Interleukin 6 increases the in vitro expression of key proteins associated with steroidogenesis in the bovine adrenal zona fasciculata.

In this study, the in vitro effects of interleukin 6 (IL-6) on the messenger RNAs (mRNAs) and proteins for key steroidogenic factors in the bovine adrenal zona fasciculata (ZF) were determined. Bovine adrenal glands were obtained from an abattoir, and the ZF was isolated. Strips of ZF were then exposed to different concentration of murine IL-6 and/or adrenocorticotropic hormone (ACTH) for various intervals, the protein and mRNA extracted, and the mRNA and protein expression determined by real-time polymerase chain reaction and Western blots. Exposure (1 h) to IL-6 increased in a concentration-dependent manner (10-pg IL-6/mL, P < 0.05 vs control; 100-pg IL-6/mL, P < 0.01 vs control) the relative expression of the mRNAs and proteins for steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), 3ß hydroxysteroid dehydrogenase type 2 (3ß HSD), 17α-hydroxylase/17,20-lyase/17,20-desmolase (P450 17OH), steroid 21-hydroxylase (P450 21OH), steroid 11-ß-hydroxylase type 1 (P450 11ßOH), and steroidogenic factor 1 (SF-1), a nuclear factor that increases StAR and steroidogenic enzymes (SEs) expression. Similarly, IL-6 (10 pg/mL) increased the relative expression of proteins and mRNAs for StAR, P450scc, 3ß HSD, P450 17OH, P450 21 OH, P450 11ßOH, and SF-1 in a time-dependent manner (30 min, P < 0.05 vs control; 60, 120, and 240 min, P < 0.01 vs control). In contrast, IL-6 decreased in a concentration-dependent (P < 0.01 vs control for 1, 10, and 100 pg IL-6/mL) and time-dependent (P < 0.05 vs control for 30, 60,120, and 240 min of 10 pg IL-6/mL) manner the relative expression of the mRNA and protein for adrenal hypoplasia congenita-like protein (DAX-1), a nuclear factor that decreases expression of StAR and SEs. Incubation (1 h) of ZF with 100-nM ACTH increased (P < 0.05 vs control) the relative expression of StAR, P450scc, 3ß HSD, P450 17OH, P450 21OH, P450 11ßOH, and SF-1 and decreased (P < 0.01 vs control) the relative expression of DAX-1. Murine IL-6 (10 pg/mL) augmented (P < 0.05 vs ACTH) both the stimulatory and inhibitory effects of ACTH. Bovine IL-6 (100 pg/mL, 1-h incubation) also increased (P < 0.01 vs control) the relative expression of the proteins for StAR, P450scc, and SF-1 and decreased (P < 0.01 vs control) the relative expression of DAX-1. In summary, IL-6 increased ZF expression of StAR and 5 SEs, which may be mediated in part by decreasing DAX-1 expression and increasing SF-1 expression.

Interleukin-6 inhibits adrenal androgen release from bovine adrenal zona reticularis cells by inhibiting the expression of steroidogenic proteins.

Interleukin-6 (IL-6) is secreted by adrenocortical cells and modifies cortisol secretion. In this study, the effects of IL-6 on adrenal androgen release were investigated. The zona reticularis (ZR) was generally isolated from bovine adrenal glands by dissection. In select experiments, the intact adrenal cortex (ie, all 3 adrenocortical zones) was dissected from the adrenal glands. For androgen release experiments, ZR and intact adrenocortical cubes were dispersed into isolated cells, the cells cultured and exposed to IL-6 and/or adrenocorticotropic hormone (ACTH), and androgen release determined by radioimmunoassay. Basal and ACTH-stimulated androgen release from the ZR was inhibited by IL-6 in a concentration-dependent (10-1000 pg/mL) and time-dependent (4-24 h) manner (P < 0.01 by 1-way analysis of variance and the Bonferroni test). In contrast, IL-6 increased basal and ACTH-stimulated androgen release from mixed adrenocortical cells (P < 0.01). The mechanism of IL-6 inhibition of androgen release was investigated by exposing ZR strips to IL-6 and measuring the expression of the messenger RNA (mRNA) and protein of steroidogenic factors. Basal and ACTH-stimulated expression of the mRNA and protein for steroidogenic acute regulatory protein, cholesterol side chain cleavage enzyme, 3-ß-hydroxysteroid dehydrogenase type 2, steroid 17-α-hydroxylase/17,20 lyase/17,20 desmolase, and the nuclear factor steroidogenic factor 1 (SF-1), that stimulates steroidogenesis, were decreased by IL-6 (P < 0.01). In contrast IL-6 increased the mRNA and protein for dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1), a nuclear factor that inhibits steroidogenesis (P < 0.01). In summary, IL-6 decreased androgen release and the expression of steroidogenic factors in the ZR, and this decrease may be mediated in part through increasing DAX-1 and decreasing SF-1.

Formation of transient non-protein calcium pores by lysophospholipids in S49 Lymphoma cells.

Palmitoyl-lysophosphatidylcholine promotes a transient calcium influx in lymphoma cells. Previously, it was observed that this influx was accompanied by a temporary increase in propidium iodide permeability that appeared linked to calcium entry. Those studies demonstrated that cobalt or nickel could block the response to lysophosphatidylcholine and raised the question of whether the calcium conductance involved specific channels. This communication describes a series of experiments to address that issue. The time dependence and structural specificity of the responses to lysophosphatidylcholine reinforced the hypothesis of a specific channel or transporter. Nevertheless, observations using patch clamp or calcium channel blockers suggested that this "channel" does not involve proteins. Alternative protein-mediated mechanisms such as indirect involvement of the sodium-calcium exchanger and the sodium-potassium ATPase were also excluded. Experiments with extracellular and intracellular calcium chelators suggested a common route of entry for calcium and propidium iodide. More directly, the ability of lysophosphatidylcholine to produce cobalt-sensitive permeability to propidium iodide was reproduced in protein-free artificial membranes. Finally, the transient nature of the calcium time course was rationalized quantitatively by the kinetics of lysophosphatidylcholine metabolism. These results suggest that physiological concentrations of lysophosphatidylcholine can directly produce membrane pores that mimic some of the properties of specific protein channels.

Mechanisms by which intracellular calcium induces susceptibility to secretory phospholipase A2 in human erythrocytes.

Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A(2) (sPLA(2)). Analysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatidylethanolamine during incubation with ionomycin and sPLA(2). Several possible mechanisms for the effect of ionomycin were considered. Involvement of intracellular phospholipases A(2) was excluded since inhibitors of these enzymes had no effect. Assessment of membrane oxidation by cis-parinaric acid fluorescence and comparison to the oxidants diamide and phenylhydrazine revealed that oxidation does not participate in the effect of ionomycin. Incubation with ionomycin caused classical physical changes to the erythrocyte membrane such as morphological alterations (spherocytosis), translocation of aminophospholipids to the outer leaflet of the membrane, and release of microvesicles. Experiments with phenylhydrazine, KCl, quinine, merocyanine 540, the calpain inhibitor E-64d, and the scramblase inhibitor R5421 revealed that neither phospholipid translocation nor vesicle release was required to induce susceptibility. Results from fluorescence spectroscopy and two-photon excitation scanning microscopy using the membrane probe laurdan argued that susceptibility to sPLA(2) is a consequence of increased order of membrane lipids.

Bovine adrenal cells secrete interleukin-6 and tumor necrosis factor in vitro.

Interleukin-6 (IL-6) and tumor necrosis factor (TNF) are secreted and/or synthesized by the rat and human adrenal cortex. In this study, the release of IL-6 and TNF from bovine adrenal cells was determined. Bovine adrenal glands were collected from an abattoir and dissected into the zona glomerulosa (ZG), zona fasciculata (ZF), zona reticularis (ZR), and medulla. The tissues were enzymatically dispersed to single cells and cultured for 4-6 days. The cells were then exposed (4 h) to angiotensin II (AII), adrenocorticotrophic hormone (ACTH), phorbol dibutyrate (PDB), interleukin-1beta (IL-1beta), interleukin-1alpha (IL-1alpha), and endotoxin (LPS). The IL-6 and TNF content of the incubation medium was determined by bioassays. The release of IL-6 and TNF from the ZG, ZF, ZR, and medulla was increased by PDB, IL-1alpha, IL-1beta, and LPS. In contrast, ACTH and AII increased IL-6 release from the ZG, ZF, and ZR but had no effect on IL-6 release from the medulla. ACTH decreased TNF release from all adrenal cortical zones but had no effect on TNF release from the medulla. Immunohistochemistry utilizing antibodies against TNFalpha demonstrated TNFalpha-containing cells throughout the adrenal gland. The majority of the cells of the ZG, ZF, and ZR contained TNFalpha. However, the cells of the ZG contained more TNFalpha than the cells of the ZR or ZF. Small patches of TNFalpha-containing cells were also found in the adrenal medulla and capsule. These findings support the hypothesis that IL-6 and TNF may have autocrine/paracrine effects on the adrenal gland.

Possible function of IL-6 and TNF as intraadrenal factors in the regulation of adrenal steroid secretion.

Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF alpha) and their mRNAs are present in the human, rat, and bovine adrenal cortex. The release of these cytokines from adrenal cells is regulated by factors that alter adrenal function (e.g., ACTH, angiotensin II, interleukin-1). IL-6 and TNF type 1 receptors are also present on adrenocortical cells. Exposure to IL-6 increases cortisol or corticosterone release from human, bovine, and rat adrenal cells. IL-6 increases basal and ACTH-stimulated aldosterone release, but inhibits angiotensin II-stimulated aldosterone secretion from bovine adrenal cells. IL-6 increases dehydroepiandrosterone (DHEA) release from human cells, but decreases DHEA secretion from bovine cells. TNF alpha inhibits corticosterone release from normal rat adrenal cells or fragments, but increases corticosterone release from cholestatic rat adrenal slices. TNF alpha decreases cortisol release from bovine and fetal human adrenal cells, but increases cortisol release from adult human adrenal cells. TNF alpha inhibits aldosterone secretion from rat and bovine adrenocortical cells. TNF alpha does not affect DHEA secretion from fetal human adrenocortical cells, but inhibits basal and ACTH-stimulated DHEA release from bovine adrenal cell. Because IL-6 and TNF alpha are produced in the adrenal gland and modify adrenal steroid secretion, these cytokines may function as intraadrenal factors in the regulation of adrenal steroid secretion.

Stimulation by interleukin-6 and inhibition by tumor necrosis factor of cortisol release from bovine adrenal zona fasciculata cells through their receptors.

Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are synthesized and released from adrenal cells. Therefore, the effects of TNF-alpha and IL-6 on cortisol release from bovine zona fasciculata (ZF) cells were investigated. IL-6 (10-1000 pg/mL) significantly increased basal and adrenocorticotropic hormone (ACTH)-stimulated cortisol release in a concentration-dependent manner. This stimulatory effect of IL-6 became apparent at intervals as short as 4 h and continued through 24 h. IL-6 also potentiated the cortisol release stimulated by the adenylyl cyclase activator forskolin. By contrast, TNF-alpha (0.1-10 ng) inhibited basal and ACTH-stimulated cortisol release in a concentration-dependent manner. The inhibitory effects of TNF-alpha on cortisol release were significant at time intervals as short as 4 h and continued through 24 h. TNF-alpha inhibited forskolin-stimulated cortisol release. Binding studies demonstrated that ZF cells have IL-6 receptors (100 receptors/cell, Kd of 7.5 x 10(-11)) and TNF receptors (200 receptors/cell, Kd of 2.4 x 10(-9) M). Immunohistochemical analysis provided evidence that the majority of ZF cells have IL-6 receptors, TNF type 1 receptors, and TNF type 2 receptors. Because IL-6 and TNF-alpha are released from the adrenal cortex and these cytokines modify the release of cortisol from the ZF, IL-6 and TNF-alpha may play a paracrine or autocrine role in the regulation of adrenal function.

Mechanisms by which elevated intracellular calcium induces S49 cell membranes to become susceptible to the action of secretory phospholipase A2.

Exposure of S49 lymphoma cells to exogenous group IIA or V secretory phospholipase A2 (sPLA2) caused an initial release of fatty acid followed by resistance to further hydrolysis by the enzyme. This refractoriness was overcome by exposing cells to palmitoyl lysolecithin. This effect was specific in terms of lysophospholipid structure. Induction of membrane susceptibility by lysolecithin involved an increase in cytosolic calcium and was duplicated by incubating the cells with calcium ionophores such as ionomycin. Lysolecithin also activated cytosolic phospholipase A2 (cPLA2). Inhibition of this enzyme attenuated the ability of lysolecithin (but not ionomycin) to induce susceptibility to sPLA2. Lysolecithin or ionomycin caused concurrent hydrolysis of both phosphatidylethanolamine and phosphatidylcholine implying that transbilayer movement of phosphatidylethanolamine occurred upon exposure to these agents but that susceptibility is not simply due to exposure of a preferred substrate (i.e. phosphatidylethanolamine) to the enzyme. Microvesicles were apparently released from the cells upon addition of lysolecithin or ionomycin. Both these vesicles and the remnant cell membranes were susceptible to sPLA2. Together these data suggest that lysolecithin induces susceptibility through both cPLA2-dependent and -independent pathways. Whereas elevated cytosolic calcium was required for both pathways, it was sufficient only for the cPLA2-independent pathway. This cPLA2-independent pathway involved changes in cell membrane structure associated with transbilayer phospholipid migration and microvesicle release.

Cytokine expression in the rat adrenal cortex.

The rat adrenal cortex produces the cytokines interleukin-6, tumor necrosis factor, interleukin-1beta, interleukin-1alpha, macrophage migration inhibitory factor, interferon-gamma inducing factor, and transforming growth factor-beta1. Interleukin-6, tumor necrosis factor, and macrophage migration inhibitory factor are localized to the zona glomerulosa. In contrast, interferon-gamma inducing factor is localized to the zona reticularis and fasciculata. Transforming growth factor-beta1 is localized to the zona fasciculata. Endotoxin and interleukin-1 increase interleukin-6 and tumor necrosis factor release from adrenal cells. In contrast, adrenocorticotrophic hormone, adenosine, serotonin, and dopamine increase adrenal interleukin-6 release, but inhibit tumor necrosis factor release. These secretagogues also increase interleukin-6 mRNA content of adrenal cells. Adrenocorticotrophic hormone decreases transforming growth factor beta1 content of adrenal glands. Endotoxin increases adrenal expression of mRNA for macrophage migration inhibitory factor, but decreases the tissue content of this protein. Endotoxin increases the expression of interleukin-1beta mRNA. Cold stress increases the expression of mRNA for interferon-gamma inducing factor. Therefore, cytokines are differentially expressed in the adrenal cortex and the release and production of these cytokines are regulated selectively. Because cytokines have effects on adrenal function and are differentially regulated, they may play autocrine/paracrine roles in regulating the adrenal gland.

Adenosine increases interleukin 6 release and decreases tumour necrosis factor release from rat adrenal zona glomerulosa cells, ovarian cells, anterior pituitary cells, and peritoneal macrophages.

Adenosine modifies interleukin 6 (IL-6) and tumour necrosis factor (TNF) release from immune tissues. Because adenosine alters endocrine function and endocrine cells secrete cytokines, its effects on IL-6 and TNF release from rat adrenals, ovaries, and anterior pituitaries were compared with its effects on cytokine release from rat peritoneal macrophages. Adenosine increased basal IL-6 release and decreased basal TNF release from adrenal zona glomerulosa and zona fasciculata/reticularis cells. IL-6 and TNF release from zona glomerulosa cells was greater (20x) than that of other adrenal cells. An A2 agonist modified adrenal IL-6 and TNF release at lower concentrations than an A1 agonist. Adenosine augmented adrenal IL-6 release stimulated by endotoxin (LPS), interleukin 1 beta (IL-1 beta), adrenocorticotrophic hormone, and angiotensin II. LPS- and IL-1 beta-stimulated adrenal TNF release was inhibited by adenosine. Adenosine increased IL-6 release and inhibited TNF release from ovarian cells. Anterior pituitary cells released IL-6, but no detectable TNF. Adenosine, via A2 receptors, stimulated IL-6 secretion from these cells. Peritoneal macrophage IL-6 release was increased and TNF release decreased by adenosine. Thus, in immune and endocrine tissues, adenosine increases IL-6 release, but inhibits TNF release.

Mechanisms by which thionin induces susceptibility of S49 cell membranes to extracellular phospholipase A2.

Whereas cells normally resist attack by PLA2, they become susceptible under certain pathological conditions. To ascertain the regulatory mechanisms that induce cellular susceptibility to PLA2, the effect of thionin on S49 cells was examined in the presence of PLA2. Thionin alone was unable to evoke hydrolysis of the lipid bilayer. Likewise, the addition of PLA2 alone caused production of only a minimal amount of free fatty acid. However, thionin and PLA2 together resulted in significant hydrolysis of the cell membrane. Thionin caused perturbation of the bilayer structure as suggested by the changes in the emission spectra of laurdan and the permeability of the membrane to propidium iodide. These changes correlated quantitatively with the susceptibility of the lipid bilayer to PLA2. Furthermore, thionin induced a modest increase in intracellular Ca2+. The source of this Ca2+ was the extracellular fluid since EDTA in the extracellular medium inhibited the Ca2+ influx. Moreover, cobalt chloride, a universal Ca2+ channel blocker, prevented the rise in intracellular Ca2+, the uptake of propidium iodide, and the susceptibility to PLA2 induced by thionin. In contrast, the changes in the laurdan emission caused by the thionin were not affected by the cobalt. Furthermore, incubation of the cells with the calcium ionophore A23187 also caused the cells to become susceptible to PLA2. We hypothesize that thionin causes S49 cell membranes to become susceptible to PLA2 by a Ca2+-dependent perturbation of the bilayer structure.

Dopamine increases interleukin 6 release and inhibits tumor necrosis factor release from rat adrenal zona glomerulosa cells in vitro.

Interleukin 6 (IL-6) and tumor necrosis factor (TNF) are released from the zona glomerulosa of rat adrenal glands. The release of these cytokines from adrenal cells is regulated by interleukin 1 beta (IL-1 beta) and lipopolysaccharide (LPS), which are involved in the immune and inflammatory responses. Adrenocorticotropic hormone (ACTH) and angiotensin II, hormones that regulate the adrenal cortex, likewise regulate release of cytokines from adrenal glands. Dopamine inhibits aldosterone release from the adrenal cortex. Therefore, effects of dopamine on IL-6 and TNF release from rat adrenal zona glomerulosa were investigated. Primary cultures of rat adrenal zona glomerulosa cells were exposed to test agents and IL-6 and TNF release determined by the 7TD1 and WEHI bioassays, respectively. Dopamine increased basal IL-6 release and potentiated IL-6 release stimulated by ACTH, LPS or IL-1 beta. Dopamine inhibited basal and secretagogue-stimulated (LPS and IL-1 beta) TNF release. These effects of dopamine were mediated by D2 receptors because N-0437, a D2 agonist, had effects on TNF and IL-6 release identical to those of dopamine. Therefore, dopamine, through D2 receptors, regulates the release of IL-6 and TNF from adrenal cells. Because TNF and IL-6 regulate adrenal steroid release, these cytokines may serve as autocrine or paracrine mediators of adrenal gland function.

Serotonin increases interleukin-6 release and decreases tumor necrosis factor release from rat adrenal zona glomerulosa cells in vitro.

Interleukin-6 (IL-6) and tumor necrosis factor (TNF) are secreted by rat adrenal zona glomerulosa cells. Serotonin increases the release of aldosterone, corti-costerone, and cortisol from the adrenal cortex. Therefore, the effects of serotonin on IL-6 and TNF release from rat adrenal zona glomerulosa cells were investigated. Cultures of rat adrenal zona glomerulosa cells were enzymatically prepared and cultured for 4-6 d. The cells were then exposed to serum-free RPMl-1640 medium containing vehicle (RPMl medium alone), serotonin, and/or endotoxin, interleukin-1ß, or adrenocorticotrophic hormone (ACTH). Following a 5-h incubation, medium was removed from the cells, and IL-6 and TNF content of this medium determined with bioassays. Serotonin (1-1000 nM) increased basal IL-6 release from zona glomerulosa cells, but inhibited basal TNF release from these cells. Endotoxin and interleukin-1ß (IL-1ß) increased IL-6 and TNF release from zona glomerulosa cells. Serotonin potentiated IL-6 release stimulated by endotoxin and IL-1ß, but inhibited TNF release stimulated by these agents. Serotonin potentiated ACTH-stimulated IL-6 release. Serotonin had no effect on IL-6 release from rat anterior pituitary cells. Because IL-6, TNF, and serotonin modify the release of aldosterone and glucocorticoids from adrenal cells, the stimulatory effects of serotonin on aldosterone and glucocorticoid release may be mediated in part by the effects of serotonin on IL-6 and TNF release from adrenal cells.

Role of the cytokines in the hypothalamic-pituitary-adrenal and gonadal axes.

Cytokines are soluble mediators of immune function that also regulate several endocrine systems. Interleukin-1 (IL-1), IL-6 and tumor necrosis factor-alpha (TNF alpha) each mediate certain aspects of inflammation. In addition, these agents regulate hormone secretion from and cellular proliferation within endocrine tissues. Thus, IL-1 and IL-6 each affect hormone release from anterior pituitary cells (e.g., growth hormone) and inhibit the proliferation of these cells. Cytokines are also localized within discrete nuclei of the hypothalamus (e.g., IL-1 in the paraventricular nucleus), where they may affect production of neuropeptides and biogenic amines (e.g., corticotropin-releasing hormone). Similarly, IL-1 and TNF alpha affect granulosa cell steroidogenesis and IL-6 production. Follicular atresia may either be augmented or inhibited by cytokines depending on their ability to regulate cellular apoptosis. Compartmentation of cytokines within adrenal tissue (e.g., IL-6 in the zona glomerulosa) allows localized effects of these factors on glucocorticoid secretion. Thus, cytokines affect via paracrine or autocrine pathways both hormone secretion from, and possibly cellular differentiation within, endocrine tissues.

Differential release of tumor necrosis factor and IL-6 from adrenal zona glomerulosa cells in vitro.

In previous studies, rat adrenal zona glomerulosa (ZG) cells were demonstrated to release interleukin-6 (IL-6). In the current study, cultures of ZG cells and bioassays for tumor necrosis factor (TNF) and IL-6 were used to determine if ZG cells release TNF and to define more fully the factors that regulate ZG IL-6 release. ZG cells released IL-6 and TNF, and this release was stimulated by lipopolysaccharide, interleukin-1 alpha, interleukin-1 beta, a protein kinase C activator, and a calcium ionophore without affecting intracellular adenosine 3', 5'-cyclic monophosphate (cAMP) content. In contrast, adrenocorticotropic hormone (ACTH) increased the intracellular cAMP content, increased basal and secretagogue-stimulated IL-6 release but decreased basal and secretagogue-stimulated TNF release. The effects of ACTH on IL-6 and TNF release may be mediated by increases in intracellular cAMP because ACTH and dibutyryl cAMP modified IL-6 and TNF release in an identical manner. Therefore, IL-6 and TNF release from ZG cells can be differentially regulated. Because IL-6 and TNF modify adrenal steroid release, the adrenal production of these cytokines may have a role in the stress response.

Vasoactive intestinal peptide increases the liberation of arachidonate from anterior pituitary cells in vitro.

Several secretagogues increase prolactin (PRL) release from anterior pituitary cells through biochemical pathways that involve the liberation of arachidonate from cellular phospholipids. Vasoactive intestinal peptide (VIP) increases PRL release from anterior pituitary cells through a mechanism involving the generation of cAMP. In this study, we determined whether VIP increases the liberation of arachidonate from anterior pituitary cells. Primary cultures of anterior pituitary cells were prepared from the anterior pituitary gland of female Sprague-Dawley rats. After four to five days in culture, the incubation medium was replaced with [3H] arachidonate containing medium, and the cells incubated for 90 min. The cells were then extensively rinsed with incubation medium without [3H] arachidonate to remove the [3H] fatty acid not associated with cellular phospholipids. The pituitary cells were then incubated with medium containing various concentrations of VIP and the release of [3H] arachidonate and PRL into the incubation medium determined. VIP (500 nM) significantly increased [3H] arachidonate liberation from primary cultures of anterior pituitary cells at 30 min (p < 0.5) and 60 min (p < 0.01), but had no significant effect on the liberation of this fatty acid at 15 or 120 min. PRL release was significantly increased by VIP at 30, 60, and 120 min. VIP (60 min exposure) at concentrations of 100 and 500 nM significantly increased PRL release and arachidonate liberation in a concentration-dependent manner. Similarly, VIP increased [3H] arachidonate liberation from a preparation of anterior pituitary cells enriched in lactotropes. Since the increment in [3H] arachidonate liberation was greater in the lactotrope-enriched population than in the anterior pituitary cell preparation, it is highly probable that the lactotropes are the primary source of [3H] arachidonate liberated by VIP. These experiments provide evidence that [3H] arachidonate liberation may play a role in VIP-stimulated PRL release.

Tumor necrosis factor increases interleukin-6 release from adrenal zona glomerulosa cellsin vitro.

Rat adrenal zone glomerulosa cells secrete tumor necrosis factor (TNF) and interleukin-6 (IL-6). We have extended previous studies to determine if TNFα can modify the release of adrenal IL-6. Primary cultures of rat adrenal zone glomerulosa cells were prepared by enzymatic techniques and cultured for 4-6 days. The cells were then exposed to serum-free RPMI 1640 incubation medium containing vehicle (RPMI-1640 medium alone), TNFα and/or selected agents known to stimulate adrenal IL-6 release. Following a 5 h incubation, the incubation medium was removed from the cells and the IL-6 content of the medium measured with the 7TD1 bioassay. TNFα (0.5-50 ng/mL) increased basal adrenal IL-6 release in a concentration-dependent manner. Furthermore, TNFα potentiated in a more than additive manner the adrenal IL-6 release stimulated by lipopolysac-charide (LPS), interleukin-1ß, angiotensin II and ACTH. TNFα potentiated the IL-6 release stimulated by a wide range of concentration of IL-lß (0.01-100 ng/mL) and ACTH (0.1-100 nM). Because IL-6 and TNFα modify the steroid secretion from adrenal cells, these cytokines may interact together to regulate the function of the adrenal cortex.

Pyrularia thionin increases arachidonate liberation and prolactin and growth hormone release from anterior pituitary cells.

Pyrularia thionin is a 47 amino acid peptide isolated from the nuts of Pyrularia pubera. This peptide does not have intrinsic phospholipase A2 activity, but it increases the liberation of arachidonate from several tissues. Exposure of anterior pituitary cells to this toxin increases the liberation of arachidonate, increases the cellular levels of lysophospholipids, and decreases cellular phospholipids. Thus, phospholipase A2 is involved in the liberation of arachidonate stimulated by this peptide. Because this toxin also increases stearate liberation from the pituitary cells, either diacylglycerol lipase, phospholipase A1 or lysophospholipase may be directly or indirectly activated by this toxin. In addition to increasing fatty acid liberation, Pyrularia thionin increases the release of prolactin and growth hormone from anterior pituitary cells over the identical concentration ranges that this toxin liberates the fatty acids. Pyrularia thionin increased arachidonate liberation and prolactin release from perifused pituitary cells within 2 min, and following withdrawal of the toxin, arachidonate liberation and prolactin release returned to near basal levels within 6 min. Dopamine, a physiological inhibitor of prolactin release that closes calcium channels, decreased prolactin release stimulated by Pyrularia thionin. However, dopamine had no effect on the arachidonate liberation stimulated by this peptide. Similarly, D-600, an organic calcium channel blocker, decreased the prolactin and growth hormone release stimulated by the toxin without affecting the toxin-stimulated arachidonate liberation. Therefore, Pyrularia thionin increases arachidonate liberation through the rapid activation of phospholipase A2 by a mechanism that is not dependent on calcium uptake via D-600-inhibitable calcium channels. In contrast, the prolactin and growth hormone release stimulated by this toxin requires calcium uptake via D-600 inhibitable calcium channels.

Thyrotropin-releasing hormone and lysine-bradykinin stimulate arachidonate liberation from rat anterior pituitary cells through different mechanisms.

TRH and lysine-bradykinin (Lys-bradykinin) increase PRL release and arachidonate liberation from anterior pituitary cells. We investigated whether the arachidonate liberation stimulated by TRH and Lys-bradykinin originates in pituitary lactotropes and whether these events are accomplished through similar mechanisms. Lys-bradykinin and TRH rapidly (0.5 min) increased the intracellular [3H]arachidonate content of rat anterior pituitary cells. Lys-bradykinin also increased [3H]arachidonate liberation and PRL release from lactotrope-enriched pituitary cells, but not from a pituitary cell preparation with a diminished number of lactotropes. In contrast, TRH increased [3H]arachidonate liberation from both lactotrope-enriched and lactotrope-diminished preparations; this increased [3H]arachidonate liberation stimulated by TRH in the lactotrope-diminished cells may originate in the thyrotropes. The effects of TRH and Lys-bradykinin on [3H]arachidonate and [14C]stearate liberation in perfused pituitary cells also were determined. Both secretagogues increased arachidonate and stearate liberation in a biphasic manner, characterized by a transient spike, followed by a lower magnitude wave of fatty acid release. The spike phase produced by Lys-bradykinin was more pronounced than that produced by TRH. The calcium dependence of TRH- and Lys-bradykinin-stimulated arachidonate liberation also was investigated. Cobalt and the low calcium medium containing ionomycin were used to block the secretagogue-induced increase in intracellular calcium concentrations. These conditions blocked TRH-stimulated arachidonate liberation, but only marginally decreased Lys-bradykinin-stimulated arachidonate liberation, indicating that the two peptides act through different mechanisms. Therefore, TRH stimulation of arachidonate liberation is linked to an increase in intracellular calcium. In contrast, Lys-bradykinin increases arachidonate liberation through a calcium-independent intracellular mediator. This calcium-independent increase in arachidonate liberation may involve the bradykinin receptor being coupled directly to a phospholipase, a G-protein that provides a link between the bradykinin receptor and the phospholipases that liberate arachidonate, or bradykinin-induced activation of a protein kinase-C that activates the phospholipases and subsequently liberates arachidonate.

Adrenocorticotropin increases interleukin-6 release from rat adrenal zona glomerulosa cells.

Interleukin-6 (IL-6) is produced by adrenal zona glomerulosa cells; its release is stimulated by several secretagogues, including IL-1 alpha, IL-1 beta, and angiotensin II. The present study reports that ACTH (0.1-100 nM) increased the release of IL-6 from primary cultures of rat adrenal cells in a concentration-dependent manner. This increase was accompanied by an increase in cAMP content in cell extracts and in the incubation medium. The dynamics of IL-6 release from the adrenal cells also were investigated using a perifusion system; approximately 50 min were required for the effects of IL-1 alpha, IL-1 beta, and ACTH on IL-6 release to become apparent. Following withdrawal of the secretagogues, IL-6 release returned to basal levels within 90-120 min. In some experiments, the adrenal zona glomerulosa was separated from the zona fasciculata/reticularis to determine the origin of secretagogue-stimulated IL-6 release. PGE2 and forskolin increased IL-6 release from both cell types, but maximal release from zona glomerulosa cells was more than 10-fold greater than that from zona fasciculata/reticularis cells. ACTH (0.1-100 nM) increased intracellular cAMP levels in cells from both cell types in a concentration-dependent manner, but increased IL-6 release only from zona glomerulosa cells. Dexamethasone, an inhibitor of IL-6 production in several tissues, had no effect on either basal or stimulated IL-6 production in the adrenal. Because IL-1 beta is produced primarily by tissues of the immune system, whereas ACTH is a classical endocrine hormone, we investigated the effect of interaction of these proteins on IL-6 release from the adrenal. Together, IL-1 beta and ACTH stimulation of IL-6 release was greater than the sum of the effects of each substance separately; however, IL-1 beta did not potentiate the effect of ACTH on cAMP levels. Similarly, IL-1 beta potentiated IL-6 release stimulated by forskolin and (Bu)2cAMP. Thus, the adrenal may be an important convergence point between the immune and endocrine systems, and because IL-6 release is regulated by IL-1 alpha, IL-1 beta, ACTH, and angiotensin II, and this cytokine stimulates corticosterone release, IL-6 may play an important paracrine role in integrating the signals derived from these systems.