Interleukin-1 beta enhances corticosterone secretion by acting directly on the rat adrenal gland.

Interleukin-1 (IL-1), a monokine released by activated monocytes during the acute phase of the inflammatory responses, has been reported to enhance hypophyseal ACTH release mainly by stimulating hypothalamic CRF secretion. We investigated a possible direct effect of IL-1 beta on the adrenal gland of the rat. IL-1 beta was found to dose-dependently (4-8 micrograms/kg) raise corticosterone (B) blood concentration in hypophysectomized rats, without inducing any significant increase in the level of circulating ACTH. IL-1 beta did not affect B production by either isolated rat inner adrenocortical cells or fragments of adrenocortical autotransplants lacking chromaffin cells, but dose-dependently (10(-8)-10(-6) M) enhanced that by adrenal slices including both cortex and medulla. The secretory effect of IL-1 beta (10(-6) M) was completely blocked by both alpha-helical-CRF (10(-6) M) and corticotropin-inhibiting peptide (10(-6) M), two competitive inhibitors which (at these concentrations) were able to annul B response of adrenal slices to CRF (10(-6) M) and ACTH (10(-8) M), respectively. In light of many findings indicating that adrenal medulla contains and releases CRF and numerous POMC-derived peptides (including ACTH), the hypothesis is advanced that the mechanism underlying the direct secretory effect of IL-1 beta on the adrenal gland may involve the activation of an intraadrenal CRF/ACTH system.     

Role of arachidonic acid in the regulation of adrenocorticotropin release from rat anterior pituitary cell cultures

In addition to cAMP-dependent mechanisms, stimulation of pituitary ACTH secretion by various stimuli, including CRF, may involve phospholipid and arachidonic acid turnover. To determine the role of phospholipase A2 activation in corticotroph function, we studied the effect of exogenous arachidonic acid, phospholipase A2, and the phospholipase A2 activator melittin on ACTH release in cultured rat anterior pituitary cells. Incubation with 1-100 micron arachidonic acid, 0.01-1 micron melittin, 0.1-10 U/ml phospholipase A2, and 0.01-10 nM CRF caused dose-dependent increases in ACTH release to 8.1 +/- 1.1- (+/- SE), 16.2 +/- 0.9-, 13.6 +/- 1.2-, and 2.9 +/- 0.3-fold; respectively. The participation of the major pathways of arachidonic acid metabolism in the control of ACTH release was analyzed in cells treated with nordihydroguaiaretic acid, a lipoxygenase inhibitor; indomethacin, a cycloxygenase inhibitor; and 5,8,11,14-eicosatetraynoic acid, an inhibitor of both pathways. The effects of arachidonic acid, melittin, and CRF were partially blocked by 10 micron nordihydroguaiaretic acid and 5,8,11,14-eicosatetraynoic acid, but were significantly enhanced by 10 micron indomethacin. These results suggest that arachidonic acid is mainly metabolized through the lipoxygenase pathway to a stimulatory metabolite and, to a lesser extent, through the cycloxygenase pathway to an inhibitory metabolite. Arachidonic acid release from anterior pituitary cells labeled with [3H]arachidonic was analyzed during cell column perifusion and stimulation by CRF and other secretagogues. Two-minute pulses of CRF (10 nM), vasopressin (10 nM) and phorbol 12-myristate 13-acetate (100 nM) caused immediate 1.5- to 2-fold increases in [3H]arachidonic acid release, and melittin (100 nM) caused a 5-fold increase in [3H]arachidonic acid release. The ability of both exogenously added and endogenously generated arachidonic acid to stimulate ACTH secretion, together with the stimulation of arachidonic acid release by ACTH secretagogues and the attenuation of stimulated ACTH release by lipoxygenase blockers, indicate that lipoxygenase products of arachidonic acid metabolism participate in the control of ACTH secretion.     

Angiotensin II and adrenocorticotropin release: mediation by endogenous corticotropin-releasing factor

Recent reports indicate that the main effect of systemically administered angiotensin II (AII) on ACTH release is probably due to some central nervous system mechanism. The present studies were designed to investigate whether the central action of AII on ACTH release is directly mediated through CRF. In order to test the participation of endogenous CRF in the AII-induced ACTH release in vivo, intact and pharmacologically blocked (pretreated with chlorpromazine-morphine-nembutal) female rats were injected iv with AII (8 nmol/100 g BW). Plasma levels of ACTH as well as CRF content in the median eminence (ME) and medial basal hypothalamus (MBH) were evaluated before and 1, 2.5, and 5 min after treatment. These responses were compared with the effect of 1 min ether exposure on hypothalamic CRF content and plasma ACTH levels in unanesthetized animals. AII injection and ether exposure increased plasma ACTH levels several-fold at both 2.5 and 5 min post treatment in intact rats. Conversely, AII failed to induce any significant increase in plasma ACTH levels in centrally blocked rats at any interval studied. On the other hand, AII injection and ether inhalation acted in similar fashion on CRF content in ME, inducing fast depletion at 1 min post treatment, recovering to control values at 2.5 min after injection, and finally, accumulating peptide at 5 min post treatment. In addition, CRF content in the MBH decreased significantly at 5 min, under both experimental conditions; AII had no effect on hypothalamic CRF content in centrally blocked rats. In vitro experiments using whole MBH (containing ME) fragments incubated with either neural peptides or high K+ solutions indicate that AII possesses a CRF releasing effect at concentrations of 10(-6) M or more. Conversely, other hypothalamic peptides, such as LHRH, TRH, and somatostatin did not induce significant release of CRF at any of the concentrations assayed (10(-7) to 10(-5) M). On the other hand, high K+ solutions released CRF in a concentration-related manner (15-60 mM). These studies suggest that the central effect of AII stimulation on ACTH release in vivo could be, at least in part, through the release of hypothalamic CRF into the portal circulation.     

Role of arachidonic acid or its metabolites in growth-hormone-releasing factor-induced release of somatostatin from the median eminence

The possible involvement of arachidonic acid (AA) release in growth-hormone-releasing factor (GRF)-induced somatostatin (SRIF) release from the median eminence (ME) of the hypothalamus was evaluated in adult male rats using an in vitro incubation system. The MEs were preincubated with [14C]-AA, then washed and incubated with vehicle or test agents, and the release of SRIF and [14C]-AA into the medium was measured. In the experiments designed only to determine SRIF release, the MEs were first preincubated for 30 min. The medium was then discarded and replaced with fresh buffer or test substances and incubated for 10, 20 and/or 30 min. GRF (10(-10) M) stimulated both AA and SRIF release significantly within 20 min, with maximum release occurring at 30 min. The stimulatory effect of GRF on AA release was coincident with the release of SRIF. A phospholipase A2 inhibitor (10(-6) M, quinacrine) completely abolished the stimulatory effect of GRF on both AA and SRIF release. The release of SRIF induced by GRF was also inhibited by both indomethacin (10(-6) M, a cyclooxygenase inhibitor) and metyrapone (10(-6) M, a cytochrome P-450 inhibitor). On the other hand, nordihydroguaiaretic acid (10(-6) M, a lipoxygenase inhibitor) had no effect on GRF-evoked SRIF release. The data presented here suggest that an important GRF-mediated event leading to SRIF secretion is an elevated release of AA from ME fragments in vitro. In conclusion, our data are suggestive that the stimulatory effect of GRF on SRIF release is due, in part, to the release and subsequent metabolism of AA to one or more metabolites.     

Role of nitric oxide in interleukin 2-induced corticotropin-releasing factor release from incubated hypothalami.

Stimulation of corticotropin-releasing factor (CRF) release from the hypothalamus by interleukin 2 (IL-2) was recently demonstrated. Cytokines induce nitric oxide synthase (NOS), an enzyme that converts L-arginine into L-citrulline and nitric oxide (NO). NO is believed to be responsible for the cytotoxic action of these agents. The constitutive form of NOS occurs in neurons in the central nervous system and NO appears to play a neurotransmitter role in cerebellar and hippocampal function. We explored the probability that IL-2 and synaptic transmitters might release CRF via NO. The effects of L-arginine, the substrate for NOS, and NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, on IL-2-induced CRF release were studied using mediobasal hypothalami (MBHs) incubated in vitro in Krebs-Ringer bicarbonate buffer. L-Arginine did not alter basal and IL-2-induced CRF release after 30 min of incubation but significantly elevated both basal and IL-2-induced CRF release when MBHs were incubated 30 min longer, presumably because the endogenous substrate had been depleted after the initial 30-min incubation period. In 30-min incubations, both carbachol, an acetylcholineomimetic drug, and norepinephrine stimulated CRF release. There was an additive effect of incubation of the MBHs in the presence of carbachol (10(-7) M) and IL-2 (10(-13) M). On the other hand, coincubation of MBHs with norepinephrine (10(-6) M) and IL-2 (10(-13) M) did not produce any additive effect. Addition of NMMA, an inhibitor of NOS, at 1 or 3 x 10(-4) M completely suppressed IL-2-induced release of CRF as well as that caused by IL-2 plus carbachol. In contrast, the release of CRF induced by norepinephrine was not blocked by 3 x 10(-4) M NMMA. The data indicate that IL-2 can activate constitutive NOS leading to increased NO release, which activates CRF release. It appears that NO is also involved in the release of CRF induced by carbachol but not by norepinephrine.     

Changes in the hypothalamo-corticotrope axis after bilateral adrenalectomy: evidence for a median eminence site of glucocorticoid action.

Bilateral adrenalectomy (ADX) leads to increased ACTH synthesis and secretion. It is thought that endogenous glucocorticoids exert a feedback mechanism at both pituitary and brain levels. The present study has been performed in order to determine the effect of ADX on the release of hypothalamic neuropeptides with corticotropin-releasing activity (CRA) and if there exists a median eminence site of glucocorticoid action to regulate hypothalamic-pituitary-adrenal (HPA) function. Adrenalectomized and sham-operated male rats were killed at different periods after surgery (2, 5, 7 and 14 days) and trunk blood was collected for ACTH and corticosterone (B) concentrations measurement. Brain (median eminence, ME; and medial basal hypothalamus, MBH) and pituitary (anterior lobe, AP; and neurointermediate lobe, NIL) tissues were dissected in order to evaluate either peptide content or in vitro hormone release. The results indicate that ADX blunted plasma B levels and increased AP ACTH content and secretion in a time-related fashion up to the 14th day. ADX significantly decreased both CRF and CRA contents in the ME at all periods studied; ME arginine-vasopressin (AVP) increased 7 and 14 days after ADX. MBH CRF decreased after ADX, but returned to sham value 2 weeks later; similarly, MBH AVP decreased at all periods after ADX. Removal of endogenous glucocorticoids did not vary neither oxytocin (OXY) content in the ME and MBH nor AVP and OXY contents in the NIL. In our superfusion experiments, we found that ADX increased basal AVP release and did not change spontaneous CRF secretion from ME terminals. Dexamethasone (Dxm, 10 nM) diminished AVP but not CRF output by ME tissues from adrenalectomized rats. A direct relationship was found between ME CRF and 28 mM KCl (hK+)-induced CRF release by MEs from adrenalectomized rats. ME fragments from adrenalectomized rats were hyperresponsive to kH+ stimulation of AVP release. Dxm (10 nM) decreased the hK(+)-evoked CRF and AVP release by MEs from adrenalectomized rats. ADX and dexamethasone treatment did not influence basal and hK(+)-elicited ME OXY release. Additionally, a rapid glucocorticoid inhibitory effect on ACTH secretion by isolated AP cells from both sham and adrenalectomized rats was found, and an in vitro corticotrope hyporesponse to 0.63 nM CRF and 9.25 nM AVP stimulation during several days after ADX.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rapid as well as delayed inhibitory effects of glucocorticoid hormones on pituitary adrenocorticotropic hormone release are mediated by type II glucocorticoid receptors and require newly synthesized messenger ribonucleic acid as well as protein

Glucocorticoid hormones suppress the release of ACTH by the anterior pituitary gland: rapid feedback inhibits hormone secretion within 30 min of steroid application, delayed feedback is most effective at 1-2 h, and slow feedback becomes manifest in several hours. The aim of the present study was to determine the type of glucocorticoid receptor that mediates the rapid and delayed feedback actions of glucocorticoids and whether genomic activation occurs during the rapid and delayed time domains. Rat anterior pituitary cell columns were perfused with Dulbecco's minimum essential medium, 41-residue CRF (10(-9) M) was used as the secretagogue, which stimulated ACTH secretion to a peak of about 8- to 10-fold of basal release. The amount of ACTH released upon repeated 5 or 10 min stimulation with CRF was constant. Treatment with 10(-7) M corticosterone for 20 min immediately before and for 10 min during stimulation with CRF reduced ACTH release by about 50% (rapid feedback), while at 1 h and 2 h after the initial exposure to corticosterone the secretory response was 33% and 15% of control, respectively. The effect of corticosterone was prevented by the type II glucocorticoid/progesterone antagonist RU 38486 (10(-6) M). The selective type II receptor agonist RU 28362 (10(-7) M) was even more potent than corticosterone in inhibiting ACTH release; the time course of action was similar. When actinomycin D (10(-4) M) was applied in conjunction with RU28362 or corticosterone, no inhibitory effects appeared up to 2 h after the exposure to steroid. Puromycin (10(-4) M), given during and for 1 h after the administration of the steroid prevented the rapid as well as the delayed (1 h) inhibitory action of RU28362. When puromycin was removed from the system, a 75% inhibition of stimulated ACTH release developed at 2 h after the application of the steroid, indicating that translatable messenger RNA (mRNA) was still present in the cells. Cycloheximide (10(-4) M) was only partially effective at inhibiting rapid or delayed feedback, and increasing its concentration impaired the ACTH response to CRF-41. In summary, at the pituitary level the rapid as well as the delayed feedback inhibition of ACTH secretion by adrenal corticoids is exerted via type II glucocorticoid receptors. Furthermore, both rapid and delayed feedback require the synthesis of new mRNA and protein.     

Effects of synthetic ovine corticotropin-releasing factor (CRF) on ACTH release in vitro

The effect of the synthetic ovine corticotropin-releasing factor (CRF) on adrenocorticotropin (ACTH) release was examined by the perifusion method using rat anterior pituitary tissue and rat monolayer cultured pituitary cells. Quartered anterior pituitaries were placed in a chamber and perifused at a rate of 400 microliters/min with Dulbecco's modified Eagle Medium (DMEM, pH 7.4) bubbled with a mixture of 95% O2 and 5% CO2. The perifused medium was fractionated, and the ACTH concentration was measured by radioimmunoassay. In the monolayer cultured pituitary cells, the amount of ACTH released in the culture medium during three hours incubation was assayed by radioimmunoassay. ACTH was released from the perifused anterior pituitary in a dose-related manner by the pulse administration of CRF or arginine vasopressin (AVP) at the concentration of 1 ng/ml, 10 ng/ml and 100 ng/ml. A significant difference was not found between CRF- and AVP-induced ACTH release. In the monolayer cultured pituitary cells, synthetic ovine CRF induced ACTH release in a dose-related manner between 30 pg/ml and 30 ng/ml, but AVP induced a slight ACTH release. ACTH release was pulsatile during the continuous administration of 2.5 ng/ml of CRF for 150 min, although if gradually increased during the continuous administration of 10 ng/ml or 20 ng/ml of CRF. The continuous administration of AVP also caused pulsatile ACTH release at 10 ng/ml, but the ACTH release gradually decreased during the continuous administration of AVP. The interaction between CRF and AVP on ACTH release was examined by two methods. When CRF and AVP were given simultaneously, a mainly additive effect on ACTH release was observed. However, a low concentration of CRF seemed to potentiate AVP-induced ACTH release. These results show that both CRF and AVP have a significant CRF activity on the perifusion system, that AVP induced a slight ACTH release in monolayer cultured pituitary cells, and that CRF acts additively or potently with AVP to control the ACTH release from the anterior pituitary gland.     

Role of arachidonic acid metabolism in ACTH-stimulated cortisol secretion by bovine adrenocortical cells

We have studied the effects of inhibitors of arachidonic acid (AA) metabolism, nordihydroguaiaretic acid (NDGA), a lipoxygenase (LPX) inhibitor, and indomethacin (INDO), a cyclooxygenase (COX) inhibitor, on cortisol secretion and StAR protein in primary cultures of bovine adrenal zona fasciculata (ZF) cells. NDGA inhibited cortisol secretion in response to both 10(-12) M and 10(-8) M ACTH. AA (10(-4) M) partially reversed the inhibition of cortisol secretion by NDGA at 10(-12) M ACTH but not at 10(-8) M ACTH. On the other hand, INDO potentiated the cortisol response to 10(-12) M ACTH. Neither NDGA nor INDO significantly affected StAR protein levels. These results suggest a StAR protein-independent role for the LPX and COX pathways in acute cortisol secretion, and support the hypothesis that LPX products of AA metabolism are key cellular signals when bovine ZF cells are acutely stimulated by physiological concentrations of ACTH (10(-12) M).     

Increased pituitary sensitivity to glucocorticoid feedback during the stress nonresponsive period in the neonatal rat

Neonatal rats show a diminished response to stress [the stress-nonresponsive period (SNRP)] from day 2-3 until day 14 of age; the physiological bases for the SNRP are unknown. We examined whether enhanced sensitivity of the brain or pituitary to the inhibitory feedback effects of circulating glucocorticoids (GC) contributes to the SNRP. Age-related changes in the ability of corticosterone (CORT) and dexamethasone (DEX) to inhibit the ACTH secretion induced by urethane or CRF were studied. We also examined the ACTH response to ether stress or CRF in intact or 24 h-adrenalectomized 5-day-old rats. Plasma ACTH did not increase in intact rats after ether stress (basal: 64.6 +/- 9.1 pg/ml vs. stressed: 66.8 +/- 8.9 pg/ml; P greater than 0.05), whereas small elevations occurred after CRF challenge (184.6 +/- 40 pg/ml; P less than or equal to 0.01). Five-day-old adrenalectomized rats, which had elevated basal ACTH concentrations, increased ACTH secretion after exposure to ether or CRF. Thus, negative feedback appears to mediate critically the SNRP. Furthermore, sensitivity to such feedback was enhanced during the SNRP since the capacity of CORT to inhibit urethane-induced ACTH secretion in vivo declined with age; 1 mg/kg BW was the minimal dose that inhibited ACTH secretion at day 10, whereas at day 18, the threshold for a similar inhibition was 5 mg/kg BW. In contrast, at both ages, a dose of 10 micrograms/kg BW DEX inhibited ACTH release. In vitro dose response studies in whole pituitaries further demonstrated the enhanced pituitary sensitivity to GC feedback during the SNRP since the IC50 for CORT inhibition of CRF-induced ACTH release increased from days 3-5 to days 22-23. A similar, although not statistically significant trend was observed for DEX inhibition. Thus, neonatal rats exhibit an enhanced pituitary sensitivity to GC during the SNRP and removal of this inhibition allows ACTH secretion in response to ether stress.     

Angiotensin II potentiates corticotropin-releasing activity of CRF41 in rat anterior pituitary cells: mechanism of action

In this work the ability of angiotensin II (AII) to potentiate the corticotropin-releasing activity of ovine CRF41 (CRF) and the intracellular mechanism responsible for this effect are described. In perfused rat anterior pituitary cells, AII (10(-8)M) was found to potentiate the corticotropin-releasing activity of CRF producing a parallel shift of the dose-response curve. Similar results for ACTH release were observed in monolayer cell cultures. In this system, the concentration of cAMP was measured and was shown to be increased in the presence of CRF with a maximal value (2.5-fold greater than control) after 5-10 min incubation. On the other hand, AII at a dose inducing ACTH release (10(-7)M), had no effect on basal cAMP concentration, but when given simultaneously with CRF, potentiated the CRF-induced cAMP production (1.9-fold greater than CRF value). These results indicate that AII potentiates the corticotropin-releasing activity of CRF and that this effect is preceded by a similar increase in the CRF-induced cAMP production.     

Neuropeptide Y stimulates the release of luteinizing hormone-releasing hormone from medial basal hypothalamus in vitro: modulation by ovarian hormones

These studies investigated the effects of neuropeptide Y (NPY) on in vitro release of luteinizing hormone-releasing hormone (LHRH) from the medial basal hypothalmus (MBH) and tested whether ovarian steroids modulate the LHRH response to NPY. Ovariectomized rats were implanted with 20-mm-long Silastic capsules containing a low concentration of estradiol (E2) (150 micrograms/ml oil), a high concentration of E2 (250 micrograms/ml oil), or sesame oil vehicle. Additional animals received high-dose E2 capsules plus an injection of progesterone (15 mg) concomitantly. Two days later, individual MBH fragments were incubated in medium alone for a 30-min period to obtain the basal rate of LHRH release, followed by a second 30-min period in medium containing NPY or saline. Exposure to NPY (10(-6) M) increased the release of LHRH from MBH of ovarian hormone-treated, but not from hormonally untreated rats. The LHRH response was most pronounced from the MBH of rats treated with either high-dose E2 or E2 plus progesterone. The increase in LHRH release was also elicited by 10(-7) M, but not by 10(-8) M NPY concentrations, using MBH from E2 plus progesterone-treated rats. In addition, NPY markedly potentiated the KCl-evoked release of LHRH from MBH of ovariectomized, hormonally untreated or low-dose E2-treated rats, under conditions when there was little or no effect of NPY on the basal LHRH release. Further, the release of LHRH stimulated by NPY was not accompanied by increase in the release of norepinephrine or of dopamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible involvement of lipoxygenase pathway of arachidonic acid in rat pituitary hormone release in vitro

The roles of arachidonic acid (AA) and its lipoxygenase products in control of secretion of anterior pituitary hormones were studied in vitro using cultured cells. AA (10(-4)M) and 5-hydroxy-eicosatetraenoic acid (5HETE) (5 x 10(-6)M) significantly (p less than 0.05) stimulated the releases of LH, TSH, GH, PRL, ACTH and beta-endorphin (beta-E). Added leukotriene B4 (LTB4) (5 x 10(-6)M) also caused significant increases in the secretions of LH, GH, ACTH and beta-E. The other lipoxygenase metabolites tested, 12HETE, 15HETE, LTA4, LTC4 and LTD4, had no effect on the releases of anterior pituitary hormones. These results suggest that AA and 5-lipoxygenase metabolites may be involved in the control of the releases of anterior pituitary hormones.     

Regulation of corticotropin-releasing factor (CRF) receptors in the rat pituitary gland: effects of adrenalectomy on CRF receptors and corticotroph responses

The stimulation of ACTH release from anterior pituitary cells by corticotropin-releasing factor (CRF) is mediated by specific, high affinity receptors with a Ka of 10(9) M-1 for ovine CRF. The relationship between ACTH secretion and CRF receptor activation was analyzed in normal and adrenalectomized rats by comparison of ACTH release with changes in CRF receptors and adenylate cyclase activity. The marked increase in plasma ACTH levels that occurred after adrenalectomy (from 71 to 478 pg/ml after 4 days) was accompanied by a progressive decrease in pituitary CRF receptor concentration [by 29 +/- 1%, 75 +/- 2%, 77 +/- 6%, and 80 +/- 4% (+/- SE) after 1, 2, 3, and 4 days, respectively]. Most of this decrease was due to receptor down-regulation rather than occupancy by endogenous CRF, since high dose infusions of CRF (300-500 ng/min) for 30 min before pituitary membrane preparation reduced CRF-binding sites by only 40%. The marked reduction in CRF receptors after adrenalectomy was accompanied by comparable decreases in maximal CRF-stimulated adenylate cyclase activity and sensitivity to CRF (ED50, 3.8 +/- 2.8 vs. 58 +/- 3.7 X 10-9 M CRF in control and 2-day-adrenalectomized rats, respectively). Fluoride-stimulated adenylate cyclase activity was unchanged at 24 h, but was decreased by 28 +/- 7% at later times. Such decreases in CRF receptors and adenylate cyclase activity in adrenalectomized rats were prevented by dexamethasone treatment. In cultured anterior pituitary cells from 4-day-adrenalectomized rats, CRF-stimulated cAMP production was decreased by 40%. However, in contrast to the decreases in CRF receptors and cAMP production, there was a 3-fold increase in CRF-stimulated ACTH release, with no change in sensitivity to CRF. The ability of corticotrophs to maintain increased ACTH release, in conjunction with reduced CRF receptors and CRF-stimulated adenylate cyclase, indicates that elevated ACTH secretion can be maintained by occupancy and activation of only a small number of CRF receptors. This finding also suggests that synergistic interactions between CRF and other regulators of ACTH release may contribute to the sustained increase in ACTH secretion that follows adrenalectomy.     

Induction of interleukin-6 release by interleukin-1 in rat anterior pituitary cells in vitro: evidence for an eicosanoid-dependent mechanism

We have reported previously that a subpopulation(s) of anterior pituitary cells released IL-6 and that this release was stimulated by interleukin-1 (IL-1) through a non-cAMP-dependent mechanism. We now report that IL-1 induces IL-6 release from anterior pituitary cells in an eicosanoid-dependent manner. Dispersed rat anterior pituitary cells were briefly prelabeled (2-3 h) with [3H]arachidonic acid (AA) to esterify the fatty acid within the lipid pool. Incubation of these prelabeled cells with 25 ng/ml IL-1 beta caused an increase only within 1-2 min in the amount of free [3H]AA detected in the extracts of the cells. During 15- to 30-min incubations, IL-1 beta (25 ng/ml) caused an increased accumulation of [3H]AA in the incubation medium which reached levels similar to those induced by 100 nM TRH. Perifused anterior pituitary cells responded to IL-1 beta (25 ng/ml) with a rapid (less than 2 min), biphasic, and reversible efflux of [3H]AA. The [3H]AA appears to have been derived from choline phospholipids, as formation of [3H]glycerophosphorylcholine was substantially increased by exposure of [3H]choline-prelabeled cells to either IL-1 alpha (171%) or IL-1 beta (236%); in addition, the complete deacylation of phosphatidylcholine suggests that other fatty acid species are liberated as a consequence of IL-1 receptor activation and, thus, may also contribute to the actions of IL-1 alpha and IL-1 beta. However, the levels of [3H]phosphorylcholine and [3H]choline were unchanged as well as those of catabolites of other lipid species. These data suggested an involvement of phospholipase-A2 (PLA2) in mediating the IL-1 induction of IL-6 release. Subsequently, we used inhibitors of the PLA2, cyclooxygenase, and lipoxygenase enzymes to investigate a possible role for the generation of AA and its subsequent enzymatic conversion in the signal transduction pathway activated by IL-1. The PLA2 inhibitor aristolochic acid (10 microM) blocked IL-1 beta-induced IL-6 release and the release of IL-6 caused by Pyrularia pubera thionin (5 micrograms/ml), a stimulator of PLA2 activity. The cyclooxygenase inhibitor indomethacin (10 microM) did not inhibit IL-1 beta-induced IL-6 release. In contrast, the general lipoxygenase inhibitor nordihydroguaiaretic acid (10 microM) and the more specific 5-lipoxygenase inhibitors AA861 and RHC5901 (both 10 microM) reduced basal and blocked IL-1 beta-induced IL-6-release.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interleukin-2 induces corticotropin-releasing hormone release from superfused rat hypothalami: influence of glucocorticoids.

The present work shows that interleukin-2 (IL-2) is able to increase in a dose-dependent manner (25-100 U/ml) CRF release from continuous perifused hypothalami. The effects of IL-2 and IL-1 on CRF secretion are potentiated by the simultaneous action of the two cytokines at the hypothalamus. The stimulatory effect of IL-2 on CRF secretion is significantly inhibited by the presence of dexamethasone in the perifusion medium. However, the CRF response to IL-2 was similar in adrenalectomized animals and sham-operated rats. It is suggested that the action of IL-2 on hypothalamic CRF secretion is integrated in the communication between the immune system and the hypothalamic-pituitary-adrenocortical axis, and that such action is subjected to glucocorticoid negative feedback modulation. The mechanism underlying the effect of IL-2 on CRF release is unknown, but arachidonic acid metabolites do not seem to be involved, since neither a lipooxygenase (nordihidrogueretic acid) nor a cyclooxigenase (indomethacin) inhibitor affected the hypothalamic secretory response to IL-2.     

Effects of interleukin-1 on hormone release from normal rat pituitary cells in primary culture

The present study was performed mainly to determine whether interleukin-1 (IL-1), a polypeptide produced by immunologically activated monocytes, plays a physiological role in the regulation of adrenocorticotropic hormone (ACTH) using primary monolayer cultures of rat anterior pituitary cells. Neither human IL-1 alpha nor IL-1 beta stimulated the ACTH release from normal pituitary cells in concentrations ranging from 0.01 to 10 nM. IL-1 beta caused a slight, but significant, increase in ACTH release at a concentration of 100 nM, while IL-1 alpha did not, even at the highest dose tested. IL-1 beta exhibited a synergistic action with corticotropin-releasing factor (CRF) in ACTH secretion at 10 and 100 nM of CRF, but the interaction was not striking. Both of the monokines failed to cause any change in the secretions of growth hormone, prolactin, follicle-stimulating hormone and luteinizing hormone throughout concentrations ranging from 0.01 to 100 nM. The effects of possible sex-related differences and prolonged preincubation of cultured pituitary cells in serum-free medium prior to assay incubation were also tested, providing no significantly different findings. These results suggest that the physiological significance of IL-1 as a tissue CRF is indeed questionable and should be further clarified.     

Changes in corticotrophin releasing factor of the stalk median eminence in rats with various cuts around the medial basal hypothalamus.

The activity of corticotrophin releasing factor (CRF) in extracts of the stalk median eminence (SME) complex proper (average protein content, 30.6 micrograms) of male rats was assayed by monolayer cultures of anterior pituitary cells using the release of immunoreactive ACTH. Extracts which were equivalent to 0.025 SME of control rats usually had detectable CRF activity, while there was no detectable activity in extracts of 0.4 SME equiv. taken 8 days after complete surgical isolation of the medial basal hypothalamus (MBH). The activity of CRF in extracts from rats with an anterolateral cut around the MBH was at least ten times less than that in the control rats. One day after placing an anterolateral cut around the MBH the ACTH releasing activity of the SME was not significantly different from that of the control animals but activity decreased significantly 3 days after the operation and was at least ten times less than in the control animals on day 7 after the operation. It is suggested that most of the CRF activity of the SME is contained in nerve fibres entering the neurohaemal region from outside the MBH and that transection of these fibres produced the fall in CRF content of the SME in rats with partial or total surgical isolation of the MBH.     

A central mechanism is involved in the secretion of ACTH in response to IL-6 in rats: comparison to and interaction with IL-1 beta.

The cytokines interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha are known to be potent effectors of ACTH secretion. Some of the peripheral effects of IL-1 beta appear to be related to the secretion of IL-6 induced by IL-1 beta. Thus, we evaluated the effect of IL-6 on ACTH secretion and its interaction with IL-1 beta. Rats received recombinant human (rhIL-6) or murine (rmIL-6) IL-6 through indwelling jugular cannulae. rhIL-6 (200 ng or 2 micrograms/rat) produced peak plasma ACTH levels which were 3- to 4-fold greater than basal levels. rmIL-6 produced similar responses. Neither species of IL-6 affected plasma prolactin levels. Comparison of rhIL-1 beta (200 ng) to rhIL-6 (200, 100 or 50 ng) showed that IL-6 elevated ACTH in a dose-dependent manner and that IL-1 beta was significantly more effective. IL-1 beta was also administered concomitantly with or 10 min after IL-6. Delivered together, IL-1 beta (100, 30 or 10 ng) and IL-6 (100 ng) produced significantly higher ACTH levels than when given alone. This additivity was also evident when IL-6 was given 10 min prior to IL-1 beta. The coadministration of IL-6 (2 micrograms) with corticotropin-releasing factor (CRF, 1 micrograms/kg, b.w.) also had an additive effect on ACTH secretion (at 20 min: 300 +/- 40 pg/ml for CRF; 320 +/- 83 pg/ml for IL-6; and 540 +/- 44 pg/ml for CRF + IL-6), whereas a higher dose of CRF (10 micrograms/kg b.w.) yielded ACTH levels of 1,000 +/- 107 pg/ml at 20 min, with no further enhancement by IL-6. Incubation of pituitary cells with IL-6 alone (0.1, 1.0 or 3.0 nM) produced a slight but significant stimulation of ACTH secretion within 2 h in response to the higher doses of IL-6 only (p < 0.05), but did not modify the effect of CRF in vitro. To determine if the action of IL-6 was at a site(s) within the brain, IL-6 (30 or 100 ng/0.5 microliters) was injected into the third cerebroventricle of alert rats. 100 ng IL-6 elicited peak plasma ACTH levels (300 +/- 65 pg/ml) within 30 min; these were significantly higher than the buffer responses (90 +/- 25 pg/ml, p < 0.01), and lower than the responses to 30 ng IL-1 beta (530 +/- 50 pg/ml, p < 0.001). 30 ng IL-6 was ineffective.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of various blockers of arachidonic acid metabolism on release of beta-endorphin- and adrenocorticotropin-like immunoreactivity induced by phospholipase A2 from rat adenohypophysis in vitro

Anterior pituitary quarters were incubated in vitro and the release of beta-endorphin-like (beta-End-IR) and adrenocorticotropin-like immunoreactivity (ACTH-IR) was determined. The effect of phospholipase A2 as well as the effect of various compounds known to influence arachidonic acid metabolism under certain conditions were examined. Phospholipase A2 increased the release of beta-End-IR and ACTH-IR. This effect was reversible, concentration-dependent (1-400 ng/ml) and inhibited in calcium-free medium and in the presence of CoCl2 (5 mM) or phospholipase A2 inhibitors (p-bromophenacylbromide, 21 microM; mepacrine, 1 mM). The phospholipase A2-induced beta-End-IR release was accompanied by the release of prostaglandin E2. Inhibition of cyclooxygenase activity by indomethacin (14 or 140 microM) did not change beta-End-IR release induced by phospholipase A2 (5 ng/ml). The effects of blockers of lipoxygenase (nordihydroguaiaretic acid, NDGA; AA861) or lipoxygenase plus cyclooxygenase (BW755C; eicosatetraynoic acid, ETYA) on phospholipase A2-induced release of beta-End-IR were diverse. BW755C (up to 250 microM) and AA861 (up to 100 microM) produced no effect. However, NDGA or ETYA inhibited phospholipase A2-induced beta-End-IR release. NDGA (100 microM) produced a maximum inhibition by about 40% (p less than 0.05), whereas ETYA (100 microM) produced a maximum inhibition by about 85% (p less than 0.001). These data are consistent with the view that phospholipase A2 releases endogenous arachidonic acid which is transformed into products which stimulate ACTH and beta-endorphin release from the corticotrophs; the metabolizing enzyme (possibly a lipoxygenase or epoxygenase) is sensitive to NDGA and especially to ETYA.