Formyl peptide receptors and the regulation of ACTH secretion: targets for annexin A1, lipoxins, and bacterial peptides.

The N-formyl peptide receptors (FPRs) are a family of G-protein coupled receptors that respond to proinflammatory N-formylated bacterial peptides (e.g., formyl-Met-Leu-Phe, fMLF) and, thus, contribute to the host response to bacterial infection. Paradoxically, a growing body of evidence suggests that some members of this receptor family may also be targets for certain anti-inflammatory molecules, including annexin A1 (ANXA1), which is an important mediator of glucocorticoid (GC) action. To explore further the potential role of FPRs in mediating ANXA1 actions, we have focused on the pituitary gland, where ANXA1 has a well-defined role as a cell-cell mediator of the inhibitory effects of GCs on the secretion of corticotrophin (ACTH), and used molecular, genetic, and pharmacological approaches to address the question in well-established rodent models. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis identified mRNAs for four FPR family members in the mouse anterior pituitary gland, Fpr-rs1, Fpr-rs2, Fpr-rs6, and Fpr-rs7. Functional studies confirmed that, like dexamethasone, ANXA1 and two ANXA1-derived peptides (ANXA1(1-188) and ANXA1(Ac2-26)) inhibit the evoked release of ACTH from rodent anterior pituitary tissue in vitro. Fpr1 gene deletion failed to modify the pituitary responses to dexamethasone or ANXA1(Ac2-26). However, lipoxin A4 (LXA4, 0.02-2 microM, a lipid mediator with high affinity for Fpr-rs1) mimicked the inhibitory effects of ANXA1 on ACTH release as also did fMLF in high (1-100 microM) but not lower (10-100 nM) concentrations. Additionally, a nonselective FPR antagonist (Boc1, 100 microM) overcame the effects of dexamethasone, ANXA1(1-188), ANXA1(Ac2-26), fMLF, and LXA4 on ACTH release, although at a lower concentration (50 microM), it was without effect. Together, the results suggest that the actions of ANXA1 in the pituitary gland are independent of Fpr1 but may involve other FPR family members, in particular, Fpr-rs1 or a closely related receptor. They thus provide the first evidence for a role of the FPR family in the regulation of neuroendocrine function.

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.

Post-translational modification plays an essential role in the translocation of annexin A1 from the cytoplasm to the cell surface.

Annexin A1 (ANXA1) has an important role in cell-cell communication in the host defense and neuroendocrine systems. In both systems, its actions are exerted extracellularly via membrane-bound receptors on adjacent sites after translocation of the protein from the cytoplasm to the cell surface of adjacent cells. This study used molecular, microscopic, and pharmacological approaches to explore the mechanisms underlying the cellular exportation of ANXA1 in TtT/GF (pituitary folliculo-stellate) cells. LPS caused serine-phosphorylation of ANXA1 (ANXA1-S27-PO4) and translocation of the phosphorylated protein to the cell membrane. The fundamental requirement of phosphorylation for membrane translocation was confirmed by immunofluorescence microscopy on cells transfected with wild-type or mutated (S27/A) ANXA1 constructs tagged with enhanced green fluorescence protein. The trafficking of ANXA1-S27-PO4 to the cell surface was dependent on PI3-kinase and MAP-kinase. It also required HMG-coenzyme A and myristoylation. The effects of HMG-coenzyme A blockade were overcome by mevalonic acid (the product of HMG-coenzyme A) and farnesyl-pyrophosphate but not by geranyl-geranylpyrophosphate or cholesterol. Together, these results suggest that serine-27 phosphorylation is essential for the translocation of ANXA1 across the cell membrane and also identify a role for isoprenyl lipids. Such lipids could target consensus sequences in ANXA1. Alternatively, they may target other proteins in the signal transduction cascade (e.g., transporters).

Perinatal glucocorticoid treatment disrupts the hypothalamo-lactotroph axis in adult female, but not male, rats.

This study aimed to test the hypothesis that the tuberoinfundibular dopaminergic neurons of the arcuate nucleus and/or the lactotroph cells of the anterior pituitary gland are key targets for the programming effects of perinatal glucocorticoids (GCs). Dexamethasone was administered noninvasively to fetal or neonatal rats via the mothers' drinking water (1 mug/ml) on embryonic d 16-19 or neonatal d 1-7, and control animals received normal drinking water. At 68 d of age, the numbers of tyrosine hydroxylase-positive (TH+) cells in the arcuate nucleus and morphometric parameters of pituitary lactotrophs were analyzed. In control animals, striking sex differences in TH+ cell numbers, lactotroph cell size, and pituitary prolactin content were observed. Both pre- and neonatal GC treatment regimens were without effect in adult male rats, but in females, the overriding effect was to abolish the sex differences by reducing arcuate TH+ cell numbers (pre- and neonatal treatments) and reducing lactotroph cell size and pituitary prolactin content (prenatal treatment only) without changing lactotroph cell numbers. Changes in circulating prolactin levels represented a net effect of hypothalamic and pituitary alterations that exhibited independent critical windows of susceptibility to perinatal GC treatments. The dopaminergic neurons of the hypothalamic periventricular nucleus and the pituitary somatotroph populations were not significantly affected by either treatment regimen in either sex. These data show that the adult female hypothalamo-lactotroph axis is profoundly affected by perinatal exposure to GCs, which disrupts the tonic inhibitory tuberoinfundibular dopaminergic pathway and changes lactotroph morphology and prolactin levels in the pituitary and circulation. These findings provide new evidence for a long-term disruption in prolactin-dependent homeostasis in females, but not males, after inappropriate GC exposure in perinatal life.

Time-specific effects of perinatal glucocorticoid treatment on anterior pituitary morphology, annexin 1 expression and adrenocorticotrophic hormone secretion in the adult female rat.

Perinatal glucocorticoid (GC) treatment is increasingly associated with long-term disturbances in hypothalamo-pituitary-adrenocortical function. In the male rat, such treatment induces profound molecular, morphological and functional changes in the anterior pituitary gland at adulthood. To determine whether these effects are sex-specific, we have examined the effects of perinatal dexamethasone treatment on the female pituitary gland, focusing on (i) the integrity of the annexin 1 (ANXA1) dependent regulatory effects of GCs on adrenocorticotrophic hormone (ACTH) release and (ii) corticotroph and folliculo-stellate (FS) cell morphology. Dexamethasone was given to pregnant (gestational days 16-19) or lactating (days 1-7 post partum) rats via the drinking water (1 microg/ml); controls received normal drinking water. Pituitary tissue from the female offspring was examined ex vivo at adulthood (60-90 days). Both treatment regimes reduced the intracellular and cell surface ANXA1 expression, as determined by western blot analysis and quantitative immunogold electron microscopic histochemistry. In addition, they compromised the ability of dexamethasone to suppress the evoked release of ACTH from the excised tissue in vitro, a process which requires the translocation of ANXA1 from the cytoplasm to the cell surface of FS cells. Although neither treatment regime affected the number of FS cells or corticotrophs, both altered the subcellular morphology of these cells. Thus, prenatal dexamethasone treatment increased while neonatal treatment decreased FS cell size and cytoplasmic area. By contrast, corticotroph size was unaffected by either treatment, as also was the size of the secretory granules. Corticotroph granule density and margination were, however, increased markedly by the prenatal treatment, while the neonatal treatment had no effect on granule density but decreased granule margination. Thus, perinatal dexamethasone treatment exerts long-term effects on the female pituitary gland, altering gene expression, cell morphology and the ANXA1-dependent GC regulation of ACTH secretion. The changes are similar but not identical to those reported in the male.

Lack of annexin 1 results in an increase in corticotroph number in male but not female mice.

Annexin 1 (ANXA1) is a member of the annexin family of phospholipid- and calcium-binding proteins with a well demonstrated role in early delayed (30 min to 3 h) inhibitory feedback of glucocorticoids in the pituitary. We have examined corticotrophs in wild-type and ANXA1 knockout mice to determine the effects of lack of ANXA1 in male and female animals. Anterior pituitary tissue from ANXA1 wild-type, heterozygote and null mice was fixed and examined (i) by confocal immunocytochemistry to determine the number of corticotrophs and (ii) by electron microscopy to examine the size, secretory granule population and secretory machinery of corticotrophs. No differences in these parameters were detected in female mice. In male ANXA1 null mice, there were approximately four-fold more corticotrophs than in wild-type animals. However, the corticotrophs in ANXA1 null mice were smaller and had reduced numbers of secretory granules (the reduction in granules paralleled the reduction in cell size). No differences in the numerical density of folliculo-stellate, gonadotroph, lactotroph or somatotroph cells were detected in male ANXA1 null mice. Plasma corticosterone, adrenocorticotrophic hormone (ACTH) and pituitary pro-opiomelanocortin mRNA were unchanged but pituitary ACTH content was increased in male ANXA1 null mice. Interleukin (IL)-6 pituitary content was significantly elevated in male and reduced in female ANXA1 null mice compared to wild-type. In conclusion, these data indicate that ANXA1 deficiency is associated with gender-specific changes in corticotroph number and structure, via direct actions of ANXA1 and/or indirect changes in factors such as IL-6.

Perinatal glucocorticoid treatment produces molecular, functional, and morphological changes in the anterior pituitary gland of the adult male rat.

Stress or glucocorticoid (GC) treatment in perinatal life can induce long-term changes in the sensitivity of the hypothalamo-pituitary-adrenocortical axis to the feedback actions of GCs and, hence, in GC secretion. These changes have been ascribed largely to changes in the sensitivity of the limbic system, and possibly the hypothalamus, to GCs. Surprisingly, the possibility that early life stress/GC treatment may also exert irreversible effects at the pituitary level has scarcely been addressed. Accordingly, we have examined the effects of pre- and neonatal dexamethasone treatment on the adult male pituitary gland, focusing on the following: 1) the integrity of the acute annexin 1 (ANXA1)-dependent inhibitory actions of GCs on ACTH secretion, a process requiring ANXA1 release from folliculostellate (FS) cells; and 2) the morphology of FS cells and corticotrophs. Dexamethasone was given to pregnant (d 16-19) or lactating (d 1-7 postpartum) rats via the drinking water (1 microg/ml); controls received normal drinking water. Pituitary tissue from the offspring was examined ex vivo at d 90. Both treatment regimens reduced ANXA1 expression, as assessed by Western blotting and quantitative immunogold labeling. In particular, the amount of ANXA1 located on the outer surface of the FS cells was reduced. By contrast, IL-6 expression was increased, particularly by the prenatal treatment. Pituitary tissue from untreated control rats responded to dexamethasone with an increase in cell surface ANXA1 and a reduction in forskolin-induced ACTH release. In contrast, pituitary tissue from rats treated prenatally or neonatally with dexamethasone was unresponsive to the steroid, although, like control tissue, it responded readily to ANXA1, which readily inhibited forskolin-driven ACTH release. Prenatal dexamethasone treatment reduced the size but not the number of FS cells. It also caused a marked reduction in corticotroph number and impaired granule margination without affecting other aspects of corticotroph morphology. Similar but less marked effects on pituitary cell morphology and number were evident in tissue from neonatally treated rats. Our study shows that, when administered by a noninvasive process, perinatal GC treatment exerts profound effects on the adult pituitary gland, impairing the ANXA1-dependent GC regulation of ACTH release and altering the cell profile and morphology.

Altered mesencephalic dopaminergic populations in adulthood as a consequence of brief perinatal glucocorticoid exposure.

Early exposure to stressors is strongly associated with enduring effects on central nervous system function, but the mechanisms and neural substrates involved in this biological 'programming' are unclear. This study tested the hypothesis that inappropriate exposure to glucocorticoid stress hormones (GCs) during critical periods of development permanently alters the mesencephalic dopaminergic populations in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Using a rat model, the synthetic GC dexamethasone was added to the maternal drinking water during gestational days 16-19 or over the first week of postnatal life. In adulthood, the effects upon tyrosine hydroxylase immunopositive (TH+) cell numbers in the midbrain, and monoamine levels in the forebrain, of the adult offspring were assessed and compared with control offspring whose dams received normal drinking water. In the VTA, both prenatal and postnatal dexamethasone treatment increased TH+ cell numbers by approximately 50% in males and females. Although prenatal dexamethasone treatment also increased TH+ cell numbers in the SNc by 40-50% in males and females, postnatal treatment affected females only by increasing TH+ cell numbers by approximately 30%. In comparison, similar changes were not detected in the monoamine levels of the dorsolateral striatum, nucleus accumbens or infralimbic cortex of either males or females, which is a feature likely to reflect adaptive changes in these pathways. These studies demonstrate that the survival or phenotypic expression of VTA and SNc dopaminergic neurones is profoundly influenced by brief perinatal exposure to GCs at times when endogenous levels are normally low. These findings are the first to demonstrate permanent changes in the cytoarchitecture within midbrain dopamine nuclei after perinatal exposure to stress hormones and implicate altered functionality. Thus, they have significance for the increasing use of GCs in perinatal medicine and indicate potential mechanisms whereby perinatal distress may predispose to the development of a range of psychiatric conditions in later life.

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.

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.

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.

Annexin 1 (lipocortin 1) mediates the glucocorticoid inhibition of cyclic adenosine 3',5'-monophosphate-stimulated prolactin secretion.

Our previous studies have identified a role for annexin 1 (also called lipocortin 1) in the regulatory actions of glucocorticoids (GCs) on the release of PRL from the rat anterior pituitary gland. In the present study we used antisense and immunoneutralization strategies to extend this work. Exposure of rat anterior pituitary tissue to corticosterone (1 nM) or dexamethasone (100 nM) in vitro induced 1) de novo annexin 1 synthesis and 2) translocation of the protein from intracellular to pericellular sites. Both responses were prevented by the inclusion in the medium of an annexin 1 antisense oligodeoxynucleotide (ODN; 50 nM), but not by the corresponding sense and scrambled ODN sequences. Unlike the GCs, 17beta-estradiol, testosterone, and aldosterone (1 nM) had no effect on either the synthesis or the cellular disposition of annexin 1; moreover, none of the steroids or ODNs tested influenced the expression of annexin 5, a protein closely related to annexin 1. The increases in PRL release induced in vitro by drugs that signal via cAMP/protein kinase A [vasoactive intestinal polypeptide (10 nM), forskolin (100 microM), 8-bromo-cAMP (0.1 microM)] or phospholipase C (TRH, 10 nM) were attenuated by preincubation of the pituitary tissue with either corticosterone (1 nM) or dexamethasone (100 nM). The inhibitory actions of the steroids on the secretory responses to vasoactive intestinal polypeptide, forskolin, and 8-bromo-cAMP were specifically quenched by inclusion in the medium of the annexin 1 antisense ODN (50 nM) or a neutralizing antiannexin 1 monoclonal antibody (antiannexin 1 mAb, diluted 1:15,000). By contrast, the ability of the GCs to suppress the TRH-induced increase in PRL release was unaffected by both the annexin 1 antisense ODN and the antiannexin 1 mAb. In vivo, interleukin-1beta (10 ng, intracerebroventricularly) produced a significant increase in the serum PRL concentration (P < 0.01), which was prevented by pretreatment of the rats with corticosterone (100 microg/100 g BW, sc). The inhibitory actions of the steroid were specifically abrogated by peripheral administration of an antiannexin 1 antiserum (200 microl, sc); by contrast, when the antiserum was given centrally (3 microl, intracerebroventricularly), it was without effect. These results support our premise that annexin contributes to the regulatory actions of GCs on PRL secretion and suggest that it acts at point distal to the formation of cAMP.

Two distinct corticotrophin releasing activities of vasopressin.

The effect of various corticotrophin releasing factors (CRFs) on the secretion of corticotrophin (ACTH) by segments of rat anterior pituitary tissue has been studied in vitro. ACTH release was stimulated by CRF-41 (5.0 X 10(-9) - 2.0 X 10(-7) M), hypothalamic extracts (0.2 - 1.6 HE ml-1) and arginine vasopressin (AVP, 5.0 X 10(-9) - 8.0 X 10(-7) M). The slopes of the dose-response lines of CRF-41 were greater than those of AVP, less than those of hypothalamic extracts from control animals and resembled those of hypothalamic extracts from Brattleboro rats. Simultaneous addition of AVP (10(-10) M) to the incubation medium enhanced the response to CRF-41 and increased the slope of its dose-response lines. The adrenocorticotrophic response to CRF-41 was enhanced similarly by pretreatment of the tissue with AVP (1.25 X 10(-11) - 2.0 X 10(-10) M) as also was the response to hypothalamic extracts. In contrast, pretreatment of the tissue with CRF-41 (2.3 X 10(-11) - 2.3 X 10(-8) M) depressed the subsequent response to CRF-41 and to hypothalamic extracts. The marked difference between the concentrations of vasopressin required to facilitate maximally the response to CRF-41 and those necessary to evoke ACTH release suggests that the two effects may be mediated by different types of vasopressin receptor.

A cytochemical bioassay method for the determination of luteinizing hormone in biological fluids and tissues.

1 The ability of luteinizing hormone (LH) to alter the reducing activity of corpora lutea in rat ovarian sections has been exploited to develop a new cytochemical bioassay for the hormone. 2 Sections of ovaries, removed from mature rats during the second day of dioestrus, were incubated with either standard LH or samples diluted for assay, stained immediately for reducing potential and the intensity of the stain measured by scanning and integrating microdensitometry. 3 An inverse linear relationship existed between the density of the stain and the logarithm of the concentration of standard LH (68/40) and the dose-response lines of serial dilutions of rat or human plasma were parallel with those of the standard. 4 The method was found to be accurate, specific, sensitive, precise and suitable for the determination of LH in the rat and in man.

Hypothalamo-pituitary adrenocortical function in the rat after treatment with betamethasone.

1 Hypothalamo-pituitary-adrenocortical (HPA) activity was suppressed in rats treated with betamethasone. 2 Recovery of normal HPA function occurred after corticosteroid withdrawl. 3 Although corticotrophin release was rapidly restored to its basal rate there was a delay in the return of the normal adrenocorticotrophic response to stress and normal adrenocortical function was evident only after the plasma adrenocorticotrophic hormone had reached 'supra-normal' levels. 4 The physiological significance and possible clinical relevance of the results are discussed.

The inhibition of nicotine-evoked relaxation of the guinea-pig isolated basilar artery by some analgesic drugs and progesterone.

1. The purpose of this study was to investigate the mechanism of nicotine-evoked relaxation of the guinea-pig isolated basilar artery and to study the effects of drugs associated with the aetiology or treatment of migraine on the nicotine response. 2. The guinea-pig isolated basilar artery, pre-contracted with prostaglandin F2alpha (PGF2alpha), in the presence of atropine (3 microM) and guanethidine (3 microM), relaxed on addition of nicotine (0.1 mM) in approximately 50% of preparations. The responses to nicotine were of short duration and blocked in preparations pre-treated for 10 min with capsaicin (1 microM) and are therefore probably a consequence of the stimulation of trigeminal C fibre terminals. 3. Responses to nicotine were reduced in the presence of 5-carboxamidotryptamine, 5-hydroxytryptamine and sumatriptan in that order of potency. This is consistent with a 5-HT1 receptor mechanism. These agonists evoked small additional contractions in vessels pre-contracted with PGF2alpha. 4. Indomethacin (0.3-10 microM), aspirin (10-30 microM), and nitro-L-arginine methyl ester (L-NAME, 0.1 mM) reduced nicotine-evoked relaxation of the basilar artery, suggesting the involvement of both nitric oxide and cyclo-oxygenase products in this response. 5. Progesterone (1 microM) markedly reduced the response to nicotine, a possible reflection of the ion channel blocking activity of high concentrations of this compound. 6. The guinea-pig basilar artery is a preparation in which the effects of drugs on responses to stimulation of trigeminal nerve terminals can be studied in vitro and may thus be of interest in assessing the actions of drugs used in treatment of headache.

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.

Evidence from immunoneutralization and antisense studies that the inhibitory actions of glucocorticoids on growth hormone release in vitro require annexin 1 (lipocortin 1).

1. Our previous studies have identified a role for annexin 1 as a mediator of glucocorticoid action in the neuroendocrine system. The present study centred on growth hormone (GH) and exploited antisense and immunoneutralization strategies to examine in vitro the potential role of annexin 1 in effecting the regulatory actions of glucocorticoids on the secretion of this pituitary hormone. 2. Rat anterior pituitary tissue responded in vitro to growth hormone releasing hormone, forskolin, 8-Bromo-cyclic adenosine 3'5'-monophosphate (8-Br-cyclic AMP) and an L-Ca(2+) channel opener (BAY K8644) with concentration-dependent increases GH release which were readily inhibited by corticosterone and dexamethasone. 3. The inhibitory actions of the steroids on GH release elicited by the above secretagogues were effectively reversed by an annexin 1 antisense oligodeoxynucleotide (ODN), but not by control (sense or scrambled) ODNs, as also were the glucocorticoid-induced increases in annexin 1. Similarly, a specific anti-annexin 1 monoclonal antibody quenched the corticosterone-induced suppression of secretagogue-evoked GH release while an isotype matched control antibody was without effect. 4. Transmission electron micrographs showed that the integrity and ultrastructural morphology of the pituitary cells were well preserved at the end of the incubation and unaffected by exposure to the ODNs, antibodies, steroids or secretagogues. 5. The results provide novel evidence for a role for annexin 1 as a mediator of the inhibitory actions of glucocorticoids on the secretion of GH by the anterior pituitary gland and suggest that its actions are effected at a point distal to the formation of cyclic AMP and Ca(2+) entry.

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.

Opposing influences of glucocorticoids and interleukin-1beta on the secretion of growth hormone and ACTH in the rat in vivo: role of hypothalamic annexin 1.

1. This study exploited established immunoneutralization protocols and an N-terminal annexin 1 peptide (annexin 1(Ac2 - 26)) to advance our knowledge of the role of annexin 1 as a mediator of acute glucocorticoid action in the rat neuroendocrine system in vivo. 2. Rats were treated with corticosterone (500 microg kg(-1), i.p.) or annexin 1(Ac2 - 26) (0.1 - 10 ng rat(-1), i.c.v.) and 75 min later with interleukin 1beta (IL-1beta, 10 ng rat(-1), i.c.v. or 500 microg kg(-1), i.p). Blood was collected 1 h later for hormone immunoassay. Where appropriate, anti-annexin 1 polyclonal antiserum (pAb) was administered subcutaneously or centrally prior to the steroid challenge. 3. Corticosterone did not affect the resting plasma corticotrophin (ACTH) concentration but suppressed the hypersecretion of ACTH induced by IL-1beta (i.p. or i.c.v.). Its actions were quenched by anti-annexin 1 pAb (s.c. or i.c.v) and mimicked by annexin 1(Ac2 - 26). 4. By contrast, corticosterone provoked an increase in serum growth hormone (GH) which was ablated by central but not peripheral administration of anti-annexin 1 pAb. IL-1beta (i.c.v. or i.p.) did not affect basal GH but, when given centrally but not peripherally, it abolished the corticosterone-induced hypersecretion of GH. Annexin 1(Ac2 - 26) (i.c.v.) also produced an increase in serum GH which was prevented by central injection of IL-1beta. 5. The results support the hypothesis that the acute regulatory actions of glucocorticoids on hypothalamo-pituitary-adrenocortical function require annexin 1. They also provide novel evidence that the positive influence of the steroids on GH secretion evident within this timeframe is effected centrally via an annexin 1-dependent mechanism which is antagonized by IL-1beta.