Biomedical subjects

W Vale

Publications and source records attributed to W Vale.

At least 73 records · Page 4Linked to original sources

PubMed1994

Association of human corticotropin-releasing hormone to its binding protein in blood may trigger clearance of the complex.

Late in the last trimester of human pregnancy, as plasma CRH levels rise, the concentration of circulating CRH-binding protein (CRH-BP) falls. We have investigated, using nonpregnant subjects, the hypothesis that CRH has a negative effect on plasma levels of CRH-BP. A specific RIA developed with the aid of recombinant binding protein has been used to measure CRH-BP. Subjects given iv infusions of human CRH for 10 h showed a sustained fall in plasma CRH-BP for the duration of the infusion. Intravenous bolus injection of human CRH produced a rapid reduction in CRH-BP levels to 54% of the basal value, whereas ovine CRH was without effect, even though both peptides are cleared from the plasma at similar rates and have similar effects on the pituitary-adrenal axis. The rapid clearance was concluded to be related to ligand affinity, as ovine CRH has a 200-fold lower affinity than human CRH for CRH-BP. We suggest that the rising levels of CRH are responsible for the reduction in CRH-BP concentrations observed in late pregnancy, and that this reduction is triggered by the binding of CRH-BP to its ligand.

Animals↗

PubMed1994

Characterization of gonadal sex cord-stromal tumor cell lines from inhibin-alpha and p53-deficient mice: the role of activin as an autocrine growth factor.

Inhibin-alpha-deficient mutant mice have been generated by a targeted deletion of the inhibin-alpha gene through homologous recombination in murine embryonic stem cells. Essentially all of the homozygous mutants develop gonadal sex cord-stromal tumors. To investigate their endocrine and proliferative characteristics, gonadal tumor cells were maintained in vitro. Cells from inhibin-alpha-deficient mice multiplied poorly; however, cells from mice deficient in both inhibin-alpha and p53 proliferated rapidly and showed higher saturation density and plating efficiency, thus allowing the establishment of clonal tumor cell lines. Although negligible estrogen and testosterone was produced by the clonal cells, high levels of progesterone were secreted. A clonal testis tumor cell line (inhibin-alpha/p53 deficient) showed no response to exogenous FSH, human CG (hCG), or inhibin A but exhibited a 6- to 8-fold increase in progesterone production in response to forskolin treatment. The stimulatory effect of forskolin was, however, partially blocked by activin treatment. Northern blot analysis revealed inhibin beta A and beta B mRNA expression in these cells. Furthermore, Western blot analyses indicated the secretion of the beta A-subunit protein. We further tested the role of activin on tumor cell growth. Treatment with follistatin, an activin-binding protein, inhibited tumor cell replication in a dose-dependent manner. In contrast, treatment with activin A stimulated tumor cell growth by itself and partially blocked follistatin action. Incorporation of thymidine into DNA of these cells was also stimulated by activin. In addition, treatment with antiactivin A serum inhibited tumor cell replication and blocked the stimulatory action of activin on cell growth. The activin action is likely mediated by specific receptors because cross-linking of [125]activin to the 50-55 kilodalton type I and 75-80 kilodalton type II receptors was found using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Northern blot analysis also revealed follistatin mRNA expression in the tumor cells, suggesting these cells are related to granulosa cells. Our findings indicate that activin can act as an autocrine growth factor in stimulating the proliferation of gonadal tumor cell lines derived from inhibin-alpha and p53-deficient mice and inhibits progesterone production. These tumor cell lines are useful for studies on the regulation of gonadal cell proliferation and steroidogenesis as well as the signaling pathway mediating activin action.

Activins↗

PubMed1994

Activin at parturition: changes of maternal serum levels and evidence for binding sites in placenta and fetal membranes.

OBJECTIVE: To evaluate maternal serum activin A levels in pregnant women at parturition, correlated to the mode of delivery, and to localize activin receptor messenger RNA in human placenta and fetal membranes. METHODS: A specific two-site enzyme-linked immunosorbent assay (ELISA) was used to measure maternal activin A levels. Activin receptor mRNA was localized in placenta and fetal membranes by in situ hybridization, using ActRII or ActRIIB antisense riboprobes. RESULTS: Serum activin A levels increased significantly in pregnant women during vaginal or cesarean delivery after spontaneous labor. No significant changes of serum activin A were found in patients undergoing elective cesarean delivery. Syncytiotrophoblast and amnion cells hybridized to radiolabeled ActRIIB probe, whereas few cells within the structure of the villi and decidual cells hybridized to radiolabeled ActRII probe. CONCLUSION: The present studies indicate that vaginal or cesarean delivery following spontaneous labor is characterized by increased activin A levels and that activin receptors are present on trophoblast and fetal membranes.

Activin Receptors↗

PubMed1994

Characterization of a novel and potent corticotropin-releasing factor antagonist in rats.

The present study examined the ability of two analogs of human/rat corticotropin-releasing factor (h/rCRF), [Met18, Lys23, Glu27,29,40, Ala32,41, Leu33,36,38] h/rCRF9-41 (alpha-hel CRF9-41) and [D-Phe12, Nle21,38, C alpha MeLeu37] h/rCRF12-41 (D-Phe CRF12-41), to antagonize CRF- and stress-induced behavioral changes in rats. The potency and duration of action of D-Phe CRF12-41 in vivo was compared with that of alpha-hel CRF9-41, the most effective CRF antagonist studied to date. When administered i.c.v., both CRF antagonists dose-dependently reduced the locomotor activity induced by rat CRF (0.5 microgram/rat i.c.v.) and attenuated the social stress-induced anxiogenic-like effect, as measured by the elevated plus-maze. However D-Phe CRF12-41 was 5 times more potent and remained effective for a longer period (> 1.5 hr) than alpha-hel CRF9-41. High doses of alpha-hel CRF9-41 (25 micrograms/rat), by itself, exhibited weak agonist effects, which were not observed with D-Phe CRF12-41 at the same doses. These results demonstrate that a N-terminus-shortened CRF antagonist that encompasses the substitution D-Phe12 has significantly higher biological potency and an extended duration of action without intrinsic agonist effects in these in vivo systems. The availability of potent and effective CRF antagonists will provide valuable tools for exploring the functional role of brain CRF and may ultimately lead to an understanding of the biological basis of stress-related illnesses.

Amino Acid Sequence↗

PubMed1993

Chlordiazepoxide attenuates stress-induced accumulation of corticotropin-releasing factor mRNA in the paraventricular nucleus.

Corticotropin-releasing factor (CRF) plays a role in coordinating endocrine, autonomic and behavioral responses to stressful stimuli. Benzodiazepines exert many effects which are antithetical to those of CRF, including anxiolysis and suppression of the pituitary-adrenal axis. Although there is evidence that benzodiazepines can modulate several electrophysiological and behavioral responses to exogenous CRF, we questioned whether this class of drug might also affect CRF biosynthesis as well. We have shown previously that footshock stress increases CRF mRNA levels as monitored by in situ hybridization histochemical techniques in the paraventricular nucleus (PVN) and Barrington's nucleus (the pontine micturition center). We report here the effects of the potent benzodiazepine, chlordiazepoxide (CDP), on stress-induced CRF mRNA accumulation in these two regions. Male albino rats were exposed to electrical footshock (1.5 mA, 1-s duration, 60 times/30 min) twice daily for 4 days and sacrificed 24 h after the last shock session. Either CDP (1, 2.5, 5 or 10 mg/kg) or saline was given i.p. 30 min before each stress. Sections were hybridized with an 35S-labeled prepro-CRF cRNA probe. Relative levels of CRF mRNA were quantified by densitometry of the autoradiography with X-ray film. CRF mRNA concentrations were significantly increased in both the PVN and Barrington's nucleus after stress, and CDP attenuated these increases in the PVN. By contrast, CDP did not affect CRF mRNA accumulation in Barrington's nucleus after stress. The results suggest that the benzodiazepine, CDP, suppresses stress-induced pituitary adrenal activation at least in part through inhibition of CRF production in the PVN.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Single point D-substituted corticotropin-releasing factor analogues: effects on potency and physicochemical characteristics.

In an attempt to determine which conformational parameters are important for the biological activity of ovine corticotropin-releasing factor (oCRF), we have synthesized in significant amounts (50-200 mg) and characterized chemically, structurally (CD), and biologically, oCRF analogues with substitution of each amino acid by its corresponding D-isomer. Out of 37 of these analogues, three were found to be equipotent to, or twice as potent as, oCRF, 13 had potencies in the range from 10 to 60%, 17 had potencies ranging from 1 to 10%, and the four others had potencies less than 0.5%. None of the analogues antagonized oCRF-induced release of ACTH in vitro at concentrations > or = 1000 oCRF. Since antagonists to CRF action can be generated by deletion of the first 8-14 residues, a series of CRF antagonists which exhibit significantly higher in vitro and in vivo biological potency than [Met18,Lys23,Glu27,29,40,Ala32,41,-Leu33,3 6,38] h/rCRF, [alpha-helical-CRF9-41], is also described. [D-Phe12,Nle21,38,Arg36]h/rCRF, in particular, was found to be ca. 15 times more potent than alpha-helical-CRF9-41 in vitro. In the rat, however, this analogue was about as effective as alpha-helical-CRF9-41 in blocking CRF-induced decrease in mean arterial blood pressure and increase in heart rate. Its potency in blocking epinephrine release by CRF was not significantly different from that of alpha-helical-CRF9-41. In the adrenalectomized rat, [Lys36]alpha-helical-CRF(9-41) (1.7 mg/kg) blunted the effect of endogenous CRF over a 90-min period; by comparison, a similar dose of alpha-helical-CRF9-41 was effective for less than 1 h.

Adrenocorticotropic Hormone↗

PubMed1993

Synthesis and relative potencies of new constrained CRF antagonists.

Two series of CRF antagonists with N alpha- and C alpha-methylated alanine and leucines were evaluated for their biological activities in vitro and in vivo in several systems. The poly-N-methylated analogue of alpha-helical-CRF9-41, [N alpha MeLeu10,15,27,37,N alpha MeAla22,32,41]-alpha-Hel-CRF9-41, was found to be considerably less potent than the parent non-N-methylated analogue. This result was expected on the basis that alpha-helicity was thought to be required for biological activity and the prediction that backbone substitutions on the nitrogen have a tendency to break alpha-helices (a hypothesis that was confirmed by circular dichroism). Next, a series of constrained analogues of the potent CRF antagonist, [DPhe12,Nle21,38]h/rCRF12-41, was synthesized that contained C alpha-methylleucine and/or C alpha-methylalanine (Aib) residues at selected positions. Because C alpha-methylation is recognized to increase alpha-helicity, and because there is now strong NMR data suggesting that residues 6-36 assume a well-defined alpha-helix, it was expected that these analogues would be more potent. Although usual solid-phase peptide synthesis procedures were followed, success in coupling the C alpha-methyl amino acids was obtained only with a 1:1 mixture of BOP/HOBt. In vitro potencies of the synthesized compounds were measured in a collagenase-dispersed anterior pituitary cell culture bioassay. Monosubstituted analogues were shown to be twice to one fourth as potent as the parent compound; while the pluri-substituted peptides were slightly less potent. This decrease in potency might be correlated to an unexpected lower helical content of the pluri-substituted compounds (as determined by CD spectroscopy), as it was suggested that the bioactive conformation of the CRF was predominantly alpha-helical. Interestingly, one analogue, [DPhe12,Nle21,38,C alpha-MeLeu37]h/rCRF12-41, was found to be more potent and longer acting than the parent compound in two in vivo assays measuring ACTH release after intravenous administration to adrenalectomized rats and reversal of stress-induced delay in gastric emptying in the rat after intracisternal administration. The molecular basis for this increased duration of action and potency is being investigated.

Adrenalectomy↗

PubMed1993

The functional neuroanatomy of corticotropin-releasing factor.

Descriptions of the central distribution of corticotropin-releasing factor (CRF) have been taken as generally supporting the proposition that this neuropeptide is involved in the mediation of complementary neuroendocrine, autonomic and behavioural responses to stress. The hypothalamic paraventricular nucleus (PVN) is recognized as the principal source of CRF in hypophysial portal plasma; CRF mRNA and peptide expression in parvocellular neurosecretory neurons are regulated negatively by adrenal steroids and positively by many stressors. Consistent with the latter, the hypophysiotropic zone of the PVN receives a rich, and biochemically differentiated, afferent supply that provides visceral, somatic and special sensory systems with access to the 'CRF neuron'. Within the PVN, CRF is also expressed, and differentially regulated, in oxytocinergic magnocellular neurosecretory neurons and in autonomic-related projection neurons. CRF expression in at least some extrahypothalamic cell groups (olfactory bulb, Barrington's nucleus) is responsive to certain stressful stimuli, but not to perturbations of the steroid environment. Refinement of our understanding of the central distribution of CRF has been provided by the recognition that most CRF antisera cross-react with an amidated dipeptide encoded by the melanin-concentrating hormone precursor, and by the likelihood that some central sites of CRF peptide expression may be muted or masked by the presence of a CRF-binding protein (CRF-BP). The CRF-BP is expressed prominently in the telencephalon, where it is co-localized with CRF in some neurons, and in anterior pituitary corticotrophs.

Afferent Pathways↗

PubMed1993

Immunocytochemical evidence for the presence of inhibin and activin-like proteins and their localization in goldfish gonads.

In previous studies, we have demonstrated that inhibin and activin are stimulatory to goldfish gonadotropin release. In the present study, the distribution of immunoreactive inhibin and activin subunits (alpha, beta A, and beta B) in goldfish gonads was examined with domain-specific antibodies against mammalian inhibin and activin subunits, using the avidin-biotin-peroxidase complex method. In the ovary, follicle cells surrounding the oocyte were heavily stained with anti-porcine beta A and human beta B subunits. The cytoplasm of previtellogenic oocytes, at all stages of the ovarian cycle, also showed strong reactions with anti-beta A and beta B. As ovarian recrudescence progressed, oocytes that started to accumulate cortical vesicles gradually lost their immunoreactivity, with the reaction intensity inversely proportional to the amount of cortical vesicles in the oocyte; when oocytes became full of cortical vesicles, the oocytoplasm was no longer immunoreactive to anti-beta A and beta B. The staining with antiporcine alpha subunit was similar to that with anti-beta A and beta B but the reaction intensity was weaker than that with anti-beta A and beta B. In the testis, the mature sperm in the tubular lumen exhibited a strong immunoreaction to anti-alpha antibody, while the interstitial tissue was completely negative. However, the immunoreactivities with anti-beta A and beta B in the testis were exclusively restricted to the interstitial tissues. These results provide evidence for the presence of inhibin and activin-like molecules in the goldfish and reveal the distribution of these molecules in the goldfish gonads.

Activins↗

PubMed1993

Corticotropin-releasing factor release from the mediobasal hypothalamus of the rat as measured by microdialysis.

Procedures were developed to permit the measurement of corticotropin-releasing factor in perfusate collected from microdialysis probes implanted in various brain areas of anesthetized and awake rats. Initially in vitro experiments were carried out to optimize the recovery of corticotropin-releasing factor and the radioimmunoassay conditions. Addition of a specific antiserum against corticotropin-releasing factor to the perfusion medium (artificial cerebrospinal fluid) increased the relative in vitro recovery over a range of different flow rates (1-10 microliters/min) using commercially available microdialysis probes with a membrane cutoff of 20,000 mol. wt. This procedure increased recovery from 3% to 6% at flow rate of 2.5 microliters/min, and from 4% to 8% at a flow rate of 5 microliters/min. In vivo experiments were performed with a flow rate of 3.3 microliters/min and 50-microliters fractions were used for radioimmunoassay. In each experiment, the standard curve of the radioimmunoassay was constructed from aliquots of the same medium used to perfuse the probe. Basal levels of corticotropin-releasing factor in dialysate collected from the mediobasal hypothalamus of anesthetized rats were estimated to be 0.75 +/- 0.07 fmol/50 microliters. Raising the concentration of potassium (60 mM) in the perfusate increased corticotropin-releasing factor levels to 2.04 +/- 0.37 fmol/50 microliters. Hypertonic stress induced by intraperitoneal injection of 1.5M NaCl (20 ml/kg) elevated the levels to 1.32 +/- 0.07 fmol/50 microliters. A marked increase of corticotropin-releasing factor levels was also produced by a 10-min pulse of the potassium-channel blocker 4-aminopyridine (10 mM) included in the perfusate. A second stimulation pulse with 4-aminopyridine, administered 2 h after the first pulse again increased the levels, with a mean ratio between the first and second pulse of 0.97. Corticotropin-releasing factor efflux produced by the second stimulation pulse was completely inhibited by perfusion with calcium-free medium containing calcium-chelating agent ethyleneglycol tetraacetic acid (10 mM). In separate experiments, microdialysis probes were implanted in several brain areas of anesthetized rats. Basal and potassium-evoked levels of corticotropin-releasing factor were measured in dialysate collected from the amygdala (1.20 +/- 0.22 and 2.05 +/- 0.48 fmol/50 microliters, respectively) and frontal cortex (0.51 +/- 0.10 and 1.64 +/- 0.15 fmol/50 microliters, respectively). Corticotropin-releasing factor levels in the dorsal part of the third ventricle and in the striatum were below the detection limits. In awake rats, corticotropin-releasing factor levels in the mediobasal hypothalamus were 0.98 +/- 0.03 fmol/50 microliters.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenocorticotropic Hormone↗

PubMed1993

Rapid regression of uterine leiomyomas in response to daily administration of gonadotropin-releasing hormone antagonist.

OBJECTIVE: The efficacy of acute and sustained pituitary gonadotropin down-regulation by the Nal-Glu GnRH antagonist (Nal-Glu) was evaluated in the treatment of uterine leiomyomas. DESIGN: Prospective, open clinical trial. PATIENTS: Seven normally cycling women with symptomatic leiomyomas. INTERVENTIONS: Nal-Glu (50 micrograms/kg per day) was administered subcutaneously for 3 months. MAIN OUTCOME MEASURES: Baseline ultrasound examinations were obtained and repeated monthly throughout treatment. Each leiomyoma was mapped and measured in three dimensions. Blood samples were drawn daily for 7 days, weekly for 4 weeks, and monthly for the remaining 2 months. RESULTS: Mean leiomyoma size decreased 52.8 +/- 7.3% (means +/- SD) after 1 month of therapy and remained unchanged for the remainder of the study. Serum levels of E2 (35.9 +/- 11.8 to 9.3 +/- 0.8 pg/mL, 131.7 +/- 43.3 to 34.0 +/- 1.4 pmol/L), estrone (37.3 +/- 7.5 to 13.0 +/- 2.5 pg/mL, 138.1 +/- 27.7 to 48.1 +/- 9.1 pmol/L), and P (1.6 +/- 1.1 to 0.3 +/- 0.01 ng/mL, 5.0 +/- 3.6 to 0.9 +/- 0.04 nmol/L) declined rapidly (within 48 hours) and remained suppressed throughout treatment. Serum LH, FSH, androstenedione, T, and DHEA levels did not change significantly. In two subjects who did not have surgical removal, leiomyomas grew to original size within the 1st month off drug. Six patients remained amenorrheic and the other subject spotted during the last 2 months of therapy. CONCLUSIONS: Continuous treatment with Nal-Glu induces immediate and sustained pituitary-gonadal down-regulation that results in regression in leiomyoma size. By circumventing GnRH agonist-induced pituitary-ovarian up-regulation, GnRH antagonists may prove to be superior tools in the medical management of leiomyomas.

Adult↗

PubMed1993

Coupling of hormonal stimulation and transcription via the cyclic AMP-responsive factor CREB is rate limited by nuclear entry of protein kinase A.

Cyclic AMP (cAMP) regulates a number of eukaryotic genes by mediating the protein kinase A (PKA)-dependent phosphorylation of the CREB transcription factor at Ser-133. In this study, we test the hypothesis that the stoichiometry and kinetics of CREB phosphorylation are determined by the liberation and subsequent translocation of PKA catalytic subunit (C subunit) into the nucleus. Using fluorescence imaging techniques, we observed that PKA was activated in a stimulus-dependent fashion that led to nuclear entry of C subunit over a 30-min period. The degree of CREB phosphorylation, assessed with antiserum specific for CREB phosphorylated at Ser-133, correlated with the amount of PKA liberated. The time course of phosphorylation closely paralleled the nuclear entry of the catalytic subunit. There was a linear relationship between the subsequent induction of the cAMP-responsive somatostatin gene and the degree of CREB phosphorylation, suggesting that each event--kinase activation, CREB phosphorylation, and transcriptional induction--was tightly coupled to the next. In contrast to other PKA-mediated cellular responses which are rapid and quantitative, the slow, incremental regulation of CREB activity by cAMP suggests that multifunctional kinases like PKA may coordinate cellular responses by dictating the kinetics and stoichiometry of phosphorylation for key substrates like CREB.

Animals↗

PubMed1993

Protooncogene junB as a target for activin actions.

Activin, a member of the transforming growth factor-beta family of peptides, is implicated in the regulation of cell growth and differentiation in a variety of biological systems. We have sought to identify immediate early genes whose altered expression may provide a common nuclear event involved in activin-regulated phenotypic changes in many cell types. In both human K562 myelogenous leukemia and rat PC12 pheochromocytoma cells, activin treatment caused transient transcription-dependent and protein synthesis-independent increases of junB messenger RNA (mRNA) within 1 h, whereas neither c-jun nor c-fos mRNA were inducible. In K562 cells, this selective junB mRNA induction was synergistically augmented by treatment with 12-O-tetradecanoyl phorbol-13-acetate but not affected by forskolin. Furthermore, in PC12 cells, the up-regulation of junB mRNA by activin was observable even after high-dose treatment with 12-O-tetradecanoyl phorbol-13-acetate for 48 h, indicating that junB mRNA expression by activin is independent of both A- and C-kinases. Our report suggests that induction of this ubiquitous gene product may be a critical event shared by a set of activin-responsive tissues.

Activins↗

PubMed1993

Inhibin and activin in human fetal membranes: evidence for a local effect on prostaglandin release.

Trophoblast and fetal membranes (amnion and chorion) are the tissues forming the anatomical/functional interface between the mother and the fetus. The aim of the present study was to evaluate the presence and a possible effect of inhibin and activin in human amnion and chorion. The expression of inhibin/activin alpha, beta A, and beta B subunit messenger RNA (mRNA) and the localization of immunoreactive material were evaluated in amnion and chorion collected at term pregnancy by a Northern blot analysis and by immunohistochemistry. Amnion cells expressed the mRNA of the three inhibin/activin subunits, with the beta B message the most abundantly expressed. The epithelial layer of the amnion showed an intense fluorescent staining of beta B inhibin/activin subunit, and positive signals were also observed for the alpha and beta A subunits. Inhibin/activin alpha and beta A subunit mRNAs were found highly expressed in chorion. The cytotrophoblast of the chorion showed a positive staining for the three inhibin/activin subunit antisera. The addition of activin A increased the release of prostaglandin E2 from human amnion-derived cultured cells. The effect was dose and time dependent. Inhibin A did not induce significant changes of prostaglandin E2 release from amnion cells. The present results show that amnion and chorion produce inhibin and activin subunits and that activin stimulates the release of prostaglandin E2 from cultured amnion cells, suggesting a possible role of inhibin and activin in fetal membranes.

Activins↗

PubMed1993

Corticotropin-releasing factor-binding protein is produced by human placenta and intrauterine tissues.

CRF circulates in high concentration in pregnant woman. It is produced by the placenta and the other intrauterine tissues (maternal decidua, amnion, and chorion). Recently, a CRF-binding protein (CRF-BP) has been identified and cloned. It binds the circulating CRF, reducing its biological action during pregnancy. Liver is the major source of CRF-BP. The aim of the present study was to evaluate whether human placenta and intrauterine tissues produce CRF-BP. The localization of mRNA and immune CRF-BP by in situ hybridization and immunohistochemistry, respectively, was performed. Antisense and sense riboprobes synthesized from a fragment of human CRF-BP cRNA and a specific rabbit anti-hCRF-BP serum was used. The syncytial layer of placental villi at term intensely expressed CRF-BP mRNA and immunoreactivity, whereas rare positively hybridized cells were observed within the cytotrophoblasts and mesenchymal cells. Large decidual cells, amniotic epithelial cells, and chorionic cytotrophoblast stained positively for CRF-BP mRNA and protein. Control sections collected from the same tissues failed to show any positive localization of sense strand cRNA probe and antiserum preadsorbed with immunogen. Finally, the addition of recombinant CRF-BP to human cultured placental cells significantly decreased CRF-induced ACTH release, with a dose-dependent effect. The present data show that local production of CRF-BP occurs in human trophoblast and intrauterine tissues and may represent one of the major mechanisms used by targets tissues to control CRF activity during pregnancy.

Adrenocorticotropic Hormone↗

PubMed1993

Corticotropin-releasing factor activates c-fos, NGFI-B, and corticotropin-releasing factor gene expression within the paraventricular nucleus of the rat hypothalamus.

Studies examining the regulation of hypothalamic CRF biosynthesis have provided substantial information regarding the relevance of this peptide in neuroendocrine homeostasis. However, the consequences of elevated CRF levels within the mammalian central nervous system on regulation of CRF production within the paraventricular nucleus (PVN) of the hypothalamus remain unclear. The expression of the immediate early gene c-fos has been used and validated as a marker for neural systems activated by a variety of extracellular stimuli and has been especially useful when examining activation of central neuroendocrine systems such as those involved in the response to stressful stimuli. The present study investigates the effects of injecting CRF into the lateral ventricle of conscious rats, firstly on the expression of two separate immediate early genes, c-fos and NGFI-B within the hypothalamic PVN, and secondly on the expression of CRF mRNA itself, as determined by quantitative in situ hybridization. Expression of Fos protein was also examined by immunohistochemical techniques. Intracerebroventricular (icv) injection of CRF increased the gene expression of both c-fos and NGFI-B in the parvocellular division of the PVN 30 min after injection. Fos immunoreactivity increased in this same region between 30-60 min, whereas expression of the CRF gene itself increased 2-fold 60 min after injection and remained elevated 2 h after treatment. A positive hybridization signal for CRF was observed over Fos-immunoreactive neurons within the parvocellular division of the PVN. Finally, we observed that all CRF-induced changes in gene expression were abolished by pretreatment with the competitive CRF antagonist alpha-helical CRF-(9-41). The time-related changes in expression of the genes measured imply that the expression of both c-fos and NGFI-B occurs before a significant increase in the expression of CRF. The results also suggest that CRF may act in a positive manner to regulate its own biosynthesis.

Animals↗

PubMed1992

Alanine series of ovine corticotropin releasing factor (oCRF): a structure-activity relationship study.

Previous structure-activity relationship studies of CRF have shown that residues 1-4 were not necessary for receptor binding or transduction, that residues 4-8 were important for activation, and that residues 12-41 were mostly responsible for binding. Finally it was proposed that CRF assumed an alpha-helical structure when interacting with its receptor. By systematic substitution of each residue (except residues 1-4) in ovine CRF (oCRF) by Ala, we have investigated the role played by individual side chains in receptor recognition and activation. Out of 33 analogues (synthesized using SPPS on an MBHA resin, purified by RPHPLC and characterized by amino acid and mass spectral analyses), a significant loss of biological potency (less than 1% potency of native) was observed for 6 analogues ([Ala6], [Ala8], [Ala10], [Ala12], [Ala14], and [Ala38]); 12 analogues had biological potencies ranging from 1% to 60% and ranked as follows: [Ala35] less than [Ala16] less than [Ala9] less than [Ala19] less than [Ala15] less than [Ala13] less than [Ala7] less than [Ala23] less than [Ala11] less than or equal to [Ala21] less than [Ala27] less than or equal to [Ala18]; 8 analogues were found to be equipotent (greater than 60% and less than 150%) ([Ala5], [Ala17], [Ala26], [Ala29], [Ala30], [Ala34], [Ala36], and [Ala37]; and 7 analogues were found to be approximately 2-5 times more potent than native oCRF ([Ala25] = [Ala40] less than or equal to [Ala39] less than or equal to [Ala33] less than [Ala20] less than [Ala22] less than [Ala32], in an in vitro pituitary cell culture assay. In summary, the Ala substitutions which showed the greatest loss of potency (less than 1% of native oCRF) were those replacing hydrophobic residues while those showing the greatest increase in potency were replacing hydrophilic residues. Of the 22 Ala-containing analogues in the C-terminal half of the molecule, 17 analogues have equal or greater potencies than native oCRF. Substitution of Ala in the N-terminal region (residues 5-19) on the other hand is generally detrimental to biological activity. These results suggest that the side chains of residues 5-19 are very important for receptor binding and activation while, in the C-terminal region, the amino acid side chains may be more responsible for structural conservation than for functional expression.

Adrenocorticotropic Hormone↗

PubMed1992

The melanin-concentrating hormone system of the rat brain: an immuno- and hybridization histochemical characterization.

In addition to a nonadecapeptide homologous to the teleost melanin-concentrating hormone (MCH), the amino acid sequence predicted from a rat prepro-MCH (ppMCH) cDNA suggested that at least one (neuropeptide EI, or NEI), and possibly a second (NGE), additional neuropeptide may be encoded by this precursor. Cross-reactivity with epitopes of NEI or NGE can account for reported localization of alpha-MSH, rat CRF, and human GRF in rat dorsolateral hypothalamic neurons. We have used antisera raised against rat MCH and NEI in immunohistochemical studies at the light and electron microscopic levels, along with hybridization histochemical localization of ppMCH mRNA, to define the organization of this system. As expected, ppMCH mRNA is prominently expressed in cells in the lateral hypothalamic area and zona incerta. The MCH and NEI peptides were extensively colocalized in neurons in both of these areas. In addition, smaller cell groups in the olfactory tubercle and pontine tegmentum were also positively hybridized for ppMCH mRNA and immunostained for MCH and NEI. Fibers stained for MCH and NEI were similarly, and very broadly, distributed throughout the central nervous system in patterns that generally conformed with known projection fields of the lateral hypothalamic area and zona incerta. A differential distribution was seen in at least one region, the interanterodorsal nucleus of the thalamus, which contained a prominent terminal field stained for MCH but not NEI. At the electron microscopic level, MCH-stained perikarya displayed a prominent staining associated with the Golgi apparatus; this was not encountered in NEI-stained cells. Both peptides were distributed similarly in terminals in the lateral hypothalamic area and median eminence, with staining associated principally with dense-cored vesicles. The results suggest that ppMCH-derived peptides may serve as neurotransmitters or modulators of prominence in a surprisingly expansive projection field of incerto-hypothalamic neurons. The terminal distributions of this system seem most compatible with functional roles in generalized arousal and sensorimotor integration, processes previously implicated as being subject to modulation by the lateral hypothalamic area.

Animals↗