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J C Buckingham

Publications and source records attributed to J C Buckingham.

At least 37 records · Page 2Linked to original sources
PubMed1999

Localisation and semi-quantitative measurement of lipocortin 1 in rat anterior pituitary cells by fluorescence-activated cell analysis/sorting and electron microscopy.

Lipocortin 1 (LC1, also called annexin 1), a Ca2(+)- and phospholipid-binding protein, is an important mediator of glucocorticoid action in the anterior pituitary gland. Previous studies based on immunoprecipitation and Western blot analysis suggest that LC1 is found intracellularly both in the cytoplasm and in association with membranes and also on the cell surface where it attaches to the membrane by a Ca2(+)-dependent mechanism. However, as yet it is unclear which anterior pituitary cell types express the protein. Accordingly, we have developed a method based on a combination of fluorescence activated cell (FAC) analysis/sorting and electron microscopy to detect and quantify intracellular LC1 in rat anterior pituitary cells and to identify the cell types in which it is expressed. In addition, we have measured cell surface LC1 and examined the influence of glucocorticoids on the cellular disposition of the protein. Anterior pituitary cells were dispersed with collagenase. For experiments measuring intracellular LC1, three cell fixation/permeabilisation methods were examined initially, i.e. (1) Zamboni's fluid (30 min) and Triton-X-100 (0.12%, 1 or 12 h); (2) paraformaldehyde (2%, 1 h) and Triton-X-100 (0.2%, 10 min); and (3) paraformaldehyde (0.2%, 15 min) and saponin (0.1%, 5 min). The protocol using paraformaldehyde/Triton-X-100 provided optimal preservation of cell ultrastructure and of LC1 immunoreactivity (ir-LC1) while also effectively permeabilising the cells; it was therefore used in subsequent studies. Using an anti-LC1 monoclonal antibody as a probe, 82+/-5% of the secretory cells in the heterogeneous anterior pituitary cell preparation were shown by FAC analysis to display specific fluorescence for intracellular ir-LC1. Morphological analysis and immunogold-histochemistry of cells separated by FAC sorting identified corticotrophs, lactotrophs, somatotrophs and gonadotrophs in the population displaying LC1 immunofluorescence. LC1 was also detected on the surface of anterior pituitary cells by FACS analysis. Incubation of anterior pituitary cells with dexamethasone or corticosterone (0.1 and 1.0 microM) prior to fixation and analysis produced a significant, concentration-dependent decrease in intracellular ir-LC1 and a concomitant increase in the amount of ir-LC1 detected on the surface of the cells; the effects of the two steroids were indistinguishable quantitatively. In conclusion, we report a novel method which permits (1) the detection and semi-quantitative measurement of intracellular and surface LC1 in anterior pituitary cells; and (2) the identification of the cell types in which the protein is found.

Animals↗
PubMed1999

Regulation of the hypothalamo-pituitary-adrenal axis by cytokines.

Many of the pro-inflammatory cytokines which are released in response to immune/inflammatory insults exert marked stimulatory influences on the hypothalamo-pituitary-adrenocortical (HPA) axis; they thus provoke the release of glucocorticoids which, in turn, temper the ensuing immune-inflammatory response and thereby complete a homeostatic neuroendocrine-immune regulatory loop. This article reviews the putative mechanisms by which cytokines, released acutely in response to such insults, activate the HPA axis, placing particular emphasis on the actions and interactions of tumour necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and interleukin-6 (IL-6) and on the counter-regulatory mechanisms that are in place.

Animals↗
PubMed1999

Lipocortin 1 (annexin 1): a candidate paracrine agent localized in pituitary folliculo-stellate cells.

It is now well established that lipocortin 1 (LC1) plays an important role as a mediator of early delayed glucocorticoid feedback action in the hypothalamo-hypophysial system. In both the hypothalamus and anterior pituitary gland, LC1 mimics some of the actions of glucocorticoids; moreover, glucocorticoids stimulate the synthesis of LC1 and cause the translocation of intracellular LC1 to the outer cell surface. The mechanism by which LC1 acts in these tissues is only partially understood, but may involve paracrine and/or autocrine actions. To address these possibilities we have investigated the localization of LC1 in the rat pituitary gland, using double labeling immunohistochemistry to identify the pituitary cell types that express LC1. At the light microscopic level LC1 was not detected in the endocrine cells in cryosections of the pituitary, but it was found in abundance in the surrounding folliculo-stellate (FS) cells. In the anterior and interme diate pituitary lobes, there was a near total colocalization of LC1 and S100, a specific marker of FS cells. By contrast, in the posterior pituitary gland, LC1 immunoreactivity was not colocalized with S100 which labeled most pituicytes, or with OX-42 monoclonal antibody, a marker of the microglial cells. Immunogold electron microscopy confirmed that LC1 is present in the nongranulated FS cells. LC1 im munoreactivity was also present in a mouse pituitary FS-like cell line (TtT/GF), particularly in the periphery of the cytoplasm. The localization of LC1 in the FS cells of the anterior pituitary gland defines LC1 as a new marker of the FS cell population. These results support our hypothesis that LC1 acts as one of the paracrine agents liberated by FS cells that modulate the release of pituitary hormones.

Animals↗
PubMed1998

Glucagon-like peptide-1 stimulates luteinizing hormone-releasing hormone secretion in a rodent hypothalamic neuronal cell line.

To examine the influence of the putative satiety factor (GLP-1) on the hypothalamo-pituitary-gonadal axis, we used GT1-7 cells as a model of neuronal luteinizing hormone- releasing hormone (LHRH) release. GLP-1 caused a concentration-dependent increase in LHRH release from GT1-7 cells. Specific, saturable GLP-1 binding sites were demonstrated on these cells. The binding of [125I]GLP-1 was time-dependent and consistent with a single binding site (Kd = 0.07+/-0.016 nM; binding capacity = 160+/-11 fmol/mg protein). The specific GLP-1 receptor agonists, exendin-3 and exendin-4, also showed high affinity (Ki = 0.3+/-0.05 and 0.32+/-0.06 nM, respectively) as did the antagonist exendin-(9-39) (Ki = 0.98+/-0.24 nM). At concentrations that increased LHRH release, GLP-1 (0.5-10 nM) also caused an increase in intracellular cAMP in GT1-7 cells (10 nM GLP-1: 7.66+/-0.4 vs. control: 0.23+/-0.02 nmol/mg protein; P < 0.001). Intracerebroventricular injection of GLP-1 at a single concentration (10 microg) produced a prompt increase in the plasma luteinizing hormone concentration in male rats (GLP-1: 1.09+/-0.11 vs. saline: 0.69+/-0.06 ng/ml; P < 0.005). GLP-1 levels in the hypothalami of 48-h-fasted male rats showed a decrease, indicating a possible association of the satiety factor with the low luteinizing hormone levels in animals with a negative energy balance.

Animals↗
PubMed1998

Blockade of the classical pathway of protein secretion does not affect the cellular exportation of lipocortin 1.

The mechanism by which lipocortin 1 (LC1) is extruded from cells in the brain and periphery in response to a glucocorticoid challenge is unknown. This study examined the influence of three inhibitors of the classical endoplasmic reticulum-Golgi pathway of protein secretion on the dexamethasone-induced (0.1 microM, 2-3 h) cellular exportation of LC1 in vitro in brain (cortex, hippocampus, hypothalamus), anterior pituitary tissue and peritoneal macrophages. In all instances, the steroid-induced exportation of LC1 was unaffected by brefeldin A (1.4 microM), monensin (10 microM) and nocodazole (3.3 microM); however, these drugs readily blocked the release of corticotrophin from pituitary tissue. These data suggest that LC1 is exported by a mechanism distinct from the classical pathway of protein secretion.

Animals↗
PubMed1998

D-amino acid-substituted analogs of corticotropin-releasing hormone (CRH) and urocortin with selective agonist activity at CRH1 and CRH2beta receptors.

The activities of corticotropin-releasing hormone (CRH)-related peptides and several analogs were examined in cells transfected with either CRH1 or CRH2beta receptors, in suppression of heat-induced rat paw edema in pentobarbital-anesthetised animals and in stimulation of release of immunoreactive corticotropin (ir-ACTH) from rat anterior pituitary tissue in vitro. The peptides tested were human/rat (h/r)-CRH, r-urocortin, h-urocortin, white sucker fish or maggy sole urotensin I and some analogs of these peptides substituted with D-amino acids at residues 4 (urocortin), 5 (CRH and urotensin I) and 20 (CRH). In cells transfected with CRH1 receptors, these peptides were similar in potency in stimulation of cAMP accumulation. By contrast, at CRH2beta receptors peptides of the urocortin and urotensin series were more potent than h/r-CRH while [D-Glu20]-h/r-CRH was 6.5-fold less active than h/r-CRH. I.v. administration of h/r-CRH or related peptides 10 min prior to a thermal stimulus produced a significant dose-dependent inhibition of rat paw edema formation. Comparison of the ED50's showed that urocortins ([D-Ser4]-h-urocortin, h-urocortin, [D-Pro4]-r-urocortin, r-urocortin) were approximately 2 to 3 times more active than h/r-CRH, but [D-Glu20]-h/r-CRH was 18.5-fold less active. In the assay for ir-ACTH release, the activity of h/r-CRH and [D-Glu20]-h/r-CRH was similar but [D-Pro5]-h/r-CRH and [D-Pro4]-r-urocortin was less potent than the native peptide. These results provide further evidence that D-amino acid substitution at residue 20 reduces the potency of h/r-CRH at endogenous (anti-edema effect) and transfected (cAMP accumulation) CRH2beta receptors whilst activity at the CRH1 receptor is retained (ACTH-release and cAMP accumulation). On the other hand substitutions at residues 4 or 5 in r-urocortin or h/r-CRH respectively appear to decrease activity at CRH1 but not CRH2beta receptors The modified CRH and urocortin analogs described here may provide clues for the further design of receptor selective ligands.

Adrenocorticotropic Hormone↗
PubMed1997

Glucocorticoids modulate the cellular disposition of lipocortin 1 in the rat brain in vivo and in vitro.

The role of glucocorticoids in the regulation of lipocortin 1 (LC1) mRNA/protein expression in the brain is uncertain. This study has examined the influence of dexamethasone on the disposition of LC1 protein in various central and peripheral tissues in the rat. LC1 was readily detectable in all tissues studied by Western blot analysis. Exposure to dexamethasone in vitro (0.1 microM, 3 h) or in vivo (20 micrograms/100 g i.p., 24 h before autopsy) had no discernible effects on intracellular LC1 levels but increased the amount of LC1 attached to the outer surface of cells (pericellular LC1) in cortex, hippocampus, hypothalamus, pituitary gland and peritoneal macrophages (in vitro only). The results suggest that in central tissues, as in the periphery, glucocorticoids promote the translocation of LC1 from intracellular to pericellular sites.

Animals↗
PubMed1997

The role of phospholipase C in arginine vasopressin secretion by rat hypothalami in vitro.

The role of phosphatidylcholine (PC) and phosphatidylinositol (PI) specific phospholipase C (PLC) enzymes in the release of immunoreactive arginine vasopressin (ir-AVP) from rat hypothalami in vitro was examined. PC-PLC (0.05-01 U ml-1) increased ir-AVP release but PI-PLC (0.01-0.5 U ml-1) did not. The response to a submaximal concentration of PC-PLC (0.075 U ml-1) was inhibited by the protei kinase C (PKC) inhibitor Ro 31-8220 (40 microM) and by removal of extracellular Ca2+ but was unaffected by the nitric oxide (NO) precursor L-arginine (1 mM), the NO synthase inhibitor N omega-nitro-L-arginine benzyl ester (1 mM) and the phospholipase A2 (PLA2) inhibitors quinacrine (100 microM) and dexamethasone (1 microM). The results suggest that PC-PLC plays an important role in AVP secretion. The responses to PC-PLC appear to be mediated by PKC but not by changes in NO synthase or PLA2 activity.

Animals↗
PubMed1997

Lipocortin 1: a second messenger of glucocorticoid action in the hypothalamo-pituitary-adrenocortical axis.

The secretion of hydrocortisone by the adrenal cortex is crucial in balancing the reaction of the body to injury or stress. In the periphery, hydrocortisone inhibits inflammation, downregulates the immune system and produces many other crucial physiological and metabolic changes. Within the neuroendocrine system, hydrocortisone inhibits the release of adrenocorticotrophic hormone (and other pituitary hormones), thereby governing its own secretion. The manifold actions of hydrocortisone are mediated through induction or repression of many genes but one pathway, mediated by the inducible protein lipocortin 1 (LC-1, also known as annexin 1), mediates several important effects both within the hypothalamo-pituitary-adrenocortical axis itself and in the periphery.

Animals↗
PubMed1997

Stimulation of the hypothalamo-pituitary-adrenal axis in the rat by three selective type-4 phosphodiesterase inhibitors: in vitro and in vivo studies.

1. Previous studies in our laboratory have shown that the synthetic xanthine analogue denbufylline, a selective type 4 phosphodiesterase (PDE-4) inhibitor, is a potent activator of the hypothalamo-pituitary-adrenal (HPA) axis when given orally or intraperitoneally (i.p.) to adult male rats. This paper describes the results of experiments in which well established in vivo and in vitro methods were used to compare the effects of denbufylline on HPA function with those of two other selective PDE-4 inhibitors, rolipram and BRL 61063 (1,3-dicyclopropylmethyl-8-amino-xanthine). For comparison, parallel measurements of the immunoreactive- (ir-) luteinising hormone (LH) were made where appropriate. 2. When injected intraperitoneally, rolipram (40 and 200 micrograms kg-1, P < 0.005), denbufylline (0.07-0.6 microgram kg-1, P < 0.05) and BRL 61063 (30 micrograms kg-1, P < 0.005) each produced marked rises in the serum ir-corticosterone concentrations. However, lower doses of rolipram (1.6 and 8 micrograms kg-1) and BRL 61063 (0.25-6 micrograms kg-1) were without effect (P > 0.05). By contrast, intracerebroventricular (i.c.v.) injection of rolipram (8 ng-1 micrograms kg-1) or denbufylline (50 ng-1 microgram kg-1) failed to influence the serum ir-corticosterone concentration. BRL 61063 (8-120 ng kg-1, i.c.v.) was also ineffective in this regard although at a higher dose (1 microgram kg-1, i.c.v.) it produced a small but significant (P < 0.05) increase in ir-corticosterone release. Denbufylline also increased the serum ir-LH concentration when given peripherally (0.2-0.6 microgram kg-1, i.p., P < 0.05) or centrally (100 ng kg-1, i.c.v., P < 0.05) but rolipram (1.6-200 micrograms kg-1, i.p. or 8 ng-1 microgram kg-1, i.c.v.) and BRL 61063 (0.25-30 micrograms kg-1, i.p. or 1 ng-1 microgram kg-1, i.c.v.) did not (P > 0.05). 3. In vitro rolipram (10 microM, P < 0.01), denbufylline (1 mM, P < 0.001) and BRL 61063 (1 and 10 microM, P < 0.05) stimulated the release of corticotrophin releasing hormone (ir-CRH-41) but lower concentrations of the drugs were without effect as also was BRL 61063 at 100 microM (P > 0.05); the rank order of potency was thus BRL 61063 > rolipram > denbufylline. The adenylyl cyclase activator forskolin (100 microM, P < 0.01) also stimulated the release of ir-CRH-41, producing effects which were additive with those of rolipram and denbufylline but not with those of BRL 61063. The secretory responses to forskolin (100 microM) were accompanied by a highly significant increase in the cyclic AMP content of the hypothalamic tissue (P < 0.005). Rolipram (10 microM) also significantly (P < 0.05) elevated the hypothalamic cyclic AMP but denbufylline (10 mM) and BRL 61063 (10 microM) did not. However, all three PDE-inhibitors potentiated the rise in cyclic AMP induced by forskolin (P < 0.05). None of the drugs tested, alone or in combination, modified the release of arginine vasopressin (ir-AVP) from the hypothalamus. 4. Rolipram (100 microM), denbufylline (100 microM) and BRL 61063 (100 microM) stimulated the release of corticotrophin (ir-ACTH) from pituitary tissue in vitro (P < 0.05) but in lower concentrations they were without significant effect. In addition, rolipram (10 microM, P < 0.05), denbufylline (0.1 microM, P < 0.05) and BRL 61063 (10 microM, P < 0.05) potentiated the significant (P < 0.05) rises in ir-ACTH secretion induced by forskolin (100 microM). Forskolin (100 microM) also produced a highly significant increase (P < 0.01) in the tissue cyclic AMP content which was further potentiated by rolipram (10 microM), denbufylline (10 microM) and BRL 61063 (10 microM) which, alone did not affect the cyclic AMP content of the tissue. 5. Since both denbufylline and BRL 61063 possess significant adenosine A1 receptor blocking activity, further studies examined the potential influence of these receptors on the secretion in vitro of CRH-41, AVP and ACTH. The release of ir-CRH-41 was increased significantly by adenosine deaminase (ADA, 5microml-1, P<0.05) and the A1-receptor antagonist, 1,3-dicyclopropyl-8-cyclopentylxanthine (DPCPX, 0.1-10nM, P<0.05). The responses to ADA were abolished by the A1 receptor agonist N6-cyclo-hexyladenosine (CHA, 100nM, P<0.05) which alone had no significant effect on ir-CRH-41 release. ADA (0.1-10microml-1) and DPCPX (1nM) had weak stimulant and inhibitory effects, respectively, on the release of ir-ACTH from the pituitary gland while CHA (0.1-10nM) was without effect. Ligand binding studies with [3H]-DPCPX as a probe demonstrated the presence of specific high affinity A1 binding sites in the hypothalamus (Kd=0.7nM; Bmax=367+/-32fmolmg-1 protein) and in the hippocampus (Kd=1nM; Bmax=1165 +/-145fmolmg-1 protein). In both tissues binding of the ligand was displaced by CHA (IC50=1nM (hypothalamus) and 2nM (hippocampus)), BRL 61063 (IC50=80nM (hypothalamus) and 100nM (hippocampus)) and denbufylline (IC50=5microM (hypothalamus) and 9microM(hippocampus)) but not by rolipram. 6.The results suggest that rolipram, denblufylline and BRL 61063 stimulate the HPA axis in the rat, acting at the levels of both the hypothalamus and the pituitary gland. Their actions may be explained, at least in part, by inhibition of PDE-4 but additional actions including blockade of hypothalamic adenosine A1 receptors by denbufylline and BRL 61063 cannot be excluded.

Adrenocorticotropic Hormone↗
PubMed1997

Characterization and localization of lipocortin 1-binding sites on rat anterior pituitary cells by fluorescence-activated cell analysis/sorting and electron microscopy.

Lipocortin 1 (LC1) is an important mediator of glucocorticoid action in the anterior pituitary gland, where it appears to act via cell surface binding sites to suppress peptide release. We have exploited a combination of fluorescence-activated cell (FAC) analysis/sorting and electron microscopy to detect, characterize, and localize LC1-binding sites on the surface of dispersed rat anterior pituitary cells, using human recombinant LC1 (hu-r-LC1) as a probe. High affinity (Kd = 14 +/- 3 nM) hu-r-LC1-binding sites were detected on approximately 80% of anterior pituitary cells dispersed with collagenase. The binding characteristics of the ligand resembled those observed in leukocytes, in that it was saturable; concentration, Ca2+, and temperature dependent; and abolished by trypsin. Functional studies demonstrated an excellent correlation between the presence of the cell surface binding protein and the capacity of an anti-LC1 monoclonal antibody to abrogate the inhibitory actions of dexamethasone (10 nM) on the release of ACTH initiated in vitro by CRH-41 (1 nM). Morphological analysis of cells harvested by FAC sorting showed that 1) somatotrophs, corticotrophs, lactotrophs, thyrotrophs, and gonadotrophs were all included in the population expressing LC1 binding sites; and 2) the LC1-binding sites assume a punctate distribution across the cell surface. These data show that anterior pituitary cells express high affinity surface LC1-binding protein(s); they thus provide further evidence for a specific membrane mechanism of action of LC1 in regulating the endocrine function of the anterior pituitary.

Adrenocorticotropic Hormone↗
PubMed1997

An antisense oligodeoxynucleotide to lipocortin 1 reverses the inhibitory actions of dexamethasone on the release of adrenocorticotropin from rat pituitary tissue in vitro.

Our previous studies have demonstrated that lipocortin 1 (LC1, also called annexin 1) is an important mediator of glucocorticoid action in the neuroendocrine system, particularly with regard to the powerful inhibitory actions of the steroids on the secretion of ACTH and its hypothalamic releasing hormones. In the present study, we have used an antisense oligodeoxynucleotide (ODN) unique to LC1 to investigate further the role of this protein in the regulatory effects of dexamethasone on ACTH release in vitro from rat anterior pituitary cells. Pituitary cells dispersed with collagenase retained their functional and morphological integrity in vitro and sequestered ODNs in a time-dependent manner from the incubation medium. LC1 was readily detected in the cells by Western blot analysis or by immunoprecipitation/autoradiography after preloading with 35S-methionine/cysteine; the bulk of the protein was contained within an intracellular pool but a small amount was attached to the outer cell surface (pericellular). Dexamethasone (100 nm, 2.5 h) initiated de novo synthesis of LC1; it also increased the amount of LC1 in the pericellular pool detected by either method and caused a concomitant decrease in intracellular LC1. The responses to the steroid were prevented by the inclusion in the medium of an LC1 antisense ODN (50 nM, 3.5 h) but the corresponding sense and scrambled ODN sequences were inert. None of the ODN sequences tested influence the expression of annexin 5 in the pituitary tissue. CRH-41 (100 pM-1 mM), forskolin (1 nM-1 mM) and an L-Ca2+-channel opener BAY K8644 (100 pM-1 microM) initiated concentration dependent increases in immunoreactive- (ir-) ACTH release from the pituitary cells that were reduced (P < 0.01) by preincubation with dexamethasone (100 nM, 2.5 h). The inhibitory effects of the steroid were reversed by the LC1 antisense ODN (50 nM, P < 0.01), whereas the LC1 sense and scrambled control sequences (50 nM) were both ineffective in this respect (P > 0.05). The results add further support to the view that the acute inhibitory effects of glucocorticoids on the secretion of ACTH by the pituitary gland are dependent on the generation of lipocortin 1.

Adrenocorticotropic Hormone↗
PubMed1996

[3H]L-arginine transport and nitric oxide synthase activity in foetal hypothalamic cultures.

[3H]L-arginine uptake and the conversion of [3H]L-arginine to [3H]citrulline were characterized in foetal hypothalamic cultures. [3H]L-arginine uptake was reduced by L-ornithine (10 microM-1 mM), high extracellular K+ (56 mM), L-glutamate (100 microM) and removal of extracellular Ca2+, but was increased by the nitric oxide synthase inhibitor, Nw-nitro-L-arginine benzyl ester (L-NABE; 1 mM). [3H]citrulline formation was inhibited by L-NABE (1 mM), increased by high extracellular K+ (56 mM) and unaffected by L-glutamate (100 microM). Removal of extracellular Ca2+ reduced [3H]citrulline formation by mixed (neurones and glia) and neurone-enriched cultures but not by glial-enriched cells. The results suggest that [3H]L-arginine uptake into hypothalamic cultures is mediated by the system y+ transporter and is dependent on extracellular Ca2+. [3H]citrulline production in hypothalamic neuronal, but not glial, cells is also dependent on extracellular Ca2+.

Amino Acid Transport Systems↗
PubMed1996

In the trout, CRH and AVT synergize to stimulate ACTH release.

Anterior pituitaries of the rainbow trout (Oncorhynchus mykiss) were incubated with graded concentrations of arginine vasotocin (AVT) or synthetic rat corticotrophin-releasing hormone (rCRH-41), alone or in combination, and the ACTH secreted into the medium was measured by a sensitive cytochemical bioassay. The aim was to determine the relative potencies of the two secretogogues and whether, in this fish species, they act synergistically. Rat CRF-41 and AVT both produced concentration-dependent increases in ACTH release. The minimum effective concentration for both peptides was approximately 1 nM but, at higher concentrations, the efficacy of CRF-41 was greater than that of AVT. Clear evidence of synergy between the two peptides was obtained. The response of the trout thus falls in line with observations in mammals and contrasts with findings for the goldfish.

Adrenocorticotropic Hormone↗
PubMed1996

Activation of the HPA axis by immune insults: roles and interactions of cytokines, eicosanoids, glucocorticoids.

It is now well established that challenges to the immune system (e.g., infection, inflammation) initiate diverse changes in neuroendocrine function, the most overt of which is activation of the hypothalamo-pituitary-adrenocortical (HPA) axis. The glucocorticoids that are released as a consequence fulfill a vital role in the maintenance of homeostasis that is effected in part through their ability to quench the immune/inflammatory response and thereby prevent them accelerating to a point where they become hazardous to the host. This article discusses the putative mechanisms by which immune insults stimulate the HPA axis, with particular reference to the roles and interactions of the interleukins, eicosanoids and glucocorticoids.

Animals↗
PubMed1996

Stimulation of the hypothalamo-pituitary-adrenal axis in the rat by the type 4 phosphodiesterase (PDE-4) inhibitor, denbufylline.

1. Preliminary studies in our laboratories showed that the synthetic xanthine analogue denbufylline, a selective type 4 phosphodiesterase (PDE-4) inhibitor, is a potent activator of the hypothalamo-pituitary-adrenal (HPA) axis when given orally to adult male rats. This paper describes the results of experiments in which well established in vivo and in vitro models were used to (a) examine further the effects of denbufylline on HPA function and (b) identify the site and mode of action of the drug within the axis. 2. In vivo, administration of denbufylline (0.1-2.5 mg kg-1, i.p.) produced a significant increase in the serum corticosterone concentration; maximal responses were attained at a dose of 1.0 mg kg-1 (P < 0.01 vs. vehicle control, Scheffe's test). However, when denbufylline was administered by intracerebroventricular injection (0.05-1 micrograms kg-1) it failed to influence significantly the serum corticosterone concentration (P > 0.05 vs. vehicle control, Scheffe's test). The adrenocortical responses to peripheral injections of denbufylline (1 mg kg-1, i.p.) were reduced in rats in which the secretion of endogenous corticotrophin releasing factors (CRFs) from the hypothalamus was blocked pharmacologically (P < 0.01 vs. controls, Scheffe's test). However, denbufylline (0.1 mg kg-1, i.p.) potentiated the significant (P < 0.01) increases in serum corticosterone concentration provoked in "CRF blocked rats' by hypothalamic extract (5 hypothalamic extracts kg-1, i.v.) although it failed to influence (P > 0.05) the relatively moderate increases in corticosterone secretion evoked by CRH-41 (2 mg kg-1, i.v.). 3. In vitro, denbufylline (0.01-1 mM) evoked small but significant (P < 0.05) increases in the release of ACTH from rat anterior pituitary segments; furthermore, at these and lower concentrations (0.01 microM-1 mM), it potentiated the adrenocorticotrophic responses to sub-maximal concentrations of hypothalamic extract (P < 0.01) and forskolin (0.1 mM, P < 0.01) but not those to CRH-41 (10 nM) or 8-bromo-cyclic AMP (1-100 microM). In addition, denbufyline (0.1 mM) increased the anterior pituitary cyclic AMP content (P < 0.05) and potentiated the rises in tissue content of the cyclic nucleotide induced by hypothalamic extract (0.1 hypothalamic equivalents ml-1, P < 0.01) and forskolin (0.1 mM, P < 0.01) but not by CRH-41 (10 nM, P < 0.05). By contrast, denbufylline (1 microM-1 mM) failed to influence the release of AVP from rat isolated hypothalami and stimulated the secretion of CRH-41 (P < 0.01) release only at the highest concentration tested (1 mM). 4. The results suggest that the stimulatory actions of denbufylline on the hypothalamo-pituitary-adrenocortical axis are exerted predominantly at the level of the anterior pituitary gland and that they may be attributed, at least in part, to inhibition of type 4 phosphodiesterase enzymes.

Adrenocorticotropic Hormone↗