Signal transduction mechanisms mediating rapid, nongenomic effects of cortisol on prolactin release

While the mechanisms governing genomically mediated glucocorticoid actions are becoming increasingly understood, relatively little is known with regard to the cell signaling pathways that transduce rapid glucocorticoid actions. Studies of the cultured tilapia rostral pars distalis (RPD), a naturally segregated region of the fish pituitary gland that contains a 95-99% pure population of prolactin (PRL) cells and is easily dissected and maintained in a completely defined, serum-free media, indicate that physiological concentrations of cortisol rapidly inhibit PRL release. The attenuative action of cortisol on PRL release occurs within 10-20 min, is insensitive to the protein synthesis inhibitor, cycloheximide, and mimicked by its membrane impermeable analog, cortisol-21 hemisuccinate-conjugated bovine serum albumin (BSA). Cortisol and somatostatin, a peptide known to work through membrane receptors to inhibit PRL release, rapidly and reversibly reduces intracellular free Ca(2+) (Ca(i)(2+)), and inhibits 45Ca(2+) influx and BAYK-8644 induced PRL release. Preliminary investigations show cortisol, but not somatostatin, suppresses phospholipase C (PLC) activity in PRL cell membrane preparations. In addition, cortisol and somatostatin reduce intracellular cAMP and membrane adenylyl cyclase activity. These findings indicate that the acute inhibitory effects of cortisol on PRL release occur through a nongenomic mechanism involving interactions with the plasma membrane and inhibition of both the Ca(2+) and cAMP signal transduction pathways. Cortisol may reduce Ca(i)(2+) by inhibiting influx through L-type voltage-gated channels and possibly release through a PLC/inositol triphosphate sensitive intracellular Ca(2+) pool. In addition, it is also likely the steroid inhibits adenylyl cyclase activity in events leading to reduced cAMP production and the subsequent release of PRL.     

Cortisol rapidly suppresses intracellular calcium and voltage-gated calcium channel activity in prolactin cells of the tilapia (Oreochromis mossambicus)

Cortisol was previously shown to rapidly (10-20 min) reduce the release of prolactin (PRL) from pituitary glands of tilapia (Oreochromis mossambicus). This inhibition of PRL release by cortisol is accompanied by rapid reductions in (45)Ca(2+) and cAMP accumulation. Cortisol's early actions occur through a protein synthesis-independent pathway and are mimicked by a membrane-impermeable analog. The signaling pathway that mediates rapid, nongenomic membrane effects of glucocorticoids is poorly understood. Using the advantageous characteristics of the teleost pituitary gland from which a nearly pure population of PRL cells can be isolated and incubated in defined medium, we examined whether cortisol rapidly reduces intracellular free calcium (Ca(i)(2+)) and suppresses L-type voltage-gated ion channel activity in events that lead to reduced PRL release. Microspectrofluorometry, used in combination with the Ca(2+)-sensitive dye fura 2 revealed that cortisol reversibly reduces basal and hyposmotically induced Ca(i)(2+) within seconds (P < 0.001) in dispersed pituitary cells. Somatostatin, a peptide known to inhibit PRL release through a membrane receptor-coupled mechanism, similarly reduces Ca(i)(2+). Under depolarizing [K(+)], the L-type calcium channel agonist BAY K 8644, a factor known to delay the closing of L-type Ca(2+) channels, stimulates PRL release in a concentration-dependent fashion (P < 0.01). Cortisol (and somatostatin) blocks BAY K 8644-induced PRL release (P < 0.01; 30 min), well within the time course over which its actions occur, independent of protein synthesis and at the level of the plasma membrane. Results indicate that cortisol inhibits tilapia PRL release through rapid reductions in Ca(i)(2+) that likely involve an attenuation of Ca(2+) entry through L-type voltage-gated Ca(2+) channels. These results provide further evidence that glucocorticoids rapidly modulate hormone secretion via a membrane-associated mechanism similar to that observed with the fast effects of peptides and neurotransmitters.     

Cholesterol efflux-mediated signal transduction in mammalian sperm: cholesterol release signals an increase in protein tyrosine phosphorylation during mouse sperm capacitation.

We previously demonstrated that mouse sperm capacitation is accompanied by a time-dependent increase in protein tyrosine phosphorylation that is dependent on the presence of BSA, Ca2+, and NaHCO(3), all three of which are also required for this maturational event. We also demonstrated that activation of protein kinase A (PK-A) is upstream of this capacitation-associated increase in protein tyrosine phosphorylation. BSA is hypothesized to modulate capacitation through the removal of cholesterol from the sperm plasma membrane. In this report, we demonstrate that incubation of mouse sperm medium containing BSA results in a release of cholesterol from the sperm plasma membrane to the medium; release of this sterol does not occur in medium devoid of BSA. We next determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Blocking the action of BSA by adding exogenous cholesterol-SO-(4) to the BSA-containing medium inhibits the increase in protein tyrosine phosphorylation as well as capacitation. This inhibitory effect is overcome by (1) the addition of increasing concentrations of BSA at a given concentration of cholesterol-SO-(4) and (2) the addition of dibutyryl cAMP plus IBMX. High-density lipoprotein (HDL), another cholesterol binding protein, also supports the capacitation-associated increase in protein tyrosine phosphorylation through a cAMP-dependent pathway, whereas proteins that do not interact with cholesterol have no effect. HDL also supports sperm capacitation, as assessed by fertilization in vitro. Finally, we previously demonstrated that HCO-(3) is necessary for the capacitation-associated increase in protein tyrosine phosphorylation and demonstrate here, by examining the effectiveness of HCO-(3) or BSA addition to sperm on protein tyrosine phosphorylation, that the HCO-(3) effect is downstream of the site of BSA action. Taken together, these data demonstrate that cholesterol release is associated with the activation of a transmembrane signal transduction pathway involving PK-A and protein tyrosine phosphorylation, leading to functional maturation of the sperm.     

Inhibition by estrogen of autofeedback regulation of prolactin secretion

The effect of estradiol-17 beta (E2) on autofeedback regulation of prolactin (PRL) secretion was tested in ovariectomized rats after s.c. implantation of an (E2)-containing or empty silastic capsule, followed by i.v. injection of bovine PRL (b-PRL) or bovine serum albumin (BSA; 500 micrograms/100 g B.W.). Implantation of an E2 capsule (day 0), 2.5 mm or 5.0 mm in length, produced plasma E2 concentrations of 79 +/- 6 (9) and 140 +/- 8 pg/ml (8), respectively. Assay of PRL in plasma samples collected at 1 h intervals between 1100-1800 h on days 3, 4 and 5, after E2 capsule implantation showed a daily afternoon PRL surge. Empty capsule-treated rats did not show any afternoon PRL surge. Injection of b-PRL, but not BSA, at 1200 h on day 3 reduced basal PRL release both on days 3 and 4 in empty capsule-treated rats. In ovariectomized rats treated with a smaller E2 capsule (2.5 mm), b-PRL injection at 1200 h on day 3 reduced the amplitude of the afternoon surge of PRL and the total amount of PRL released on day 4. b-PRL, however, was ineffective in reducing PRL release in rats bearing the large E2 capsule (5.0 mm). These results suggest that high E2 levels in the blood can block the negative feedback action of PRL on PRL release.     

Prolactin and epidermal growth factor stimulate adipophilin synthesis in HC11 mouse mammary epithelial cells via the PI3-kinase/Akt/mTOR pathway

The aim of the present study is to estimate the role played by cortisol, prolactin (PRL) and epidermal growth factor (EGF) in the synthesis of adipocyte differentiation-related protein (ADRP) as compared to the well-studied regulation of? β-casein synthesis by these hormones in the mammary epithelial cell line HC11. This comparison between a cytoplasmic lipid droplet-associated protein, which is strictly specific to both lipid accumulation and secretion by lactating mammary epithelial cells, and an archetypal milk protein is useful for evaluating the extent to which a mechanistic relationship exists between biosynthesis, transport and secretion of these two major milk components. We found that cortisol inhibits PRL-stimulated ADRP synthesis, as opposed to its known stimulating effect on β-casein synthesis. The involvement of PRL and EGF in ADRP synthesis was explored by means of a battery of inhibitors. The Jak2 inhibitor AG490 provoked a stimulation of ADRP synthesis whereas it totally suppressed that of β-casein. The use of AG1478, a specific inhibitor of EGF receptor phosphorylation, or of PD98059, a specific MEK inhibitor, revealed that the Ras/Raf/MEK/ERK1/2 pathway has no significant influence on ADRP levels. Inhibition of JNK was also ineffective. In contrast, incubation of the cells with SB 203580, a specific inhibitor of p38, slightly stimulated ADRP synthesis and induced a proportional dose-response inhibition of PRL-induced β-casein synthesis. Finally, cell treatment with wortmannin or LY294002 revealed that both PRL and EGF positively regulate ADRP and β-casein synthesis through PI3-kinase signaling. Because both the Akt inhibitor MK-2206 and the mTOR inhibitor rapamycin provoked a strong diminution of PRL-induced synthesis of the two proteins, and because oleate induced phosphorylation of Akt, we concluded that, in the mammary epithelial cell line HC11, the PI3-kinase/Akt/mTOR signaling pathway strongly participates in β-casein synthesis and is a main regulator of ADRP expression.     

Direct binding and activation of protein kinase C isoforms by steroid hormones

The non-genomic action of steroid hormones regulates a wide variety of cellular responses including regulation of ion transport, cell proliferation, migration, death and differentiation. In order to achieve such plethora of effects steroid hormones utilize nearly all known signal transduction pathways. One of the key signalling molecules regulating the non-genomic action of steroid hormones is protein kinase C (PKC). It is thought that rapid action of steroids hormones results from the activation of plasma membrane receptors; however, their molecular identity remains elusive. In recent years, an increasing number of studies have pointed at the selective binding and activation of specific PKC isoforms by steroid hormones. This has led to the hypothesis that PKC could act as a receptor as well as a transducer of the non-genomic effects of these hormones. In this review we summarize the current knowledge of the direct binding and activation of PKC by steroid hormones.     

Dependence of electrical activity and calcium influx-controlled prolactin release on adenylyl cyclase signaling pathway in pituitary lactotrophs

Pituitary lactotrophs in vitro fire extracellular Ca2+-dependent action potentials spontaneously through still unidentified pacemaking channels, and the associated voltage-gated Ca2+influx (VGCI) is sufficient to maintain basal prolactin (PRL) secretion high and steady. Numerous plasma membrane channels have been characterized in these cells, but the mechanism underlying their pacemaking activity is still not known. Here we studied the relevance of cyclic nucleotide signaling pathways in control of pacemaking, VGCI, and PRL release. In mixed anterior pituitary cells, both VGCI-inhibitable and -insensitive adenylyl cyclase (AC) subtypes contributed to the basal cAMP production, and soluble guanylyl cyclase was exclusively responsible for basal cGMP production. Inhibition of basal AC activity, but not soluble guanylyl cyclase activity, reduced PRL release. In contrast, forskolin stimulated cAMP and cGMP production as well as pacemaking, VGCI, and PRL secretion. Elevation in cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied with increased PRL release. The AC inhibitors attenuated forskolin-stimulated cyclic nucleotide production, VGCI, and PRL release. The cell-permeable 8-bromo-cAMP stimulated firing of action potentials and PRL release and rescued hormone secretion in cells with inhibited ACs in an extracellular Ca2+-dependent manner, whereas 8-bromo-cGMP and 8-(4-chlorophenylthio)-2'-O-methyl-cAMP were ineffective. Protein kinase A inhibitors did not stop spontaneous and forskolin-stimulated pacemaking, VGCI, and PRL release. These results indicate that cAMP facilitates pacemaking, VGCI, and PRL release in lactotrophs predominantly in a protein kinase A- and Epac cAMP receptor-independent manner.     

The mechanism of action of cyproheptadine on prolactin release by cultured anterior pituitary cells

The role of a direct effect of serotonin (5-HT) on PRL secretion at the pituitary level is uncertain. The present study investigated the mechanism of action of the serotonin receptor-blocking agent cyproheptadine on PRL release by normal cultured rat anterior pituitary cells. Cyproheptadine (10 nM-10 microM) and its metabolite desmethylcyproheptadine (a compound which has lost its affinity for serotonin receptors) directly inhibited PRL release, while serotonin, investigated over a wide concentration range, did not reverse this inhibition. The cyproheptadine-mediated inhibition of PRL-release could be completely prevented by 50 microM verapamil. Cyproheptadine strongly inhibited TRH-induced PRL release in the absence, but not in the presence of verapamil. Our studies suggest that cyproheptadine inhibits PRL release at the pituitary level by a blockade of calcium influx at the cell membrane, without affecting the movement of Ca2+ between intracellular compartments.     

Structural and functional analysis of the roles of influenza C virus membrane proteins in assembly and budding

Assembly and budding of the influenza C virus is mediated by three membrane proteins: the hemagglutinin-esterase-fusion glycoprotein (HEF), the matrix protein (CM1), and the ion channel (CM2). Here we investigated whether the formation of the hexagonal HEF arrangement, a distinctive feature of influenza C virions is important for virus budding. We used super resolution microscopy and found 250-nm sized HEF clusters at the plasma membrane of transfected cells, which were insensitive to cholesterol extraction and cytochalasin treatment. Overexpression of either CM1, CM2, or HEF caused the release of membrane-enveloped particles. Cryo-electron microscopy of the latter revealed spherical vesicles exhibiting the hexagonal HEF clusters. We subsequently used reverse genetics to identify elements in HEF required for this clustering. We found that deletion of the short cytoplasmic tail of HEF reduced virus titer and hexagonal HEF arrays, suggesting that an interaction with CM1 stabilizes the HEF clusters. In addition, we substituted amino acids at the surface of the closed HEF conformation and identified specific mutations that prevented virus rescue, others reduced virus titers and the number of HEF clusters in virions. Finally, mutation of two regions that mediate contacts between trimers in the in-situ structure of HEF was shown to prevent rescue of infectious virus particles. Mutations at residues thought to mediate lateral interactions were revealed to promote intracellular trafficking defects. Taken together, we propose that lateral interactions between the ectodomains of HEF trimers are a driving force for virus budding, although CM2 and CM1 also play important roles in this process.     

Latency of Human Measles Virus in Hamster Cells

A latent system employing measles virus (Schwarz strain) was developed in hamster embryo fibroblasts (HEF). Measles virus-specific antigen was detected by immunofluorescence in 30 to 50% of HEF cells, and these cells released infectious virus when co-cultivated with a susceptible monkey cell line, BSC-1 cells. No infectious virus could be detected in the cells when measures were taken to exclude passage of viable latent cells onto the indicator BSC-1 cells. Infectious center assays demonstrated that about 1 in 10 of the latently infected cells in the population could release infectious virus. Infectious virus appeared within 6 hr after co-cultivation of the HEF cells with BSC-1 cells, as compared to 24 hr required for normal replication of measles virus in the BSC-1 cells. Furthermore, labeling of progeny virus ribonucleic acid (RNA) by using tritiated uridine, and inhibition of RNA or protein synthesis by 5-azacytidine or cycloheximide suggested that neither additional RNA nor protein synthesis is required after co-cultivation of the cells to effect early virus release. It can therefore be postulated that there is a block at a late step in virus replication in the latently infected hamster cells. The most obvious site would concern maturation of infectious virions at the cell membrane.     

Rapid inhibitory effect of corticosterone on histamine release from rat peritoneal mast cells.

Glucocorticoids are steroids endowed with powerful anti-inflammatory properties, which are routinely believed to require several hours to take effect through modulation of gene expression. Our recent report has shown that glucocorticoids could inhibit allergic reaction within 10 minutes, which the classical genomic mechanism could not explain. Histamine is thought to be one of major mediators in the allergic reaction, and IgE-mediated histamine release from mast cells plays a pivotal role in allergic diseases. Here, we have determined a rapid effect of corticosterone on histamine release from rat peritoneal mast cells, using fluorometric assay. The results showed that corticosterone could inhibit antigen-induced histamine release from rat peritoneal cells within 15 minutes (p<0.05), which could be mimicked by membrane-impermeable BSA conjugated corticosterone (p<0.05). Neither glucocorticoid nuclear receptor antagonist nor protein synthesis inhibitor could block the rapid action (p<0.05). The study provided evidence that nongenomic mechanism might be involved in rapid effect of glucocorticoids on mast cells in allergic disease.     

The effect of histamine stimulation and H2 -receptor inhibition on the pituitary prolactin and ACTH release and on cortisol secretion in human males

The effects of histamine (HA) on prolactin (PRL), ACTH, and cortisol secretion in human males were investigated. Specific H2 -receptor blockade caused an immediate and significant PRL release, while specific H2 -receptor stimulation (HA and mepyramine) was without effect on PRL secretion. HA significantly stimulated both ACTH and cortisol secretion, and this stimulation was probably mediated through H2 -receptors, H2 -receptor blockade caused a late, but significant ACTH release. This release was not accompanied by a release of cortisol, and might therefore be due to a spontaneous oscillation in ACTH secretion. The results support the theory that HA acts as a modulator of human ACTH secretion. A similar effect on PRL secretion as postulated by others could not be confirmed in this study.     

Atrial natriuretic factor inhibits the early pathway of steroid biosynthesis in bovine adrenal cortex

We have previously determined that atrial natriuretic factor (ANF) is a potent inhibitor of steroid secretion in cultured bovine zona glomerulosa and fasciculata cells. The present report describes a comparison of the effect produced by ANF on aldosterone, deoxycorticosterone and progesterone secretions by zona glomerulosa cells and on cortisol, corticosterone and progesterone secretions by zona fasciculata cells. The equipotent inhibitory action of ANF on the stimulated secretion of these steroids in both cell types indicates a common site of action prior to progesterone synthesis at which ANF inhibits the steroidogenic pathway.     

Estrogen modulates the action of nitric oxide in the medial preoptic area on luteinizing hormone and prolactin secretion

Several substances work as neuromediators of the estrogen direct and indirect (through glial cells or interneurons) action on luteinizing hormone- releasing hormone (LH-RH) neurons in medial basal hypothalamus and medial preoptic area (MPOA).Angiotensin II (AII) in the MPOA stimulates the LH and it inhibits PRL secretion in some situations. On the other hand, the effect of excitatory amino acids on LH and PRL surges during proestrus as well LH surge induced by steroids depend on nitric oxide (NO). In the present study we investigated the participation of MPOA endogenous NO on gonadotropin and PRL secretion mediated by estrogen and AII. Plasma LH, FSH and PRL was determinated in estrogen primed and unprimed ovariectomized Wistar rats that received microinjection of AII or saline into the MPOA, associated or not with a previous microinjection of an inhibitor for NOS. Our results show the following: 1 - there was no change in plasma FSH in estrogen- primed or unprimed ovarictomized related with microinjections of AII or NO antagonist in the MPOA; 2- the increase in LH secretion after ovariectomy depends on, at least in part, NO activity in the MPOA; 3- estrogen may have an indirect negative feedback action on LH-RH neurons in the MPOA through NO; 4- the stimulatory action of AII in the MPOA on LH secretion in ovariectomized rats treated with estrogen depends on NO; 5 - NO in the MPOA stimulates or inhibits PRL secretion depending on the absence or presence of estrogen, respectively; 6- the inhibitory action of AII into the MPOA on PRL secretion does not seem to depend on NO.     

Stimulation of tyrosine phosphorylation by progesterone and its 11-OH derivatives: dissection of a Ca(2+)-dependent and a Ca(2+)-independent mechanism

Progesterone has previously been shown to exert non-genomic effects on human spermatozoa by opening plasma membrane ion channels and by stimulating protein tyrosine phosphorylation. Here we examined how these two activities are influenced by 11-hydroxyl substitution of the steroid molecule either in the alpha- or in the beta-configuration. Both the 11alpha-OH and the 11beta-OH derivatives of progesterone were more effective than progesterone in stimulating tyrosine phosphorylation, although 11alpha-OH-progesterone was a markedly weaker Ca(2+)-influx inducing agonist than the other two steroids. In Ca(2+)-containing medium, the agonist activity of the 11alpha-OH derivative was weaker than that of the 11beta-OH derivative, and it was completely abolished by genistein, whereas that of progesterone and its 11beta-OH derivative was inhibited only partly by this drug. In contrast, when applied in Ca(2+)-free medium, the 11alpha-OH derivative was the strongest of the three agonists tested, and the effects of all the three steroids were completely abolished by genistein. These data show that the structural motifs of steroid molecules that are responsible for the stimulation of tyrosine phosphorylation are different from those mediating the steroid action on Ca2+ influx through plasma membrane channels. The synthesis of selective agonists of both activities may lead to the development of new pharmacological agents to be used in the treatment of steroid-dependent pathologies.     

Extraneuronal Monoamine Transporter Mediates the Permissive Action of Cortisol in the Guinea Pig Trachea: Possible Involvement of Tracheal Chondrocytes

Cortisol, a member of glucocorticoids, could potentiate the action of catecholamine by a non-genomic mechanism. Although this permissive effect has been well appreciated in the anti-asthmatic medication, the underlying signaling pathway has remained mysterious. Here, we show that extraneuronal monoamine transporter (EMT), a membraneous reuptake transporter for circulating catecholamine clearance, is the direct target of cortisol in its permissive effect. We found that BSA-conjugated cortisol, which functions as a cortisol but cannot penetrate cell membrane, enhanced the spasmolytic effect of β-adrenoceptor agonist (isoprenaline) in histamine-sensitized tracheal spirals of guinea pigs, and pharmacological inhibition of EMT with famotidine was powerful enough to imitate the permissive action of cortisol. To our surprise, EMT protein expression was high in the chondrocytes of tracheal cartilage, but was undetectable in tracheal smooth muscle cells. The functionality of EMT was further confirmed with measurement of catecholamine uptake by tracheal chondrocytes. Moreover, cortisol-initiated membrane signaling could activate protein kinase C (PKC), which phosphorylates EMT and induces its internalization via a lipid raft-dependent pathway. Both of the mechanisms slow down the reuptake process by chondrocytes, leading to extracellular catecholamine accumulation and results in a more profound adrenergic signaling activation in tracheal smooth muscle cells. Thus, an EMT-centered pathway was proposed to explain the permissive action of cortisol. Collectively, our results highlight the role of EMT in the crosstalk between glucocorticoid and catecholamine. EMT may represent a promising target for adrenergic signaling modulation.     

Analgesic effect of metoclopramide and its mechanism

Metoclopramide produced a significant analgesic effect when tested by both acetic acid induced writhing and hot plate test. This effect was reduced by naloxone suggesting opioid involvement. Further, bromocriptine which inhibits the release of PRL attenuated the effect of metoclopramide indicating that this drug could act by releasing PRL. The unaltered analgesic effect of metoclopramide by yohimbine reveals that alpha-2 adrenoceptors may not be involved in this action.