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.

Expression of cyclooxygenase enzymes in rat hypothalamo-pituitary-adrenal axis: effects of endotoxin and glucocorticoids.

Prostaglandins play a key role in mediating the hypothalamo-pituitary-adrenocortical (HPA) responses to immune insults. This study aimed to provide some insight into the relative contributions of the constitutive and inducible forms of cyclooxygenase (COX-1 and COX-2) to the generation of these prostanoids by examining the effects of (1) endotoxin treatment on the expression of COX-1 and COX-2 mRNAs in the various components of the HPA axis in control and glucocorticoid pretreated rats, and (2) selective inhibition of COX-2 on the production of corticosterone by adrenal tissue in vitro. Endotoxin caused a marked rise in COX-2 mRNA in the adrenal gland that was evident 3 and 6 h after the injection and was prevented by pretreatment with dexamethasone. It also induced a modest increase in COX-2 mRNA in the hypothalamus but not in the hippocampus or anterior pituitary gland. By contrast, COX-1 mRNA was largely unaffected by the drug treatments in all tissues studied. In vitro the selective COX-2 inhibitor SC-236 caused a marked reduction in adrenocorticotropic hormone-driven corticosterone release, as did the nonselective COX inhibitor, indomethacin. These results support a role of COX-2 in the manifestation of the HPA responses to endotoxin, particularly within the adrenal gland.

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.

Roles for adenosine A1- and A2-receptors in the control of thyrotrophin and prolactin release from the anterior pituitary gland.

Adenosine has been implicated in various aspects of pituitary function but little is known of its role in the regulation of thyrotrophin (TSH) release. This study examined the effects of adenosine deaminase (ADA, which provokes adenosine breakdown) and selective adenosine-receptor ligands on the secretion of immunoreactive (ir-) TSH and prolactin (PRL) by rat anterior pituitary segments in vitro. ADA (5 U/ml) stimulated the release of both hormones (P<0.01) as also did the selective adenosine A1-receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1 & 1 nM, P<0.01); the responses to ADA were inhibited by an A1-receptor agonist, N6-cyclohexyladenosine (0.1-10 nM, P<0.01). A non-selective A1/A2-receptor agonist, N-cyclopropylcarboxamidoadenosine (1-100 nM) had mixed effects on ir-TSH release. However, the A2A-receptor selective agonist, CGS 21680 (1-100 nM) increased ir-TSH (P<0.05) and ir-PRL release (P<0.01); its effects on ir-TSH were blocked by concentrations of DPCPX (100 nM, P<0.01) sufficient to antagonize A2-receptors. These data suggest that adenosine acts via A1-receptors to tonically suppress ir-PRL and ir-TSH release but that A2A-receptor activation enhances the release of both hormones.

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.

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.

Interactions between the hypothalamo-pituitary adrenal axis and the thymus in the rat: a role for corticotrophin in the control of thymulin release.

Our recent observations in man suggested that the secretion of the thymic peptide, thymulin, is influenced by hormones of the pituitary-adrenal axis. In the present study, we have used the rat as a model in order to examine 1) the effects of corticotrophin (ACTH) and glucocorticoids on the release of thymulin in vivo and in vitro, and 2) the influence of an acute rise in plasma thymulin on the secretion of corticosterone and luteinizing hormone. Immunoreactive thymulin was readily detectable in plasma from male Sprague-Dawley rats(≃200 g). Chronic bilateral adrenalec-tomy, which effectively removed endogenous corticosterone, produced highly significant (P<0.01) increases in the plasma concentrations of both ACTH and thymulin. Treatment of the adrenalectomized rats with dexamethasone, in a dose sufficient to suppress the hypersecretion of ACTH, maintained the plasma thymulin at a low level which did not differ significantly (P > 0.2) from that in sham-operated controls. In vitro, two non-specific depolarizing agents, K(+) (56 mM) and veratridine (10 ≃M), caused significant (P<0.01) Ca(2+) -dependent increases in thymulin release from segments of rat thymic tissue. Their effects were mimicked by ACTH(1-39) . The secretory responses to ACTH (0.025 to 1 ng/ml) were concentration-dependent but a very high concentration (2 ng/ml) of the peptide was without effect. Dexamethasone (0.1 µM) reduced (P<0.05) the spontaneous release of thymulin in vitro but potentiated markedly (P<0.01) the secretory responses to ACTH (0.5 to 1.0 ng/ml). Administration of thymulin (0.1 and 10 µg/kg ip) produced, within 10 min, striking increases in the plasma thymulin concentration which were still evident at 30 min. The peptide concentration then declined rapidly and, within 24 h, was lower than that in the corresponding vehicle-treated controls. The serum concentrations of corticosterone and luteinizing hormone were unaffected by the thymulin treatment. The saline vehicle (2.0 ml/kg ip) also produced a small increase in plasma thymulin concentration which was maximal at 10 min; a further small rise was evident 6 h after the injection but thereafter the thymulin values were indistinguishable from those in uninjected controls. A similar biphasic profile of serum corticosterone was apparent after the saline injection but the serum luteinizing hormone was unaffected. The results suggest that ACTH is a physiological enhancer of thymulin release and that, in certain circumstances, its effects may be potentiated by glucocorticoids.

In vivo and in vitro studies on the opioidergic control of the secretion of gonadotrophin-releasing hormone and luteinizing hormone in sexually immature and adult male rats.

In vivo and in vitro methods have been used to compare the effects of opioid receptor blockade on the functional activity of the hypothalamo-pituitary-gonadal axis in adult (200 g) and sexually immature (50 g) male rats. In the adult, a single injection of the mu-receptor antagonist, naloxone (500 micrograms/100 g body weight, s.c.), produced hypersecretions of luteinizing hormone (LH) and testosterone. Maximal serum concentrations of the two hormones were attained within 20 and 60 min respectively. In contrast, neither ICI 174864 (100 micrograms/100 g body weight, s.c.) nor MR2266 (150 micrograms/100 g body weight, s.c.), which block delta- and kappa-receptors respectively, stimulated pituitary-gonadal activity; indeed, like the saline vehicle, both tended to depress the serum LH concentration. The injection procedure was sufficient to activate the hypothalamo-pituitary-adrenal axis and, thus, the vehicle-treated controls exhibited significant increases in the plasma adrenocorticotrophin and serum corticosterone concentrations. These effects were enhanced by naloxone (500 micrograms/100 g body weight, s.c.) and by the kappa-opioid receptor (MR2266, 150 micrograms/100 g body weight, s.c.) but not by the delta-opioid receptor antagonist (ICI 174864, 30-100 micrograms/100 g body weight, s.c.). The increases in serum corticosterone and LH concentration induced by naloxone in adult rats were not apparent in the sexually immature (50 g) animals. To the contrary, in the young rats naloxone (250 and 500 micrograms/100 micrograms body weight, s.c.) attenuated, in a dose-dependent manner, the pronounced hypersecretion of corticosterone induced by the vehicle injection. The higher dose of the antagonist (500 micrograms/100 g body weight, s.c.) also overcame the significant reductions in serum LH evident 20 (p less than 0.05) and 40 (p less than 0.01) min after the saline injection but the lower dose (250 micrograms/100 g body weight, s.c.) was ineffective in this respect. In vitro, hypothalami from both adult and sexually immature rats responded to the addition of naloxone (10(-8)-10(-6) M) to the incubation medium with significant (p less than 0.01) concentration-dependent increases in the release of gonadotrophin-releasing hormone (GnRH). In contrast, ICI 174864 (10(-7)-10(-6) M) and MR2266 (10(-7)-10(-6) M) had little effect on the secretion of the releasing hormone by hypothalami from rats of either group although, at the highest concentration tested, MR2266 (10(-6) M) precipitated a small increase in GnRH release from hypothalami from adult rats.(ABSTRACT TRUNCATED AT 400 WORDS)

A role for vasopressin in the stress-induced inhibition of gonadotrophin secretion: studies in the brattleboro rat.

Abstract The effects of stress on the secretion of adrenocorticotrophin, corticosterone and luteinizing hormone (LH) in rats congenially lacking hypothalamic vasopressin (Brattleboro rats) and in normal controls of the parent strain (Long Evans) have been compared in an attempt to examine the role of vasopressin in the stress-induced depression of gonadotrophin secretion. In the Long Evans rats, stress (0.6 mg/100g histamine, ip) initiated, within 5 and 20 min respectively, significant (P <0.01, Student's t-test) increases in the plasma adrenocorticotrophin and corticosterone concentrations. It also caused a reduction in the serum LH concentration which was maximal at 5 min. By contrast, in the vasopressin deficient Brattleboro rats, stress had no effect on the serum LH concentration and produced only modest increases in pituitary adrenocortical activity compared with those in Long Evans controls. Pretreatment of both Long Evans and Brattleboro rats with dexamethasone (20mug/100 g ip, daily for 3 days) effectively abolished the pituitary-adrenal response to stress. The steroid treatment also prevented the stress-induced suppression of LH in the Long Evans rats; indeed, these animals, unlike the vehicle-treated controls, exhibited a rise in serum LH concentration within 5 min of exposure to stress. Stress did not affect the serum LH concentrations in steroid-treated Brattleboro rats. The results confirm previous reports that vasopressin is required for the full expression of the pituitary-adrenocortical response stress. They also provide novel evidence which suggests that vasopressin released in stress contributes to the impairment of gonadotrophin secretion.

Effect of estradiol and tamoxifen on GnRH self-priming in perifused rat adenohypophysial cells.

Columns of enzymatically dispersed rat pituitary cells have been used to examine the effects of estradiol and the estrogen receptor antagonist, tamoxifen, on the responsiveness of the gonadotropes to repeated challenges with GnRH. Synthetic GnRH stimulated readily the release of LH from the pituitary cells. When challenged repeatedly with the same submaximal dose (8 nmol/l) of the releasing hormone, cells from animals in diestrus 2 or pro-estrus, like those from juvenile rats, showed a marked progressive increase in their sensitivity, but those from rats in estrus or diestrus 1 did not. Adenohypophysial cells removed from adult rats ovariectomized 14 days previously also failed to exhibit sensitization even when estradiol-17 beta (1 and 10 nmol/l) was included in the perifusion fluid. However, those from ovariectomized rats treated with appropriate doses of the steroid (1.5 microgram/day sc for 14 days or 5 micrograms sc 22 h before decapitation) did. The capacity of pituitary cells from intact rats to exhibit priming on the anticipated day of pro-estrus was unaffected by the administration of the estrogen receptor antagonist, tamoxifen (700 micrograms/100 g sc on the morning of diestrus 1 and 24 h later). Similarly, addition of tamoxifen (1 mumol/l) to the incubation medium failed to influence the capacity of pituitary cells from juvenile rats to exhibit priming in vitro. The results support the concept that the ability of GnRH to 'prime' the gonadotropes is dependent on estradiol and indicate that the exposure of the hypothalamo-pituitary complex to critical levels of estradiol prior to autopsy is necessary for the gonadotropes to retain their capacity to exhibit priming in vitro.

Effects of selective opioid-receptor blockade on the hypothalamo-pituitary-adrenocortical responses to surgical trauma in the rat.

Opioid substances have been shown to stimulate and depress the secretion of ACTH in the rat. Their opposing actions appear to be mediated in part by specific receptors in the hypothalamus which influence the secretion of corticotrophin-releasing factor (CRF). In an attempt to determine the physiological role of these receptor systems, experiments were carried out in which the plasma ACTH and serum corticosterone concentrations were determined before and after stress in rats treated s.c. with selective antagonists of mu-(naloxone, naltrexone), delta-(ICI 174864) and kappa-(MR2266) opioid receptors. Neither naloxone (25-100 micrograms/100 g) nor naltrexone (50 micrograms/100 g) influenced the resting plasma ACTH or serum corticosterone concentrations. However, both inhibited (P less than 0.01) the secretion of the two hormones elicited normally by surgical stress (laparotomy under ether anaesthesia). ICI 174864 (30-100 micrograms/100 g) also had little effect on resting hypothalamo-pituitary-adrenocortical (HPA) activity but, at the highest dose, it caused a small (P less than 0.05) potentiation of the response to surgery. In contrast, MR2266 (150-300 micrograms/100 g) produced marked activation of the HPA system and not only stimulated the resting secretion of ACTH and corticosterone but also potentiated and prolonged the HPA response to stress. The results suggest that mu- and kappa-opioid receptors mediate opposing actions of endogenous opioid peptides, both of which may be physiologically important in the regulation of CRF release.

Changes in the responsiveness of perifused rat adenohypophysial cells to repeated stimulation with luteinizing hormone releasing hormone.

The ability of luteinizing hormone releasing hormone (LRH) to stimulate the release of luteinizing hormone (LH) from columns of enzymatically dispersed perfused adenohypophysial cells is being used to study the mechanisms controlling the secretion of LH. LRH stimulated the release in vitro of LH from columns of rat pituitary cells. However, when exposed repeatedly (1 pulse every 12 min) to the same submaximal dose (8 nmol/l) of LRH the cells always exhibited a marked progressive increase and subsequent decrease in their responsiveness. Similar effects occurred when the interval between pulses was extended to 20, 30 or 45 min. The enhanced responsiveness of the cells was prevented by the inclusion of protein synthesis inhibitors, cycloheximide or puromycin, in the perifusion fluid. Cells removed from rats ovariectomized 14 days previously also failed to exhibit increased responsiveness when stimulated repeatedly with LRH. LH secretion was also elicited by K+ (50 nmol/l), 8-bromoadenosine 3'-5'-cyclic monophosphate (8-Br-cAMP, 6 nmol/l), 8-bromoguanosine 3'-5'-cyclic monophosphate (8-Br-cGMP, 6 nmol/l) and a calcium ionophore (A23187, 40 mumol/l) but the responses to these secretagogues differed markedly from those to LRH for the tachyphylaxis which resulted from repeated exposure was not preceded by an increase in responsiveness. The decreased responsiveness to K+ developed in parallel with that to LRH. Diminished responses to the cyclic nucleotides and the Ca++ ionophore developed more rapidly, but the refractory cells responded readily to stimulation with LRH or K+.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological assay of luteinizing hormone-releasing hormone (gonadorelin).

The abilities of luteinizing hormone releasing hormone (LHRH) to stimulate the release of luteinizing hormone (LH) in vitro from dispersed pituitary cells or from segments of adenohypophysial tissue have both been exploited in an attempt to develop biological assays for its determination. Columns of dispersed adenohypophysial cells were highly sensitive to LHRH. The responses appeared to be specific, but the marked continual variation in their magnitude rendered the system nonprecise. Pituitary segments were less sensitive to LHRH than the dispersed cells. However, their responsiveness was increased by pretreatment ('priming') with the releasing hormone. Over a wide range of doses, there was a linear relationship between the LH released by 'primed' segments and the logarithm of the concentration of LHRH. The dose-response lines of hypothalamic extracts were parallel with those of the synthetic peptide. The method using segments is accurate, specific, and precise and fulfils the essential requirements of a biological assay.