted from hair. We extracted successfully and dependably hair shaft DNA from eight wild Brazilian felids, ocelot, margay, oncilla, Geoffroy's cat, pampas cat, jaguarundi, puma, and jaguar, as well as the domestic cat and from three wild Brazilian canids, maned wolf, crab-eating fox, and hoary fox, as well as the domestic dog. Hair samples came mostly from feces collected at the São Paulo Zoo and were also gathered from non-sedated pet or from recently dead wild animals and were also collected from museum specimens. Fractions of hair samples were stained before DNA extraction, while most samples were not. Our extraction protocol is based on a feather DNA extraction technique, based in the phenol:chloroform:isoamyl alcohol general method, with proteinase K as digestive enzyme.

Corticotropin-releasing factor receptors in rat forebrain: autoradiographic identification.

Corticotropin-releasing factor (CRF) receptors were identified in rat forebrain by autoradiography with an iodine-125-labeled analog of ovine CRF substituted with norleucine and tyrosine at amino acid residues 21 and 32, respectively. High-affinity receptors for CRF were found in discrete areas of rat forebrain, including laminae I and IV of the neocortex, the external layer of the medium eminence, the lateral nucleus of the amygdala, and the striatum. These results are consistent with earlier findings on the immunohistochemical distribution of CRF and suggest that endogenous CRF has a physiological role in regulating activity of the central nervous system.

Genetic diversity of microsatellite loci in Leopardus pardalis, Leopardus wiedii and Leopardus tigrinus.

The microsatellite loci FCA045, FCA077, FCA008, and FCA096 are highly variable molecular markers which were used to determine the genetic diversity in 148 captive Leopardus sp. The PCR-amplified products of microsatellite loci were characterized in ABI Prism 310 Genetic Analyzer. Allele numbers, heterozygosity, polymorphism information content, exclusive allele number, and shared alleles were calculated. Sixty-five alleles were found and their sizes ranged from 116 to 216 bp in four microsatellite loci. The heterozygosity ranged from 0.36 to 0.81 in Leopardus pardalis, 0.57 to 0.67 in L. tigrinus and 0.80 to 0.92 in L. wiedii. The polymorphism information content was from 0.80 to 0.88 in L. pardalis, 0.76 to 0.88 in L. tigrinus and 0.77 to 0.90 in L. wiedii. The margay (L. wiedii) showed the highest index of polymorphism among the three species in this study. These results imply that microsatellite DNA markers can help in the study of the genetic diversity of Leopardus specimens.

Chemokines and chemokine receptors in the CNS: a possible role in neuroinflammation and patterning.

Chemokines constitute a growing family of structurally and functionally related small (8-10 kDa) proteins associated with inflammatory-cell recruitment in host defence. In addition to their well-established role in the immune system, recent data suggest their involvement in the maintenance of CNS homeostasis, in neuronal patterning during ontogeny and as potential mediators of neuroinflammation, playing an essential role in leukocyte infiltration into the brain. Chemokines and their G protein-coupled receptors are constitutively expressed at low-to-negligible levels in various cell types in the brain. Their expression is rapidly induced by various neuroinflammatory stimuli, implicating them in various neurological disorders such as trauma, stroke and Alzheimer's disease, in tumour induction and in neuroimmune diseases such as multiple sclerosis or acquired immunodeficiency syndrome (AIDS). Here, F. Mennicken, R. Maki, E. B. De Souza and R. Quirion briefly summarize recent exciting findings in the field.

Corticotrophin-releasing factor receptors: from molecular biology to drug design.

Corticotrophin-releasing factor (CRF) acts within both the brain and the periphery to coordinate the overall response of the body to stress. The involvement of the CRF systems in a variety of both CNS and peripheral disease states has stimulated great interest in this peptide as a potential site of therapeutic intervention. The recent cloning of multiple CRF receptor subtypes has precipitated a new era in CRF research that has allowed precise molecular, pharmacological and anatomical examination of mammalian CRF receptors. In this article, Derek Chalmers and colleagues highlight the major differences between the two classes of CRF receptors, CRF1 and CRF2, and a functionally related CRF-binding protein, and discuss the relevance of these sites to the ongoing development of CRF-based therapeutics.

Localization of agonist- and antagonist-binding domains of human corticotropin-releasing factor receptors.

The CRF receptors, CRFR1 and CRFR2, are members of the G protein-coupled receptor superfamily. Despite their considerable sequence similarity, CRFR1 and CRFR2 have quite different affinities for the peptide ligand rat/human CRF. Previous studies using chimeric receptors between human CRFR1 and CRFR2 have identified three potentially important regions in the second and third extracellular domains of CRF receptor for the binding of rat/human CRF. The present report further demonstrates that these same three regions also affect the binding of urocortin and sauvagine, two other members of the CRF peptide family, albeit to different extents. We also show that a fourth region in the third extracellular domain, Asp254, has been identified to be important for sauvagine but not CRF or urocortin binding. Thus, the three peptide ligands not only interact with a different set of regions on CRFR1 and CRFR2 but also differentially interact with some of the same regions. These data could, at least in part, account for the much higher affinity of CRFR2 for urocortin and sauvagine compared with rat/human CRF. We have also identified two amino acid residues, His199 in the third transmembrane domain and Met276 in the fifth transmembrane domain, that are important for binding the non-peptide high-affinity CRFR1 antagonist NBI 27914. Mutations of His199 and Met276 to the corresponding amino acids in CRFR2 each decreased the binding affinity of NBI 27914 for CRFR1 by 40- and 200-fold, respectively. This suggests that the transmembrane regions are critically important in forming the binding pocket for the nonpeptide antagonist.

Localization of ligand-binding domains of human corticotropin-releasing factor receptor: a chimeric receptor approach.

Two CRF receptors, CRFR1 and CRFR2, have recently been cloned and characterized. CRFR1 shares 70% sequence identity with CRFR2, yet has much higher affinity for rat/human CRF (r/hCRF) than CRFR2. As a first step toward understanding the interactions between rat/human CRF and its receptor, the regions that are involved in receptor-ligand binding and/or receptor activation were determined by using chimeric receptor constructs of the two human CRFR subtypes, CRFR1 and CRFR2, followed by generating point mutations of the receptor. The EC50 values in stimulation of intracellular cAMP of the chimeric and mutant receptors for the peptide ligand were determined using a cAMP-dependent reporter system. Three regions of the receptor were found to be important for optimal binding of r/hCRF and/or receptor activation. The first region was mapped to the junction of the third extracellular domain and the fifth transmembrane domain; substitution of three amino acids of CRFR1 in this region (Val266, Tyr267, and Thr268) by the corresponding CRFR2 amino acids (Asp266, Leu267, and Val268) increased the EC50 value by approximately 10-fold. The other two regions were localized to the second extracellular domain of the CRFR1 involving amino acids 175-178 and His189 residue. Substitutions in these two regions each increased the EC50 value for r/hCRF by approximately 7- to 8-fold only in the presence of the amino acid 266-268 mutation involving the first region, suggesting that their roles in peptide ligand binding might be secondary.

Serotonin and dopamine receptors in the rat pituitary gland: autoradiographic identification, characterization, and localization.

S2 serotonin and D2 dopamine receptors were identified, characterized, and localized in rat pituitary gland by quantitative light microscopic autoradiography. [3H]Spiperone was used to localize S2 serotonin and D2 dopamine receptors. A high concentration of D2 dopamine receptors [1 microM 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN)- or sulpiride-displaceable [3H]spiperone binding] was found in the rat intermediate lobe with much lower concentrations present in the anterior and posterior lobes. Significant densities of cinanserin-displaceable [3H]spiperone binding sites (i.e. S2 serotonin receptors) were present in all three lobes of the pituitary gland. [125I]Lysergic acid ([125I]LSD) was used to characterize further and selectively visualize S2 serotonin receptors in the rat pituitary. Data analysis by densitometry showed that [125I]LSD binding the rat intermediate pituitary was saturable and of high affinity with an apparent dissociation constant (Kd) of 1.2 nM. Data from competition studies using a variety of compounds showed a S2 serotonin receptor profile at this [125I]LSD binding site in rat pituitary. The highest concentration of [125I]LSD binding sites was found in the intermediate lobe with progressively lower concentrations present in the posterior and anterior lobes, respectively. There is a uniform pattern of distribution of S2 serotonin and D2 dopamine receptors within each lobe of the rat pituitary gland. The results of the present study provide the first identification of S2 serotonin receptors in the pituitary and confirm the heterogeneous distribution of D2 dopamine receptors within the rat pituitary. These data provide further evidence for the importance of dopamine in regulating pituitary function and suggest a physiological role for serotonin in regulating pituitary hormone secretion.

Rate-sensitive glucocorticoid feedback inhibition of adrenocorticotropin and beta-endorphin/beta-lipotropin secretion in rats.

In the present study, we present physiological evidence for rate-sensitive, fast feedback inhibition of secretion of ACTH and beta-endorphin (beta END)-related peptides. We used a 2 min restraint stress to physiologically increase plasma corticosterone, then examined the plasma responses of immunoreactive ACTH and beta END plus beta-lipotropin (beta END/beta LPH) to a subsequent restraint stress. After onset of this stress, plasma corticosterone increased from 2.5-10 min at a rate of 120 nM min-1, then remained at a peak from 10-15 min. A single 2 min restraint stress produced peak plasma levels of ACTH and beta END/beta LPH 2.5 min after onset of the stress, and these plasma concentrations declined after this initial stress at rates of 2.7 and 7.4 pM min-1, respectively. Application of a second restraint stress at the time of the peak corticosterone response produced plasma ACTH and beta END/beta LPH responses similar to those after the first stress. Application of a second stress during the period of significant rate-rise of corticosterone in plasma did not result in decreased incremental responses of plasma ACTH or beta END/beta LPH. However, the rates of decline of plasma ACTH and beta END/beta LPH of 7.6 and 32 pM min-1, respectively, from peak levels, were significantly greater after this second stress applied during the period of significant increase in plasma corticosterone concentration than the corresponding rates of decline observed after the initial stress or after a subsequent stress applied at the peak of plasma corticosterone. These differences in rates of decline of plasma ACTH or beta END/beta LPH appear to reflect differences in secretion rate rather than clearance, since disappearance of [125I]ACTH1-24 was not different after an initial vs. subsequent stress. In contrast to these data from intact rats, initial and subsequent stresses did not show different rates of decline of plasma ACTH or beta END/beta LPH in adrenalectomized rats. In conclusion, the stress-induced rate rise of glucocorticoid provides a negative feedback signal which serves to terminate and limit the duration, but not the peak, of the responses of POMC-derived peptides to subsequent stress.

Heterogeneity between brain and pituitary corticotropin-releasing factor receptors is due to differential glycosylation.

Chemical affinity cross-linking studies have identified brain and pituitary CRF receptors with similar pharmacological characteristics but different mol wts (anterior pituitary, 75,000; brain, 58,000). In order to determine whether the heterogeneous nature of CRF receptors was inherent in the protein, we examined the glycoprotein nature of both types of CRF receptors using lectin affinity chromatography and treatments with exo- and endoglycosidases. CRF receptors in both the cerebral cortex and anterior pituitary adsorbed to and specifically eluted from Concanavalin-A- and wheat germ agglutinin-immobilized lectin affinity columns, indicating that both forms of the receptor are glycoproteins containing complex and high-mannose carbohydrate moieties. Cerebral cortical CRF receptors were sensitive to both neuraminidase and alpha-mannosidase treatment while pituitary CRF receptors were only affected by neuraminidase treatment, suggesting that CRF receptors in brain and pituitary differed slightly in the nature of their glycosylation units. After treatment of cerebral cortical or anterior pituitary CRF receptors with the endoglycosidase, N-glycanase, the mol wts were markedly decreased; the mol wt of the anterior pituitary CRF receptor was decreased from 75,000 to approximately 40,000-45,000 while in a corresponding manner, the cortical receptor was decreased from 58,000 to approximately 40,000-45,000. Limited proteolysis after deglycosylation with N-glycanase using the proteinases Staphylococcus aureus V8 (S. aureus V8) or papain, generated virtually identical peptide fragments from anterior pituitary- or cerebral cortex- labeled CRF receptor proteins. In summary, these data support the hypothesis that the ligand binding subunit of the CRF receptor in both brain and pituitary resides on a polypeptide of 40,000-45,000 and appears to be identical in both tissues. Differences observed in the mobility of the two proteins were found to be due to differences in the posttranslational modification of the proteins in the two tissues.

D-Ala2-Met-enkephalinamide, a potent opioid peptide, alters pituitary-adrenocortical secretion in rats.

This study was designed to examine in rats the effects of systemic administration of a potent met-enkephalin analog, D-Ala2-met-enkephalinamide (DALA), on plasma ACTH and corticosterone secretion under basal conditions. Plasma radioimmunoassayable ACTH and corticosterone showed biphasic responses to intraarterial administration of DALA in chronically cannulated, conscious, freely moving, nonstressed animals. Both plasma ACTH and corticosterone increased at 5-10 min and decreased at 45-65 min after the administration of DALA (500 micrograms/kg) compared with either the basal concentrations or the corresponding responses in saline vehicle-injected control rats. Pretreatment with the specific opioid receptor antagonist naltrexone (2 mg/kg, intraarterially) completely prevented the DALA-induced increase in the plasma ACTH concentration and blunted the DALA-induced increase in the plasma corticosterone concentration. Naltrexone also increased plasma concentrations of ACTH and corticosterone above basal levels at a later time in DALA-treated animals. The DALA-induced increase in plasma corticosterone appears to be mediated by ACTH, mice DALA failed to increase plasma corticosterone in either hypophysectomized or dexamethasone-suppressed rats. DALA decreased the adrenocortical responsiveness to ACTH in hypophysectomized, but not in untreated or dexamethasone-suppressed, rats. Naltrexone increased the adrenocortical response to ACTH in DALA-treated rats. These data suggest that endogenous opioid peptides regulate pituitary-adrenocortical secretion under basal conditions by modifying both ACTH secretion and the glucocorticoid response to ACTH.

Differential plasma beta-endorphin, beta-lipotropin, and adrenocorticotropin responses to stress in rats.

This study was designed to compare the amounts of ACTH, beta-endorphin (beta END), and beta-lipotropin (beta LPH) that are present in plasma under basal conditions and after single and repeated administration of a discrete 2-min restraint stress both in intact and in chronically adrenalectomized rats. In intact rats, application of a 2-min restraint stress produced rapid parallel increases in plasma concentrations of radioimmunoassayable ACTH and beta END/beta LPH (the total of beta END-like immunoreactivities), with peaks 2.5-5 min after onset of the stress and return almost to basal concentrations by 30 min. Gel exclusion chromatography [Sephadex G-50 (fine)] and subsequent RIA revealed that plasma obtained from control nonstressed intact rats contained much greater quantities of beta END (94% of the total beta END/beta LPH immunoreactivity) than beta LPH. In contrast, equal amounts of beta END and beta LPH were present in plasma of intact rats 2.5-10 min after onset of the 2-min restraint stress. Chronically adrenalectomized rats lacking glucocorticoid-negative feedback had significantly higher basal plasma concentrations of beta END/beta LPH and ACTH than those present in intact rats. Furthermore, the plasma responses of both beta END/beta LPH and ACTH to stress were markedly enhanced in chronically adrenalectomized rats compared to the corresponding responses in intact rats. Gel exclusion chromatography revealed that both the higher basal concentration and the enhanced plasma beta END/beta LPH response to stress in adrenalectomized rats resulted primarily from increases in the beta LPH component, with lesser increases in the beta END component. In contrast to the proportion in intact rats, in chronically adrenalectomized rats, beta END represented about 27% of the total beta END/beta LPH immunoreactivity in the basal state and about 18% 5-10 min after the onset of restraint stress. In intact rats, the plasma ACTH responses to a subsequent stress applied 5 min (a time when peak plasma levels of hormones are present) or 30 min (a time when the plasma hormone concentrations have returned to prestress levels) after the initial stress and the plasma beta END/beta LPH response to a second stress applied at 30 min were equal to the corresponding hormone responses to a single stress. In contrast, the plasma beta END/beta LPH response to the subsequent stress applied 5 min after the initial stress was significantly potentiated in intact rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Alpha 1-adrenergic receptors in the neural lobe of the rat pituitary: autoradiographic identification and localization.

alpha 1-Adrenergic receptors were identified, characterized, and localized in rat pituitary gland by quantitative light microscopic autoradiography. Autoradiographic studies were carried out in slide-mounted rat pituitary sections using both [125I]2-[beta-(4-hydroxy-3-iodophenyl)ethyl-aminomethyl]tetralone ([125I]HEAT) and [3H]prazosin to localize alpha 1-adrenergic receptors. Data analysis by densitometry showed that [125I]HEAT binding in the rat neural lobe was saturable and of high affinity, with an apparent dissociation constant (Kd) of about 5 pM. Data from competition studies using a variety of compounds demonstrated an alpha 1-adrenergic receptor profile for [125I]HEAT-binding sites in the rat pituitary. A high density of alpha 1-adrenergic receptors (1 microM prazosin-displaceable [125I]HEAT binding or 10 microM phentolamine-displaceable [3H]prazosin binding) was found present only in the neural lobe, with negligible concentrations in the anterior and intermediate lobes. The regulation of [125I]HEAT-binding sites in the neural lobe was examined in pituitary stalk-transected and superior cervical ganglionectomized rats. Significant increases in [125I]HEAT-binding sites were observed after superior cervical ganglionectomy, but no changes in [125I]HEAT binding were found in pituitary stalk-transected rats compared to that in sham-operated controls. These data provide the first identification of alpha 1-adrenergic receptors in the neural lobe of the rat pituitary and suggest that these receptors may be localized primarily in blood vessels. In addition, a primary role for the peripheral sympathetic nervous system in regulating the neurohypophyseal vasculature is suggested. The precise function of alpha 1-adrenergic receptors in the neural lobe in regulating posterior lobe hormone secretion remains to be demonstrated.

Corticotropin-releasing factor receptors in mouse spleen: identification, autoradiographic localization, and regulation by divalent cations and guanine nucleotides.

The radioligand [125I-Tyro]ovine CRF [( 125I] oCRF) was used to identify, characterize, and localize CRF receptors in a crude homogenate of mouse spleen. The binding was linear with increasing membrane protein concentration, saturable, and of high affinity. Scatchard analysis of [125I]oCRF saturation curves revealed a high affinity component with an apparent dissociation constant of 0.26 nM and a maximum receptor concentration of 8.74 fmol/mg protein. The relative affinities of various CRF-related and unrelated peptides at the [125I]oCRF-binding site in spleen correlate well with their reported potencies in eliciting ACTH release from the pituitary and their relative affinities for CRF receptors in brain. Consistent with a coupling of splenic CRF receptors to a guanine nucleotide regulatory protein, the binding of [125I]oCRF was increased by magnesium ions and decreased by guanine nucleotides in both mouse spleen and brain. Saturation analysis of [125I]oCRF binding at 0, 1, and 10 mM MgCl2, 10 mM MgCl2 plus 200 microM GTP, and 10 mM MgCl2 plus 50 microM 5'-guanylimidodiphosphate indicated that high affinity [125I]oCRF binding in both brain and spleen requires Mg2+; however, subtle differences were observed in the degree of sensitivity and mechanisms by which Mg2+ and guanine nucleotides regulated specific binding of [125I]oCRF to brain and spleen. Autoradiographic studies indicated that [125I]oCRF-binding sites were primarily localized to the red pulp and marginal zone regions of the spleen, which are known to contain high concentrations of macrophages. The absence of [125I]oCRF-binding sites in the periarteriolar and peripheral follicular white pulp regions suggests that neither T nor B lymphocytes have specific CRF-binding sites. In summary, we have identified a high affinity, magnesium-dependent, and guanine nucleotide-sensitive binding site for CRF in mouse spleen which appears to be localized primarily in splenic macrophages.

Stress-induced inhibition of the plasma corticosterone response to a subsequent stress in rats: a nonadrenocorticotropin-mediated mechanism.

The present study was designed to define further the relationships between ACTH and corticosterone secretion after repeated administration of a discrete restraint stress in rats. The possibility that plasma ACTH and corticosterone responses to stress may be modified by prior exposure to stress was examined in male rats using a 2-min restraint stress. The peak plasma ACTH response to a single restraint stress occurred at 2.5-5 min after the onset of the stress, and plasma ACTH returned to the basal concentration by 30 min. The plasma corticosterone concentration after this stress peaked at 15-30 min and returned to the control range by 60-90 min. The time courses of the plasma ACTH and corticosterone responses to restraint stress after administration of three prior stresses at 90-min intervals were similar to those after a single stress. Stress-induced increments in plasma concentrations of ACTH and corticosterone were similar in rats that received either a single restraint stress or as many as seven stresses repeated at 90-min intervals. Next, we examined the plasma ACTH and corticosterone responses to repeated stress applied at intervals of less than 90 min (30 or 60 min), that is, at times at which the plasma corticosterone concentration had not yet returned to basal levels. The plasma ACTH responses to the second stress were similar in magnitude and duration to the response after a single stress, whether the second stress was applied 30, 60, or 90 min after the first stress. The plasma corticosterone response to a second stress applied at 90 min was identical to the response after the initial stress. In contrast, the plasma corticosterone responses to a second stress applied 30 or 60 min after the initial stress were markedly reduced. The decrease in plasma corticosterone response to the second stress did not result from a decrease in secretion of bioactive ACTH; no difference was found between the magnitude of the plasma ACTH response to the initial stress and a subsequent stress applied 30 min later using either bioassay or immunoassay measurements. Also, the rate of corticosterone catabolism was not increased by prior stress; the rate of disappearance of corticosterone from plasma was identical after an initial or a subsequent stress applied at 30 min. We were unable to demonstrate decreased adrenocortical responsiveness to ACTH after an initial stress in dexamethasone-suppressed rats; in these rats the plasma corticosterone response to exogenous ACTH was not decreased by prior restraint stress. These data clearly define a period of decreased adrenocortical response to subsequent stress after stress-induced activation of adrenocortical secretion. Furthermore, this altered adrenocortical response appears to be mediated by a nonadrenocorticotropin mechanism.

Upregulation of interleukin-1 receptors in mouse AtT-20 pituitary tumor cells following treatment with corticotropin-releasing factor.

Treatment of AtT-20 cell cultures with increasing concentrations of rat/human corticotropin-releasing factor (r/hCRF) for 24 h resulted in a dose-dependent 2-3 fold increase in specific 125I-labelled recombinant human IL-1 alpha (125I-IL-1 alpha) binding that was paralleled by a 70-80% decrease in 125I-Tyro-ovine CRF binding. Saturation analysis of 125I-IL-1 alpha binding in control and CRF-treated cultures indicated that CRF produced an increase in the density (Bmax) of IL-1 receptors without altering their affinity (KD). The CRF-induced upregulation of IL-1 receptors appears to be mediated through specific membrane receptors for CRF since the CRF receptor antagonist, alpha-helical oCRF (9-41), blocked the CRF-induced upregulation of IL-1 receptors without producing any effect on 125I-IL-1 alpha binding by itself. In summary, these data demonstrate complex interactions between CRF and IL-1 at the pituitary level and identify potential novel mechanisms for cytokines to alter neuroendocrine function.

Interleukin-1 receptors in mouse kidney: identification, localization, and modulation by lipopolysaccharide treatment.

The cytokine interleukin-1 (IL-1) has a variety of effects in the kidney involving induction of nephritis and renal injury. In addition, recent reports suggest that IL-1 regulates natriuresis and renin secretion in the kidney. To examine the potential sites of action of IL-1 in the kidney, we used iodine-125-labeled recombinant human interleukin-1 alpha ([125I]IL-1 alpha) to identify and characterize IL-1 receptors in crude membrane preparations of mouse (C57BL/6) kidney. The binding of [125I] IL-1 alpha was linear over a broad range of membrane protein concentrations, saturable, reversible, and of high affinity, with an equilibrium dissociation constant (Kd) of 66 +/- 10 pM and a maximum number of binding sites of 1.04 +/- 0.24 fmol/mg protein. In competition studies, recombinant human IL-1 alpha, recombinant human IL-1 beta, and a weak IL-1 beta analog (IL-1 beta+) inhibited [125I]IL-1 alpha binding to mouse kidney in parallel with their relative bioactivities in the T-cell comitogenesis assay, with inhibitory binding affinity constant (Ki) values of 28 +/- 19, 53 +/- 23, and 5560 +/- 2098 pM, respectively; rat/human CRF and human tumor necrosis factor had no effect on [125I]IL-1 alpha binding. In autoradiographic studies, IL-1 receptors were heterogeneously distributed in the kidney, with significantly higher densities present in the medulla than in the cortex. To study the effects of endogenous IL-1 in modulating [125I]IL-1 alpha-binding sites in kidney, we injected 30 micrograms of the bacterial endotoxin lipopolysaccharide (LPS) to mice ip. Autoradiographic studies demonstrated substantial decreases in [125I]IL-1 alpha binding in both the kidney cortex (control, 34.7 +/- 6.2 fmol/mg tissue equivalent; LPS, 11.3 +/- 0.3; P less than 0.05) and medulla (52.7 +/- 8.1 vs. 26.0 +/- 1.0; P less than 0.05) 24 h after injection of LPS. Saturation studies in whole kidney homogenates demonstrated that the LPS-induced decrease in [125I]IL-1 alpha binding was primarily due to a down-regulation of IL-1 receptors (i.e. decrease in the maximum number of binding sites). The identification of IL-1 receptors in kidney with characteristics similar to those of IL-1 receptors in the brain-endocrine-immune axis provides further support for a physiological role for IL-1 in regulating renal function.

Sigma-receptors in endocrine organs: identification, characterization, and autoradiographic localization in rat pituitary, adrenal, testis, and ovary.

We have used a variety of selective radioligands to identify and localize sigma- and phencyclidine (PCP)-binding sites in rat endocrine organs. [3H]Haloperidol-labeled sigma-receptors were identified in membrane homogenates of rat pituitary, adrenal, testis, and ovary which had kinetic and pharmacological characteristics similar to those of the well characterized sigma-receptors in rat cerebellum. The highest density of sigma-receptors was present in the ovary, with progressively lower densities present in the testis, pituitary, adrenal, and cerebellum, respectively. In autoradiographic studies, sigma-receptors [labeled with d-3-(3-hydroxyphenyl)N-(1-propyl-2,3-[3H]piperidine or [3H]1,3-di-(2-tolyl)guanidine] were discretely localized within the endocrine tissues. In the pituitary, the highest density of sigma-receptors was found in the anterior lobe. In the adrenal, sigma-receptors were localized primarily in the cortex. In the testis, sigma-receptors were present in highest concentrations in the ductuli efferentes and ductus epididymis; lower densities of binding sites were present in the seminiferous tubules, and no binding was seen in the interstitial tissue. In the ovary, sigma-receptors were localized in high density in the maturing follicles, and lower densities were present in resting follicles. After hypophysectomy, there were relative increases in the densities of sigma receptors in the remaining tissue in the adrenal gland and testis. In contrast, hypophysectomy resulted in a marked depletion of sigma-binding sites in the ovary. The data from hypophysectomized rats indicate that the highest densities of sigma-receptors in the ovary are localized to (LH-dependent) maturing follicles, while sigma-binding sites in adrenal and testis are localized to cells that are not dependent on trophic maintenance by the pituitary. In contrast, high affinity PCP receptors were not detected in pituitary, adrenal, testis, or ovary either by homogenate binding studies with 3,4-[3H]N-[1-(2-thienyl)cyclohexyl]piperidine or in vitro autoradiography using 3,4-[3H]N-[1-(2-thienyl)cyclohexyl]piperidine and d-[3H]5-methyl-10,11-dihydro-5H-dibenzo-[a,d] + cyclohepten-5,10-imine. In summary, the data suggest that the reported endocrine effects of PCP and the prototypic sigma-receptor agonist N-allylnormetazocine are probably mediated either through direct action on sigma-receptors in the pituitary and/or target endocrine organs or by actions on sigma- and/or PCP receptors in brain.