Interleukin 1 potentiates agonist-induced secretion of beta-endorphin in anterior pituitary cells

Interleukin 1 (IL-1) has been shown to potentiate the release of beta-endorphin induced by secretagogues, including corticotropin releasing factor (CRF) and phorbol ester (TPA), in the mouse AtT-20 pituitary tumor cell line (Fagarasan et al., PNAS, 1989, 86, 2070-2073). In cultured rat anterior pituitary cells, pretreatment with IL-1 caused only a small increase in beta-endorphin release but significantly potentiated CRF-and vasopressin-stimulated beta-endorphin secretion. Vasopressin stimulates the secretion of beta-endorphin in normal pituitary cells but not in AtT-20 cells. However, treatment of AtT-20 cells with IL-1 induced the expression of vasopressin-mediated beta-endorphin release; this effect of IL-1 was reduced after depletion of protein kinase C by prolonged treatment with TPA. The enhancement of CRF-stimulated beta-endorphin release by IL-1 was also reduced in AtT-20 cells after depletion of protein kinase C, and after treatment with staurosporine. These findings indicate that treatment with IL-1 amplifies receptor-mediated responses to the major physiological secretagogues in normal corticotrophs, and initiates a secretory response to vasopressin in AtT-20 cells.     

Influence of VIP on prolactinemia in turkey anterior pituitary cells: role of cAMP second messenger in VIP-induced prolactin gene expression

Vasoactive intestinal peptide (VIP) is the avian prolactin (PRL)-releasing factor. In the turkey, hypothalamic VIP immunoreactivity and mRNA content, as well as VIP levels in hypophyseal portal blood, are closely related to the state of prolactinemia and the reproductive stage. The present study investigated the role of VIP on prolactinemia in turkey anterior pituitary (AP) cells through PRL gene expression and the role of a cAMP second messenger system on VIP-induced PRL expression. In primary AP cells harvested from hens in different prolactinemic states, steady state promoter activities were positively correlated with secreted PRL levels. VIP increased PRL promoter activities in AP cells from hens with intermediate PRL levels (laying), but not in AP cells from hypoprolactinemic hens (nonphotostimulated reproductively quiescent). However, in AP cells from hyperprolactinemic hens (incubating), PRL promoter activity was down-regulated by VIP. PRL mRNA steady state levels were significantly decreased by the cAMP analogue, 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), and PRL secretion was down-regulated by the phosphodiesterase blocker, 3-isobutyl-1-methylxanthine (IBMX) in a dose-dependent manner, suggesting that the cAMP second messenger system might be involved in the inhibitory action of dopamine upon VIP-stimulated PRL secretion and gene expression at the pituitary level. In a study of VIP immediate and long-term effects on c-fos expression in relation to PRL expression, VIP dramatically induced c-fos mRNA expression within 5 min, suggesting that VIP-induced c-fos expression might be involved in VIP-stimulated PRL secretion and gene expression. These results provide additional evidence of the functional significance of VIP in PRL gene expression and suggest that changes in PRL promoter activity by VIP may be one of the important inductive mechanisms leading to prolactinemia.     

Opioid receptor-coupled second messenger systems

Although pharmacological data provide strong evidence for different types of opioid receptors (e.g., mu, delta, and kappa), they share many common properties in their ability to couple to second messenger systems. All opioid receptor types are coupled to G-proteins, since agonist binding is diminished by guanine nucleotides and agonist-stimulated GTPase activity has been identified in several preparations. Moreover, all three types inhibit adenylyl cyclase. This second messenger system has been identified for opioid receptors in both isolated brain membranes and in transformed cell culture. Studies with chronic treatment with opioid agonists suggest that the coupling of receptors with G-proteins and second messenger effectors may play important roles in development of opioid tolerance.     

Gonadotropin-releasing hormone stimulates annexin 5 messenger ribonucleic acid expression in the anterior pituitary cells

We previously reported that annexin 5 is found specifically in gonadotropes and that the expression is dramatically enhanced after ovariectomy. In the present study, the expression of annexin 5 was examined in the primary culture of rat anterior pituitary cells using semiquantitative RT-PCR to determine if it is under the direct control of gonadotropin-releasing hormone (GnRH). Continuous administration of GnRH analog for 1 h enhanced the expression of both FSH beta subunit and annexin 5 mRNA. The expression of annexin 5 mRNA was also augmented by phorbol 12-myristate 13-acetate but not by forskolin. Administration of recombinant rat annexin 5 to the culture increased LH beta mRNA expression. These data clearly demonstrate that the expression of annexin 5 mRNA is directly controlled by GnRH and suggest that annexin 5 is involved in mediating GnRH action in the pituitary gland.     

In vitro study on proopiomelanocortin messenger RNA levels in cultured rat anterior pituitary cells

The fundamental examination on the measurement of proopiomelanocortin (POMC) mRNA levels in cultured rat anterior pituitary (AP) cells was studied. In addition, the detailed study on time- and dose-related effects of corticotropin-releasing factor (CRF) and dexamethasone on the level of POMC mRNA in AP cells in vitro was examined. Basal levels of POMC mRNA in AP cells cultured with serum initially declined after 1-day culture, gradually increased and reached a peak after 3-day culture, and then slightly decreased after 4- and 5-day culture. These mRNA levels after 3-day culture did not change through subsequent 15-hr incubation without serum. CRF treatment caused a time- and dose-dependent increase in POMC mRNA levels. The minimum effective dose of CRF was 0.1 nM for 15-hr incubation. The significant increase in POMC mRNA levels was observed after 3 hrs of 1 nM CRF treatment with a 2-fold elevation seen after 15 hrs of exposure. Dexamethasone treatment caused a dose-dependent decrease in POMC mRNA levels in AP cells. The minimum effective dose was 0.1 microgram/ml and such mRNA levels did not decrease until 15 hrs of exposure.     

Involvement of second messenger systems in stimulation of angiotensin converting enzyme of bovine endothelial cells.

We have demonstrated previously that a variety of agents including corticosteroids, thyroid hormone, cationophores, methylxanthines, and analogues of cAMP--all of which have diversified functions in various tissues--elevate cellular angiotensin converting enzyme (ACE) activity of bovine endothelial cells in culture. In addition to these agents, we have now found that direct and receptor-mediated stimulators of adenylate cyclase, i.e., forskolin and cholera toxin, increase cellular ACE activity after 48 h incubation in culture. In an attempt to search out a more unifying concept of these stimulatory effects, we have further investigated the roles of second messengers in the stimulatory actions. Ca2+ ionophore A23187 produced significant increases in both intracellular Ca2+ and ACE of endothelial cells. In contrast to Ca2+ ionophore, agents that transiently mobilize Ca2+ from intracellular reserves such as bradykinin, acetylcholine, and ATP have no effect on the level of cellular ACE. Representative agents that elevate cellular cAMP (e.g., isobutyl methylxanthine [IBMX] and dibutyryl cAMP) elevated cellular ACE, but the slightly increased [Ca2+]i produced by these agents did not reach statistical significance. While IBMX, cholera toxin, and forskolin elevated cellular cAMP, other ACE stimulatory agents (hormones and cationophores) had no effect on cAMP. Ca2+ ionophore and the agents that elevated intracellular cAMP potentiated the effect of dexamethasone, thyroid hormone, and aldosterone in elevating cellular ACE activity. Increases in ACE activity produced by all stimulants were inhibited by the presence of 10-50 nM ouabain in the culture medium. Inhibition of ACE elevation by oubain was reversed by increasing the extracellular [K+], thereby implicating Na+, K(+)-ATPase in the ACE regulatory mechanism. These results support the presence of multiple independent mechanisms for the regulation of cellular ACE. In addition to possible involvement of intracellular Ca(2+)- and cAMP-dependent pathways, ACE is also increased by corticosteroids and thyroid hormone through mechanisms unrelated to Ca2+ and cAMP.     

Effect of clonidine on second messenger systems in rat adrenal gland.

Clonidine, an alpha 2-adrenergic agonist, also binds to non-adrenergic imidazole receptors in brain and peripheral tissues. In adrenal medulla, however, clonidine appears to bind only to imidazole receptors. To assess whether the signal transduction mechanism of imidazole receptors differs from alpha 2-adrenergic receptors, we studied the actions of clonidine on the turnover of phosphoinositide and the production of cAMP and cGMP in slices of rat adrenal gland. Clonidine did not modify basal or carbachol mediated increases in phosphoinositide turnover or production of cAMP, however it increased the production of cGMP. The increase in cGMP was slow and unaffected by the addition of the phosphodiesterase inhibitor, IBMX. We conclude that the second messenger response triggered by clonidine in adrenal differs from that usually coupled to alpha 2-adrenergic receptors. Whether the effect is mediated by cell surface imidazole receptors remains to be established.     

Neural cell adhesion molecules influence second messenger systems

We have investigated the influence of the neural cell adhesion molecules L1 and N-CAM on second messenger systems using a PC12 rat pheochromocytoma cell line as a model and triggering cell surface receptors by specific antibody binding. Antibodies directed against L1 and N-CAM, but not against other cell surface components, reduce intracellular levels of the inositol phosphates IP2 and IP3, while intracellular levels of cAMP are unaffected. Antibodies against L1 and N-CAM also reduce intracellular pH and increase intracellular Ca2+ by opening Ca2+ channels in a pertussis toxin-inhibitable manner, suggesting the involvement of a G protein in the signal transduction process. Cross-linking of the adhesion molecules on the surface membrane is not required for the effects to occur. Furthermore, adhesion of single PC12 cells to each other elicits effects on intracellular pH and Ca2+ similar to those seen after application, underscoring the physiological significance of the observed changes.     

Single calcium-dependent potassium channels in clonal anterior pituitary cells.

Single Ca2+-dependent K+-channel currents were recorded in intact and excised inside-out membrane patches of the anterior pituitary clone AtT-20/D16-16. The frequency of channel openings and lifetimes depends both on membrane potential and on the Ca2+ concentrations at the inner membrane surface. The curve of the open-state probability of the channel as a function of membrane potential appears to translate along the voltage axis with changes in internal Ca2+ concentration. For Ca2+ concentrations between 10(-7) and 10(-6) M, the shift is consistent with the hypothesis that three Ca2+ ions are required to open a Ca2+-dependent K+ channel. Single channel conductances are estimated to be 124 pS in patches with normal external K+ (5.4 mM) and 208 pS in excised patches with symmetrical K+ (145 mM) across the membrane. Tetraethylammonium (20 mM) added to the cytoplasmic surface reversibly blocks the Ca2+-dependent K+ channel.     

Interleukin 1 stimulates prostaglandin synthesis and cyclic AMP accumulation in Swiss 3T3 fibroblasts: interactions between two second messenger systems

Biochemical events elicited by interleukin 1 (IL-1) were studied in Swiss 3T3 fibroblasts. One hour after its addition, IL-1 stimulated synthesis of prostaglandin E2 (PGE2), which continued to accumulate for 4 days. IL-1 also stimulated cAMP accumulation. Indomethacin blocked cAMP accumulation in response to IL-1, suggesting that PGE2 was responsible for the increase. Addition of exogenous PGE2 to indomethacin-treated cells restored cAMP accumulation. IL-1 enhanced thymidine incorporation, and indomethacin attenuated responses to lower concentrations. Thus, PGE2 appeared to play a role in the ability of low concentrations of IL-1 to stimulate thymidine incorporation. PGE2 augmented thymidine incorporation by increasing cAMP accumulation because in the presence of indomethacin addition of exogenous cAMP enhanced thymidine incorporation in response to low concentrations of IL-1. Elevated cAMP further stimulated PGE2 synthesis. Thus, PGE2 and cAMP interacted to potentiate their mutual accumulation. In summary, IL-1 stimulates PGE2 synthesis. PGE2, in turn, stimulates cAMP accumulation which potentiates IL-1-stimulated PGE2 synthesis and thymidine incorporation.     

Microtubules in thyroidectomy cells of the rat anterior pituitary gland.

Microtubules were successfully illustrated in thyrotrophs and thyroidectomy cells of rat pituitary glands. In contrast, microfilaments were mostly seen in the nonglandular follicular cells. Numerous microtubules were observed in the early stages of development of the thyroidectomy cells. In thyroidectomy cells microtubules were located in close proximity to mitochondria, endoplasmic reticula, secretory granules, and membranes of Golgi complexes. Consequently, it is suggested that microtubules may play a role in degranulation or other processes associated with the hypersecretory state.     

Dexamethasone increases potassium channel messenger RNA and activity in clonal pituitary cells.

Glucocorticoid hormones are released as part of the stress response and regulate secretion by the pituitary. Since the activity of ion channels also influences secretion, we examined the effect of the glucocorticoid agonist dexamethasone on ion channel expression. K+ channel mRNA was detected in rat hypothalamus and anterior pituitary, with probes derived from the rat Kv1 gene, a member of the mammalian voltage-gated K+ channel superfamily. High levels were also detected in PRL-secreting clonal (GH3 and GH4C1) rat pituitary cells. Dexamethasone rapidly increased the steady state concentration of Kv1 mRNA in GH3 cells in a dose-dependent manner. This change in gene expression was accompanied by an increase in whole cell voltage-gated K+ current [lk(i)] with similar pharmacology to the Kv1 gene product. Our findings indicate that hormones may act directly on excitable cells to produce long term effects on electrical activity and secretion by regulating K+ channel expression.     

Differential expression of ionic channels in rat anterior pituitary cells.

Secretory anterior pituitary cells are of the same origin, but exhibit cell type-specific patterns of spontaneous intracellular Ca2+ signaling and basal hormone secretion. To understand the underlying ionic mechanisms mediating these differences, we compared the ionic channels expressed in somatotrophs, lactotrophs, and gonadotrophs from randomly cycling female rats under identical cell culture and recording conditions. Our results indicate that a similar group of ionic channels are expressed in each cell type, including transient and sustained voltage-gated Ca2+ channels, tetrodotoxin-sensitive Na+ channels, transient and delayed rectifying K+ channels, and multiple Ca2+ -sensitive K+ channel subtypes. However, there were marked differences in the expression levels of some of the ionic channels. Specifically, lactotrophs and somatotrophs exhibited low expression levels of tetrodotoxin-sensitive Na+ channels and high expression levels of the large-conductance, Ca2+ -activated K+ channel compared with those observed in gonadotrophs. In addition, functional expression of the transient K+ channel was much higher in lactotrophs and gonadotrophs than in somatotrophs. Finally, the expression of the transient voltage-gated Ca2+ channels was higher in somatotrophs than in lactotrophs and gonadotrophs. These results indicate that there are cell type-specific patterns of ionic channel expression, which may be of physiological significance for the control of Ca2+ homeostasis and secretion in unstimulated and receptor-stimulated anterior pituitary cells.     

The second messenger pathway for germ cell-mediated stimulation of Sertoli cells.

Treatment of cultured rat Sertoli cells with FSH or dibutyryl cAMP for 30 min resulted in phosphorylation of the same Sertoli cell proteins. Different Sertoli cell proteins were phosphorylated after calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. A23187 stimulated the phosphorylation of hsp27, while TPA alone had no effect. TPA plus A23187 resulted in phosphorylation of a 14 kDa protein, in addition to hsp27. The effect of TPA plus A23187 was identical to that of germ cells on Sertoli cell protein phosphorylation. FSH-stimulated cAMP production by Sertoli cells was reduced by prior exposure of Sertoli cells to germ cells. The results indicate that germ cells stimulate Sertoli cells by the inositol trisphosphate/diacylglycerol mediated second messenger pathway. The results also suggest that the germ cell-activated pathway interacts within Sertoli cells to modulate Sertoli cell response to FSH.     

Heat shock effects on second messenger systems of Neurospora crassa

Exposure of growing hyphae of Neurospora crassa to heat shock (44 °C) or ethanol (2.6 M) for 1 h induced a significant increase in the cAMP level, which reached a maximum approximately 2 min after the beginning of treatment and then decreased to control values despite continued heat or ethanol exposure. A 10-s heat shock or a 5-s ethanol shock also resulted in a transient cAMP increase 2 min after the pulse. Heat shock or ethanol treatment led to an increase in the amount of catalytic subunits of the cAMP-dependent protein kinase A in the nucleus almost synchronously with the increase of cAMP in the cytoplasm. The concentration of cGMP decreased a few seconds after the beginning of heat shock (44 °C) or ethanol treatment (2.6 M) to approximately 50% of the control level. Exposure to heat shock (44 °C, 1 h) led to an increase in the amount of inositol phosphates 0.5–2 min after the onset of heat shock. Thereafter, inositol phosphate levels dropped to control values despite continued heat exposure. Incubation of growing hyphae with cAMP or 8-Br-cAMP led to a two- to threefold increase of inositol phosphates 10–300 s after the beginning of incubation. Heat treatment furthermore caused a rapid release of calcium from vacuoles as determined by Fura-2 measurement of the calcium content released from isolated vacuoles. These heat-shock-dependent second messenger changes may play a role in the heat-shock-induced phase shifts of the circadian clock and heat-shock-induced conidiation. Received: 7 July 1997 / Accepted: 2 June 1998     

Estrogen-induced decrease of glucocorticoid receptor messenger ribonucleic acid concentration in rat anterior pituitary gland

Using Northern blots and hybridization techniques, we have identified an approximately 6.5 kilobase glucocorticoid receptor mRNA species in rat anterior pituitary gland. Ovariectomy resulted in an approximately 2-fold increase in glucocorticoid receptor mRNA concentrations. This effect was maximal 8 days after surgery and glucocorticoid receptor mRNA levels remained elevated for at least up to 4 weeks. Administration of 17-beta-estradiol completely reversed the ovariectomy-induced increase in glucocorticoid receptor mRNA content of pituitary gland. Treatment of rats with corticosterone did not influence the ovariectomy-induced increase in glucocorticoid receptor mRNA content, indicating that this increase is not mediated via effects on circulating glucocorticoid levels or availability. In situ hybridization experiments confirmed the ovariectomy-induced increase in glucocorticoid receptor mRNA content and indicated that this action is widely distributed throughout the anterior pituitary gland.     

Estrogen modulation of angiotensin-stimulated phosphoinositide hydrolysis in slices of rat anterior pituitary

In this study, slices of rat anterior pituitary were prelabeled with [³H]myo-inositol and the ability of angiotensins II and III to stimulate [³H]phosphoinositide hydrolysis was characterized. When using tissue derived from ovariectomized rats, dose-response experiments revealed that angiotensin II significantly increases [³H]inositol monophosphate formation (in the presence of 10 mM LiCI) at concentrations of 10 nM and above. Maximal stimulation by angiotensin II was observed at 1 μM (228% of basal) and 50% maximal stimulation was at 10.8 ± 2.7 nM. Angiotensin III was less potent when compared to angiotensin II (maximal stimulation at 10 μM; 220% of basal: 50% maximal stimulation, 475 ± 159 nM). When using normal female rats, significant stimulation by angiotensin II was not observed until 1 μM angiotensin II. When ovariectomized rats were treated for 7 days with 17β-estradiol, increases in [³H]inositol monophosphate induced by 1 μM angiotensin II were significantly reduced when compared to sesame oil vehicle controls.This study shows that estrogen down-regulates angiotensin receptor coupling in the anterior pituitary. Moreover, it illustrates the influence of the hormonal state of the animal on the regulation of the effects of angiotensins in this tissue.     

Calcitonin gene-related peptide and cyclic AMP stimulate phosphoinositide turnover in skeletal muscle cells. Interaction between two second messenger systems

Calcitonin gene-related peptide (CGRP) has previously been shown to coexist with acetylcholine in spinal cord motoneurons and to stimulate adenylate cyclase in skeletal muscle cells. We now demonstrate that in cultured chick myotubes whose phosphoinositides have been labeled with [3H]inositol, CGRP enhanced the accumulation of [3H]inositol mono-, bis-, and trisphosphates. Rat CGRP-I (rCGRP) (0.1 microM) elicited a transient increase in [3H]inositol 1,4,5-trisphosphate, as well as a more sustained elevation of [3H]inositol 1,3,4-trisphosphate levels. In the presence of Li+, rCGRP evoked an approximately 3-fold increase of [3H]inositol monophosphate levels, which persisted for up to 1 h. This effect of rCGRP was concentration-dependent, the half-maximal response being obtained at 1 nM. Since rCGRP also accelerated the rate of synthesis of [3H]inositol-containing lipids, it appears that the peptide acts by stimulating phosphoinositide turnover in chick myotubes. Agents that either mimic or elevate intracellular cyclic AMP also enhanced the synthesis of [3H]inositol-containing lipids, and the accumulation of inositol phosphates, suggesting that the effects of rCGRP are mediated, at least in part, via the activation of adenylate cyclase. This hypothesis was strengthened by the non-additivity of the inositol phosphate responses elicited by rCGRP and other cAMP-mobilizing agents, and by the sensitivity of these responses to various pharmacological treatments. The present results provide an example of positive interaction between cAMP and the phosphoinositide signaling system. They further suggest that a coexisting neuropeptide may exert pleiotropic actions upon its target cell by stimulating multiple signal transduction pathways.