Urocortin, but not corticotropin-releasing hormone (CRH), activates the mitogen-activated protein kinase signal transduction pathway in human pregnant myometrium: an effect mediated via R1alpha and R2beta CRH receptor subtypes and stimulation of Gq-proteins.

CRH and CRH-related peptides such as urocortin mediate their actions in the human myometrium via activation of two distinct classes of CRH receptors, R1 and R2. These heptahelical receptors are able to stimulate a number of different intracellular signals; one key mediator of G protein-activated intracellular signaling is the cascade of p42/p44, mitogen-activated protein kinase (MAPK). We therefore hypothesized that activation of MAPK might mediate CRH and or/urocortin actions in the myometrium. In cultured human pregnant myometrial cells, urocortin but not CRH was able to induce MAPK phosphorylation and activation, suggesting that in the human myometrium these two peptides have distinct actions and biological roles. To identify the particular receptor subtypes mediating this phenomenon, all known CRH receptors present in the human myometrial cells were stably expressed individually in HEK293 and CHO cells, and their ability to activate MAPK was tested. The R1alpha and R2beta, but not the R1beta, R1c, or R1d, receptor subtypes were able to mediate urocortin-induced MAPK activation. The signaling components were further investigated; activation of Gs, Go, or Gi proteins did not appear to be involved, but activation of Gq with subsequent production of inositol triphosphates (IP3) and protein kinase C (PKC) activation correlated with MAPK phosphorylation. Studies on Gq protein activation using [alpha-32P]-GTP-gamma-azidoanilide and IP3 production in cells expressing the R1alpha or R2beta CRH receptors demonstrated that urocortin was 10 times more potent than CRH. Moreover, urocortin (UCN) generated peak responses that were 50-70% greater than CRH in activating the Gq protein and stimulating IP3 production. In conclusion, UCN acting thought multiple receptor subtypes can stimulate myometrial MAPK via induction of the Gq/phospholipase C/IP3/PKC pathway, whereas CRH-induced activation of this pathway appears to be insufficient to achieve MAPK activation.

A novel spliced variant of the type 1 corticotropin-releasing hormone receptor with a deletion in the seventh transmembrane domain present in the human pregnant term myometrium and fetal membranes.

CRH exerts its actions via activation of specific G protein-coupled receptors, which exist in two types, CRH-R1 and CRH-R2, and arise from different genes with multiple spliced variants. RT-PCR amplification of CRH receptor sequences from human myometrium and fetal membranes yielded cDNAs that encode a novel CRH-R type 1 spliced variant. This variant (CRH-R1d) is present in the human pregnant myometrium at term only, which suggests a physiologically important role at the end of human pregnancy and labor. The amino acid sequence of CRH-R1d is identical to the CRH-R1alpha receptor except that it contains an exon deletion resulting in the absence of 14 amino acids in the predicted seventh transmembrane domain. Binding studies in HEK-293 cells stably expressing the CRH-R1d or CRH-R1alpha receptors revealed that the deletion does not change the binding characteristics of the variant receptor. In contrast, studies on the G protein activation demonstrated that CRH-R1d is not well coupled to the four subtypes of G proteins (G(s), G(i), G(o), G(q)) that CRH-R1alpha can activate. These data suggest that although the deleted segment is not important for CRH binding, it plays a crucial role in CRH receptor signal transduction. Second messenger studies of the variant receptor showed that CRH and CRH-like peptides can stimulate the adenylate cyclase system, with reduced sensitivity and potency by 10-fold compared with the CRH-R1alpha. Furthermore, CRH failed to stimulate inositol trisphosphate production. Coexpression studies between the CRH-R1d or CRH-R1alpha showed that this receptor does not play a role as a dominant negative receptor for CRH.

Activation of protein kinase C by oxytocin inhibits the biological activity of the human myometrial corticotropin-releasing hormone receptor at term.

The role of placental CRH in human pregnancy is currently unknown. The myometrium expresses CRH receptors that during pregnancy become coupled to adenylate cyclase. Oxytocin (OT) is one of the main regulators of uterine activity, acting via activation of the inositol triphosphate pathway. In view of the possible cross-talk between the CRH and OT signal transduction pathways we have sought to examine in more detail the second messenger mechanisms involved. CRH receptor binding affinity for CRH and activation of adenylate cyclase were reduced in the presence of OT in pregnant (at term, but not preterm) human myometrium. OT action was mediated via pertussis toxin-sensitive G proteins, which directly inhibit adenylate cyclase and, via activation of protein kinase C, phosphorylate the CRH receptor, leading to desensitization. Activation of protein kinase C by OT could be partially inhibited in human pregnant myometrial cells by OT antagonists (F327 and CAP476; 1 microM) or phospholipase C inhibitors (U73122; 10 microM). These results suggest that in term myometrium, CRH receptor function is modulated by OT, leading to reduced biological activity, lower cAMP levels, and a subsequent shift in favor of contractility rather than relaxation.

Basal and interleukin-1beta-stimulated prostaglandin production from cultured human myometrial cells: differential regulation by corticotropin-releasing hormone.

During pregnancy, placental CRH acts on human myometrium via specific receptors and might play a role in the regulation of myometrial contractility and hence human parturition. The myometrium is the site of production and target of several PGs, which can be activated by cytokines, especially during infection-induced preterm labor. We established primary human myometrial cell cultures that express functional CRH receptors (CRH-R1alpha, -R1beta, -Rlc, and -R2beta) to investigate the possible regulation of PG production by CRH. We studied the effect of CRH on the two major myometrial PGs, PGE2 and 6-keto PGF1alpha. Human CRH was able to partially inhibit basal, interleukin-1beta-stimulated, and oxytocin-stimulated PGE2 production (56 +/- 11%, 45 +/- 8%, and 58 +/- 6% inhibition, respectively). This effect was blocked by a specific CRH receptor antagonist in a concentration-dependent manner. Furthermore, CRH had no effect on 6-keto PGF1alpha production, indicating that the CRH inhibitory action does not involve suppression of cyclooxygenase, the enzyme responsible for the production of both PGE2 and 6-keto-PGF1alpha. These data further support the view that during pregnancy, CRH may promote myometrial quiescence and might play an important role in the regulation of human labor.

The human myometrium expresses multiple isoforms of the corticotropin-releasing hormone receptor.

Specific high affinity binding sites for CRH have been identified and characterized in the pituitary and central nervous system as well as in peripheral tissues. We recently identified and characterized a specific CRH receptor in human myometrium that changes to a high affinity state before term. In view of this, we searched for receptor heterogeneity in the pregnant and nonpregnant human myometrial CRH receptor. Myometrial membranes were prepared by differential centrifugation from either pregnant (cesarian section) or nonpregnant (hysterectomy) myometrium. Using a specific RRA followed by isoelectric focusing and autoradiography, multiple isoforms of the human myometrial CRH receptor were identified that were identical in both pregnant and nonpregnant myometrium. Five isoforms were identified (pI 4.65, 4.8, 4.95, 5.1, and 5.2). Reduction of disulfide bridges with reducing agents (dithiothreitol and cysteine) increased the specific binding of CRH to its myometrial receptor, and the action of dithiothreitol affected the two most basic receptor isoforms. These results suggest the presence of multiple isoforms of CRH receptors that may have different properties and functions and the presence of disulfide bridges within the myometrial CRH receptor, which are important, but not critical, for the receptor binding.

The identification of a human myometrial corticotropin-releasing hormone receptor that increases in affinity during pregnancy.

The concentration of immunoreactive, placentally derived CRH is increased in the peripheral circulation during the third trimester of human pregnancy. However, the function of this placental CRH is entirely unknown. A number of observations have led us to believe that CRH might influence myometrial contractility and, hence, parturition via specific receptor mechanisms. 1) In idiopathic preterm labor, plasma immunoreactive-CRH concentrations are significantly elevated compared to control values. 2) CRH and oxytocin exhibit a marked synergistic effect on myometrial contractility which is prostaglandin dependent and can be inhibited by the CRH receptor antagonist [alpha-helical CRF-(9-41)]. In view of this, we searched for specific CRH-binding sites in myometrial tissue obtained at biopsy from pregnant (cesarian section) and nonpregnant (hysterectomy) patients. To test for the presence of CRH receptors, we prepared myometrial membranes and performed binding studies using [125I]tyr-o-CRH as a ligand. The binding was found to be pH, time, temperature, and divalent cation concentration dependent and was fully reversible on addition of 1 microM unlabeled ovine CRH. In both tissues, there was a single, specific, homogenous, high affinity population of CRH receptors. Scatchard analysis of the specific binding sites revealed dissociation constants of 250-300 and 30-60 pM for the nonpregnant and pregnant myometrium, respectively. This compares with dissociation constants of 130 pM (rat anterior pituitary receptor) and 100 pM (human CRH-binding protein). This would mean that in the nonpregnant state, the equilibrium for binding is in favor of the binding protein, but during the later stages of pregnancy, the change in affinity of the receptor alters the binding in favor of the myometrial receptor.

Signal transduction characteristics of the corticotropin-releasing hormone receptors in the feto-placental unit.

Placentally derived CRH plays a major role in the mechanisms controlling human pregnancy and parturition. In this study, we sought to investigate the signal transduction mechanisms of CRH Type-1 receptors in the feto-placental unit. To clarify the signal transduction components in placenta and fetal membranes, we investigated the expression of G proteins and adenylate cyclase. Using the nonhydrolysable photoreactive analog [alpha-32P] GTP-azidoanilide and peptide antisera raised against G protein alpha-subunits, we studied coupling of CRH receptors to G proteins in both placental and fetal membranes. Treatment of placental membranes with human CRH (100 nM) increased the labeling of Gq, Go, and Gz but not Gi and Gs. Treatment of fetal membranes with human CRH (100 nM) increased the labeling of Go and Gq but not Gi, Gs, and Gz. These results were supported by experiments that showed that CRH failed to activate adenylate cyclase in these tissues, but induced an increase in inositol phosphates instead. These findings provide new insights into the components of the signal transduction machinery in both fetal and placental membranes and suggest that CRH Type-1 receptors can couple to different G proteins in different tissues. The physiological significance of these observations remains to be elucidated.

The biological activity of the corticotropin-releasing hormone receptor-adenylate cyclase complex in human myometrium is reduced at the end of pregnancy.

We recently suggested that placentally derived CRH might influence human parturition via specific receptor mechanisms. We identified a human myometrial CRH receptor that changes to a high affinity state in the later stages of pregnancy and becomes coupled to the adenylate cyclase system. The purpose of this study was to investigate the functional capacity of this receptor in myometrial tissue obtained from women being delivered electively by cesarian section at term (38-40 weeks gestation) and preterm (30-35 weeks gestation) before the onset of labor. Myometrial membrane suspensions were prepared by differential centrifugation, and the production of cAMP after stimulation with various test substances was measured by RIA. In preterm myometrium, both human CRH and cholera toxin stimulated cAMP production. This effect was significantly reduced in term myometrium. The adenylate cyclase was functionally active in term myometrium, as demonstrated by the use of forskolin. Furthermore, pertussis toxin pretreatment of term myometrial membranes did not increase the response to CRH. These results suggest that in human pregnant myometrium at term, there is a modification in the coupling mechanisms between CRH receptors and the catalytic component of adenylate cyclase, resulting in a reduction of CRH-stimulated cAMP production.

Expression of orexin-A and functional orexin type 2 receptors in the human adult adrenals: implications for adrenal function and energy homeostasis.

The hypothalamic peptides, orexin-A and orexin-B, have been implicated in the regulation of feeding behavior. In starved rats catabolic activity quickly predominates, reinforced by elevated corticosterone, independent of ACTH, implicating adrenal activity as a metabolic regulator. In view of these findings, we investigated whether orexin and orexin receptors are present in human adult adrenals and might therefore be implicated in hormonal regulation and energy homeostasis outside the central nervous system. RT-PCR, fluorescent in situ hybridization, immunoblotting, and immunostaining analysis confirmed the expression of the orexin type 2 receptor, but not of orexin type 1 receptor, in the adrenal cortex. Immunoblotting analysis also detected the presence of the prepro-orexin and its cleaved product orexin-A. Treatment of adult adrenal membranes with orexin-A increased the labeling of G(s), G(q), and, to a lesser degree, G(i), but not G(o). Stimulation with orexin-A induced cAMP and IP3 production in a dose-dependent manner. The data presented here provide conclusive evidence for the presence of orexin-A and orexin type 2 receptors in human adult adrenal glands. At the moment the functional relevance of this is uncertain. However, it is known that both orexin-A and orexin-B can induce corticosterone production in dispersed rat adrenocortical cells. Our data provide further evidence for a functional link between orexogenic signals and adrenal function. The concept that the peptide acting via these receptors in the adult adrenal is responsible for steroidogenesis and energy balance is attractive.

Human corticotropin-releasing hormone receptor: differences in subtype expression between pregnant and nonpregnant myometria.

There is increasing evidence that CRH, which is the principal neuroregulator of the hypothalamic-pituitary-adrenocortical axis, is also involved in the mechanism of human labor. The human myometrium has been shown to express several high affinity CRH receptors, although the identities of the CRH receptor subtypes have yet to be identified. To investigate further the expression of the CRH receptor in human myometrium, we used RT-PCR, fluorescent in situ hybridization and immunofluorescence to identify and localize the four subtypes, 1 alpha, 1 beta, 2 alpha, and the variant C, of the CRH receptor. Interestingly, the CRH receptor subtypes in myometrium exhibit differential expression patterns; in human pregnant myometrium at term all four receptor-subtypes were expressed, whereas only the 1 alpha- and 1 beta-receptor subtypes were found in the nonpregnant myometrium. This would suggest that CRH, acting via different receptor subtypes, is able to exert different actions on the myometrium in the pregnant state compared to the nonpregnant state. Furthermore, in the pregnant human uterus, CRH receptors were localized in both smooth muscle and fibroblasts. These findings suggest that CRH receptor expression plays an important modulatory role in myometrial and possibly in cervical function.

Expression and coupling characteristics of the CRH and orexin type 2 receptors in human fetal adrenals.

Hormones produced by the fetal adrenal regulate fetal growth, steroidogenic activity, and intrauterine homeostasis, which are essential for the maintenance of pregnancy and the preparation of the fetus for extrauterine life. There is a functional interaction between CRH and the fetal adrenal, as CRH increases dehydroepiandrosterone sulfate production in cultured fetal adrenal cells. Moreover, in a rodent model administration of orexin A induced corticosterone production. To examine this relationship in more detail we measured the expression of the different subtypes of CRH and orexin receptors and their specific coupling to G protein alpha-subunits upon activation with CRH and orexin A, respectively. Using RT-PCR and fluorescent in situ hybridization analysis, we demonstrated the presence of CRH receptors 1alpha and 2alpha, and orexin type 2 receptor mRNA. None of the other CRH receptor variants or orexin type 1 receptor were detected. Immunofluorescent analysis and Western blotting confirmed the protein expression of both receptors, which also bind fluo-CRH and fluo-orexin with high affinity. Immunoblotting analysis confirmed the expression of prepro-orexin and orexin A in fetal adrenals. Using photoaffinity labeling, we determined which G proteins are coupled to the CRH and orexin receptors in fetal adrenals when challenged with CRH or orexin. Treatment of fetal adrenal membranes with CRH (100 nM) increased the labeling of G(o) and, to a lesser extent, G(s), but not G(i) and G(q), whereas treatment with orexin A (100 nM) increased the labeling of G(s) and G(i), but not G(o) and G(q). These findings provide new insights into the components of the signal transduction machinery in human fetal adrenals and demonstrate for the first time the presence of functional orexin receptors outside of the CNS in humans.

Reduced expression of corticotropin-releasing hormone receptor type-1 alpha in human preeclamptic and growth-restricted placentas.

Placentally derived CRH seems to play a major role in the mechanisms controlling human pregnancy and parturition, via activation of specific receptors widespread in reproductive tissues. In the human placenta, CRH seems to modulate vasodilation, prostaglandin production, and ACTH secretion. It has also been suggested that CRH might act as a placental clock, determining the length of gestation. In addition, maternal plasma CRH concentrations are further elevated in pregnancies associated with abnormal placental function, such as preeclampsia and intrauterine growth retardation (IUGR). In this study, we sought to investigate the expression of CRH-R1 alpha levels in placentas from women who have undergone normal deliveries (control group) and patients who have been diagnosed as having preeclampsia or IUGR. Results showed that placental CRH-R1 alpha mRNA levels (as shown by quantitative RT-PCR) and protein levels (shown by Western blotting analysis) were significantly (P < 0.05) reduced in all of the complicated pregnancies. In contrast, levels of the angiotensin II receptor were elevated in preeclampsia and reduced in IUGR subjects, as shown by RT-PCR and Western blotting analysis. These findings might suggest that changes in receptor expression may contribute toward dysregulation of the dynamic balance controlling vascular resistance.

The human placenta and fetal membranes express the corticotropin-releasing hormone receptor 1alpha (CRH-1alpha) and the CRH-C variant receptor.

Placentally derived CRH plays a major role in the mechanisms controlling human pregnancy and parturition. It has been suggested that there is a CRH placental clock that is active from the early stages of pregnancy and determines the length of gestation and the timing of parturition. CRH can influence human reproductive tissue function via specific CRH receptors. Two distinct CRH receptors have been cloned (R1 and R2) that share 70% homology at the amino acid level and exist as two alternatively spliced forms (alpha and beta). In this study we investigated the presence of CRH receptor subtypes in human fetal membranes derived from spontaneous rupture and placental biopsies at term. Using RT-PCR, we identified the full length of the CRH-R1alpha subtype in placental and fetal membranes. In both tissues we also identified a spliced variant of the CRH receptor (CRH-Rc). We were unable to detect any CRH-R2 messenger ribonucleic acid in any of the biopsies. Fluorescent in situ hybridization and immunofluorescence in both tissues demonstrated that syncytiotrophoblast cells and amniotic epithelium are the major cell types expressing CRH-1alpha and CRH-Rc receptor messenger ribonucleic acid. Further studies are necessary to give a better insight into the role of CRH and its receptors in these tissues.

Superoxide generation from constitutive nitric oxide synthase in astrocytes in vitro regulates extracellular nitric oxide availability.

Oxygen-derived free radicals play an important role in the physiology and pathophysiology of brain cell function. Because of their labile nature, however, it has been difficult to investigate their actions directly. This problem has been addressed, in primary rat brain cell cultures, in this study by utilization of two novel electrochemical sensors. It has been demonstrated that extracellular superoxide originates from the astrocytic subpopulation in a calcium/calmodulin dependent manner and responds to constitutive nitric oxide synthase inhibition. The results indicate a novel function for the astrocytic constitutive nitric oxide synthase in regulating extracellular superoxide release and, therefore, controlling neuronal nitric oxide availability.

Direct, real-time sensing of free radical production by activated human glioblastoma cells.

Primary brain injury initiates a cascade of events which result in secondary brain damage. Although, at present, the biochemical and molecular mechanisms of nerve cell death are not well understood, sufficient evidence now exists to implicate free radicals in this brain injury response. In the light of the current understanding on the role of free radicals in cell mortality, we report on the use of two specific sensors, which we use to measure the direct, simultaneous and real time electrochemical detection of both superoxide (O2.-) and nitric oxide (NO), produced by activated glioblastoma cells. The development and application of these novel methods has enabled us to show that both the cytokine-mediated induction of the enzymes responsible for the generation of these radical species, and the metabolic requirements of the cell can modulate cell messenger release. Importantly, the data collected provides dynamic information on the time course of free radical production, as well as their interactions and their involvement in the process of cell death. In particular, one of the major advances afforded by this technology is the demonstration that suppression of one of either of the two cellular generated radical species (NO and O2.-) leads directly to a corresponding increase in the species that was not being deliberately inhibited or scavenged. This finding may indicate a mechanism involving inter-enzyme regulation of free radical production in glial cells (a phenomenon which may, in future, also be shown to operate in other relevant cell models).

Superoxide electrode based on covalently immobilized cytochrome c: modelling studies.

We have recently described an optimised electrode for the detection of enzymatic and cellular superoxide (O2*-) production based on cytochrome c immobilized covalently at a surface-modified gold electrode and applied this system to the study of free radical production by activated human glioblastoma cells. In this paper we report the development of a mathematical model for the O2*- electrode responding to enzymically produced O2*- which should enable the determination of absolute concentrations of O2*- in biological systems when this electrode is employed for direct, real-time monitoring of free radical release and interactions.

Expression and signalling characteristics of the corticotrophin-releasing hormone receptors during the implantation phase in the human endometrium.

Corticotrophin-releasing hormone (CRH) has been identified in several peripheral tissues, including the female reproductive organs. CRH is expressed in the placenta, myometrium, epithelial endometrium and the endometrial stromal cells at all phases of the menstrual cycle. Similarly, CRH receptors are present in pregnant and non-pregnant myometrium, placenta and endometrium. Putative roles of CRH in the endometrium include involvement in implantation, decidualisation and maintenance of pregnancy. In this study we sought to investigate in detail the CRH receptor repertoire expressed in the human endometrium and their signalling characteristics. Using RT-PCR we were able to demonstrate the expression of CRH receptor 1alpha (CRH-R1alpha) and CRH-R2alpha in the human endometrium. CRH-R1beta was present in 40% of endometrial cDNAs examined. No apparent expression of CRH-R2beta, CRH-R2gamma or any other CRH-R1 splice variants was detected. Chemical cross-linking studies with 125I-ovine CRH revealed that the endometrial CRH receptor has a molecular weight of 45 kDa. Using the non-hydrolysable photoreactive analogue [alpha-32P]GTP-azidoanilide and peptide antisera raised against G-protein alpha-subunits, we then studied coupling of endometrial CRH receptors to G proteins. Treatment of endometrial membranes with human CRH (100 nM) increased the labelling of Gq and Gs, but not Gi or Go. These results were supported by experiments in epithelial cells of the non-pregnant human endometrium in the secretory phase which showed that CRH induced increases in both cAMP and inositol trisphosphate levels. These results suggested that CRH may exert multiple effects in the human endometrium via distinct signalling cascades. These events are possibly mediated via different receptor subtypes.

Production and utilization of monoclonal antibodies to human/rat corticotrophin-releasing factor-41.

Murine monoclonal antibodies against human/rat corticotrophin-releasing factor-41 (CRF-41) were produced and characterized for use in the immunological and biological characterization of CRF-41. Spleen cells from BALB/c mice immunized with CRF-41 conjugated to bovine gamma-globulin were fused with a BALB/c-derived non-secretor X-63 myeloma line. Hybridomas were selected for CRF antibody production by enzyme-linked immunosorbent assay, and positive hybridomas cloned twice. Three monoclonal antibodies were obtained (KCHMB001, KCHMB002 and KCHMB003) and characterized as IgG1, IgG1 and IgG2a isotypes respectively, with affinity constants for rat CRF-41 of 30, 53 and 34 nmol/l respectively. All three monoclonal antibodies recognize an epitope contained between residues 34 and 41 of the human/rat sequence. The antibodies were able to neutralize the ACTH-releasing activity of rat CRF-41, applied to rat pituitary fragments in vitro, in a dose-dependent manner. Isoelectric focusing showed that KCHMB003 detected bands of synthetic rat CRF-41 and rat [Met(O)21,38]-CRF-41 at pH 7.1 and 6.8 respectively. Use of KCHMB003 in a two-site enzyme-amplified immunoassay showed that this antibody recognizes both synthetic rat CRF-41 and immunoreactive CRF-41 in rat hypothalamic tissue extracts.

Peripheral endotoxin induces hypothalamic immunoreactive interleukin-1 beta in the rat.

Interleukin-1 (IL-1) is a polypeptide produced by a variety of cells and contributes to the general host response to inflammation. It displays a wide spectrum of inflammatory, metabolic, physiological, haematopoietic and immunological activities. Brain cells, including neurones, microglia, endothelial cells and astrocytes can all produce IL-1 beta in response to various physiological and pathological stimuli. In this report we show that peripherally administered endotoxin stimulates the appearance of immunoreactive IL-1 beta (IL-1 beta) in the rat hypothalamus as measured by an ultrasensitive, highly specific enzyme amplified immunometric assay for rat IL-1 beta.

Effects of pyrogenic immunomodulators on the release of corticotrophin-releasing factor-41 and prostaglandin E2 from the intact rat hypothalamus in vitro.

1. The actions of the following pyrogens: lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (Poly-I:C), human interleukin (IL)-1 alpha and IL-1 beta, human IL-6 and rat interferon (INF) on corticotrophin-releasing factor-41 (CRF-41) and prostaglandin E2 (PGE2) release from the intact rat hypothalamus in vitro have been studied. 2. Rat hypothalami were incubated in vitro in an artificial cerebrospinal fluid. Immunoreactive (ir)-CFR-41 and PGE2 released into the medium were measured by two-site enzyme amplified immunometric assay (EAIA) and radioimmunoassay (RIA) respectively. 3. Human IL-6 (1 to 10,000 IU ml-1) caused a dose-dependent release of irCRF-41, rising to a maximal 3-4 fold increase over basal at the highest dose tested. Human IL-1 alpha (1 to 1000 IU ml-1), human IL-1 beta (1 to 1000 IU ml-1), poly-I:C (10 pg ml-1 to 100 micrograms ml-1) and rat INF (1 to 10,000 IRu ml-1) all failed to alter irCRF-41 release. 4. LPS (1 mg ml-1) caused a 35% decrease in irCRF-41 release; however, over the dose-range of 0.1 microgram ml-1 to 100 micrograms ml-1, LPS failed to alter irCRF-41 release. The decreased irCRF-41 release in response to LPS (1 mg ml-1) was accompanied by a decrease in the subsequent 56 mM KCl stimulation of irCRF-41. 5. Human IL-1 alpha and IL-1 beta (1000 IU ml-1) were able to stimulate the release of irPGE2 from intact hypothalami, causing a 2 fold increase over basal release. Poly-I:C (100 microg ml-1), LPS (0.1 microg ml-1 to 1 mg ml-1), rat INF (10,000 IRu ml-1) and human IL-6 (1 to 10,000 iu ml-1) all failed to alter irPGE2release.6. In conclusion, these results suggest that the in vitro release of CRF-41 and PGE2, in response to pyrogens, are mediated via different cytokines. In view of this it is possible that different cytokines may mediate the temperature, prostaglandin and hypothalamo-pituitary-adrenocortical axis activation seen during pyrogenic stimulation in vivo.