Activation of mitogen-activated protein kinase phosphatase 2 by gonadotropin-releasing hormone.

The aim of these studies was to identify the signaling mechanism(s) that contribute to GnRH-induced expression of MAPK phosphatase (MKP)-2, a dual specificity phosphatase that selectively inactivates MAPKs. GnRH receptor activation induced MKP-2 expression in both clonal (alphaT3-1) and primary gonadotropes. Activation of PKC isozymes was sufficient and required for MKP-2 induction. Inhibition of the extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) but not the p38 MAPK cascade was sufficient to block GnRH-induced MKP-2 expression. Induction of MKP-2 by GnRH was dependent on elevation in intracellular Ca(2+). Inhibition of Ca(2+) influx through L-type voltage-gated calcium channels blocked GnRH-induced MKP-2 expression. Depletion of intracellular Ca(2+) stores with thapsigargin blocked MKP-2 activation by GnRH independent of ERK and JNK activity. These results support the conclusion that MKP-2 induction by GnRH occurs via MAPK-dependent and -independent pathways. One mechanism requires GnRH-induced ERK and JNK activation, while a second MAPK-independent pathway requires a thapsigargin-sensitive calcium signal.

Willingness to pay for defense against weapons of mass destruction.

A survey assessed the willingness to pay for defense against weapons of mass destruction. The results were evaluated according to the benefit to society. The results indicated preferences for increased spending on intelligence gathering, training, and equipment. We concluded that the United States is spending less for weapons of mass destruction defense than the sample population was willing to pay.

Divergent signaling pathways requiring discrete calcium signals mediate concurrent activation of two mitogen-activated protein kinases by gonadotropin-releasing hormone.

Receptors coupled to heterotrimeric G proteins are linked to activation of mitogen-activated protein kinases (MAPKs) via receptor- and cell-specific mechanisms. We have demonstrated recently that gonadotropin-releasing hormone (GnRH) receptor occupancy results in activation of extracellular signal-regulated kinase (ERK) through a mechanism requiring calcium influx through L-type calcium channels in alphaT3-1 cells and primary rat gonadotropes. Further studies were undertaken to explore the signaling mechanisms by which the GnRH receptor is coupled to activation of another member of the MAPK family, c-Jun N-terminal kinase (JNK). GnRH induces activation of the JNK cascade in a dose-, time-, and receptor-dependent manner in clonal alphaT3-1 cells and primary rat pituitary gonadotrophs. Coexpression of dominant negative Cdc42 and kinase-defective p21-activated kinase 1 and MAPK kinase 7 with JNK and ERK indicated that specific activation of JNK by GnRH appears to involve these signaling molecules. Unlike ERK activation, GnRH-stimulated JNK activity does not require activation of protein kinase C and is not blocked after chelation of extracellular calcium with EGTA. GnRH-induced JNK activity was reduced after treatment with the intracellular calcium chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester), whereas activation of ERK was not affected. Chelation of intracellular calcium also reduced GnRH-induced activation of JNK in rat pituitary cells in primary culture. GnRH-induced induction and activation of the JNK target c-Jun was inhibited after chelation of intracellular calcium, whereas induction of c-Fos, a known target of ERK, was unaffected. Therefore, although activation of ERK by GnRH requires a specific influx of calcium through L-type calcium channels, JNK activation is independent of extracellular calcium but sensitive to chelation of intracellular calcium. Our results provide novel evidence that GnRH activates two MAPK superfamily members via strikingly divergent signaling pathways with differential sensitivity to activation of protein kinase C and mobilization of discrete pools of calcium.

Role of the cyclic AMP response element binding complex and activation of mitogen-activated protein kinases in synergistic activation of the glycoprotein hormone alpha subunit gene by epidermal growth factor and forskolin.

The aim of these studies was to elucidate a role for epidermal growth factor (EGF) signaling in the transcriptional regulation of the glycoprotein hormone alpha subunit gene, a subunit of chorionic gonadotropin. Studies examined the effects of EGF and the adenylate cyclase activator forskolin on the expression of a transfected alpha subunit reporter gene in a human choriocarcinoma cell line (JEG3). At maximal doses, administration of EGF resulted in a 50% increase in a subunit reporter activity; forskolin administration induced a fivefold activation; the combined actions of EGF and forskolin resulted in synergistic activation (greater than eightfold) of the alpha subunit reporter. Mutagenesis studies revealed that the cyclic AMP response elements (CRE) were required and sufficient to mediate EGF-forskolin-induced synergistic activation. The combined actions of EGF and forskolin resulted in potentiated activation of extracellular signal-regulated kinase (ERK) enzyme activity compared with EGF alone. Specific blockade of ERK activation was sufficient to block EGF-forskolin-induced synergistic activation of the alpha subunit reporter. Pretreatment of JEG3 cells with a p38 mitogen-activated protein kinase inhibitor did not influence activation of the alpha reporter. However, overexpression of c-Jun N-terminal kinase (JNK)-interacting protein 1 as a dominant interfering molecule abolished the synergistic effects of EGF and forskolin on the alpha subunit reporter. CRE binding studies suggested that the CRE complex consisted of CRE binding protein and EGF-ERK-dependent recruitment of c-Jun-c-Fos (AP-1) to the CRE. A dominant negative form of c-Fos (A-Fos) that specifically disrupts c-Jun-c-Fos DNA binding inhibited synergistic activation of the alpha subunit. Thus, synergistic activation of the alpha subunit gene induced by EGF-forskolin requires the ERK and JNK cascades and the recruitment of AP-1 to the CRE binding complex.

Calcium influx through L-type channels is required for selective activation of extracellular signal-regulated kinase by gonadotropin-releasing hormone.

The hypothalamic decapeptide gonadotropin-releasing hormone stimulates mobilization of two discrete pools of calcium in clonal (alphaT3-1) and primary pituitary gonadotropes. A multidisciplinary approach was implemented to investigate the effects of discrete calcium fluctuations on the signaling pathways linking the gonadotropin-releasing hormone receptor to activation of mitogen-activated protein kinases and immediate early genes. Blockade of calcium influx through nifedipine-sensitive voltage-gated calcium channels reduced buserelin-induced activation of extracellular signal-regulated kinase (ERK) and c-Fos while activation of c-Jun N-terminal kinase and c-Jun was unaffected. Inhibition of buserelin-stimulated ERK activity by nifedipine was also observed in rat pituitary cells in primary culture. Direct activation of alphaT3-1 cell L-type calcium channels with the agonist Bay-K 8644 resulted in phosphorylation of ERK and induction of c-Fos. However, simple voltage-induced channel activation did not produce a sufficient calcium signal, since depolarization with 35 mM KCl failed to induce activation of ERK. Depletion of intracellular calcium stores with thapsigargin did not affect buserelin-induced ERK activation. An inhibitor of protein kinase C decreased calcium influx through nifedipine-sensitive calcium channels and phosphorylation of ERK induced by buserelin. Pharmacological inhibition of protein kinase C did not block Bay-K 8644-induced ERK activation. These observations suggest that calcium influx through L-type channels is required for GnRH-induced activation of ERK and c-Fos and that the influence of calcium lies downstream of protein kinase C.

Homologous regulation of the gonadotropin-releasing hormone receptor gene is partially mediated by protein kinase C activation of an activator protein-1 element.

Homologous regulation of GnRH receptor (GnRHR) gene expression is an established mechanism for controlling the sensitivity of gonadotropes to GnRH. We have found that expression of the GnRHR gene in the gonadotrope-derived alpha T3-1 cell line is mediated by a tripartite enhancer that includes a consensus activator protein-1 (AP-1) element, a binding site for SF-1 (steroidogenic factor-1), and an element we have termed GRAS (GnRHR-activating sequence). Further, in transgenic mice, approximately 1900 b.p. of the murine GnRHR gene promoter are sufficient for tissue-specific expression and GnRH responsiveness. The present studies were designed to further delineate the molecular mechanisms underlying GnRH regulation of GnRHR gene expression. Vectors containing 600 bp of the murine GnRHR gene promoter linked to luciferase (LUC) were transiently transfected into alpha T3-1 cells and exposed to treatments for 4 or 6 h. A GnRH-induced, dose-dependent increase in LUC expression of the -600 promoter was observed with maximal induction of LUC noted at 100 nM GnRH. We next tested the ability of GnRH to stimulate expression of vectors containing mutations in each of the components of the tripartite enhancer. GnRH responsiveness was lost in vectors containing mutations in AP-1. Gel mobility shift data revealed binding of fos/jun family members to the AP-1 element of the murine GnRHR promoter. Treatment with GnRH or phorbol-12-myristate-13-acetate (PMA) (100 nM), but not forskolin (10 microM), increased LUC expression, which was blocked by the protein kinase C (PKC) inhibitor, GF109203X (100 nM), and PKC down-regulation (10 nM PMA for 20 h). In addition, a specific MEK1/MEK2 inhibitor, PD98059 (60 microM), reduced the GnRH and PMA responses whereas the L-type voltage-gated calcium channel agonist, +/- BayK 8644 (5 microM), and antagonist, nimodipine (250 nM), had no effect on GnRH responsiveness. Furthermore, treatment of alpha T3-1 cells with 100 nM GnRH stimulated phosphorylation of both p42 and p44 forms of extracellular signal-regulated kinase (ERK), which was completely blocked with 60 microM PD98059. We suggest that GnRH regulation of the GnRHR gene is partially mediated by an ERK-dependent activation of a canonical AP-1 site located in the proximal promoter of the GnRHR gene.

Activation of the p38 mitogen-activated protein kinase pathway by gonadotropin-releasing hormone.

Previous studies have shown that interaction of GnRH with its serpentine, G protein-coupled receptor results in activation of the extracellular signal regulated protein kinase (ERK) and the Jun N-terminal protein kinase (JNK) pathways in pituitary gonadotropes. In the present study, we examined GnRH-stimulated activation of an additional member of the mitogen-activated protein kinase (MAPK) superfamily, p38 MAPK GnRH treatment of alphaT3-1 cells resulted in tyrosine phosphorylation of several intracellular proteins. Separation of phosphorylated proteins by ion exchange chromatography suggested that GnRH receptor stimulation can activate the p38 MAPK pathway. Immunoprecipitation studies using a phospho-tyrosine antibody resulted in increased amounts of immunoprecipitable p38 MAPK from alphaT3-1 cells treated with GnRH. Immunoblot analysis of whole cell lysates using a phospho-specific antibody directed against dual phosphorylated p38 kinase revealed that GnRH-induced phosphorylation of p38 kinase was dose and time dependent and was correlated with increased p38 kinase activity in vitro. Activation of p38 kinase was blocked by chronic phorbol ester treatment, which depletes protein kinase C isozymes alpha and epsilon. Overexpression of p38 MAPK and an activated form of MAPK kinase 6 resulted in activation of c-jun and c-fos reporter genes, but did not alter the expression of the glycoprotein hormone alpha-subunit reporter. Inhibition of p38 activity with SB203580 resulted in attenuation of GnRH-induced c-fos reporter gene expression, but was not sufficient to reduce GnRH-induced c-jun or glycoprotein hormone alpha-subunit promoter activity. These studies provide evidence that the GnRH signaling pathway in alphaT3-1 cells includes protein kinase C-dependent activation of the p38 MAPK pathway. GnRH integration of c-fos promoter activity may include regulation by p38 MAPK.

Galanin activates an inwardly rectifying potassium conductance and inhibits a voltage-dependent calcium conductance in mudpuppy parasympathetic neurons.

Galanin-induced activation of an inwardly rectifying membrane potassium (K+) current and inhibition of barium current (IBa) were studied using whole cell voltage clamp recording techniques in parasympathetic neurons dissociated from the mudpuppy cardiac ganglion. Both activation of the K+ current and inhibition of IBa were concentration-dependent with an EC50 (or IC50) of approximately 35 nM and approximately 0.4 nM, respectively. Both actions of galanin were eliminated by pretreatment with pertussis toxin, which suggested involvement of Gi/Go protein activation. Galantide antagonized the galanin-induced activation of K+ current with an IC50 equal to 4 nM. By contrast, galantide, by itself, inhibited IBa with an EC50 equal to 16 nM. Another galanin analog, M40, primarily antagonized the galanin-induced activation of K+ current, but in some cells, M40 also acted as a weak agonist. M40, like galantide, inhibited IBa. The NH2-terminal fragment galanin-(1-16) activated the K+ current and inhibited IBa, indicating that the first 16 amino acids of the galanin peptide were sufficient for both actions. In summary, it is postulated that the effects of galanin on mudpuppy parasympathetic neurons might be mediated by activation of two different subtypes of galanin receptor, one that regulates membrane K+ conductance and a second that modulates calcium conductance.

Galantide distinguishes putative subtypes of galanin receptors in mudpuppy parasympathetic neurons.

The effect of the chimeric ligand galantide on the galanin-induced activation of membrane K+ conductance and inhibition of voltage-dependent Ca2+ conductance has been studied using voltage-clamped dissociated mudpuppy parasympathetic neurons. Galantide did not activate the K+ conductance but produced a concentration-dependent antagonism (IC50 = 4 nM) of the galanin-induced increase in K+ conductance. Galantide acted like galanin and inhibited the voltage-dependent Ba2+ current (IBa). The inhibition of IBa also was concentration dependent (IC50 = 16 nM) and the maximum inhibition produced by galantide was approximately 40%. We also demonstrate that the galanin-(1-16) fragment increased the membrane K+ conductance and decreased IBa, suggesting that the NH2 portion of the galanin molecule is sufficient to mediate both actions. One interpretation of these observations is that different galanin receptors mediate the different effects of galanin on the mudpuppy parasympathetic neurons.

Arachidonic-acid-activated membrane conductances in dissociated cardiac parasympathetic neurons from Necturus.

1. Characteristics of the membrane currents activated by arachidonic acid (AA) in dissociated mudpuppy parasympathetic neurons have been determined using the perforated-patch whole cell recording technique. 2. In a sodium-containing physiological solution with 12.5 mM potassium, AA (10-50 microM) increased total membrane current produced by either depolarizing or hyperpolarizing voltage steps delivered from a holding potential of -40 mV. Decreasing the external potassium concentration from 12.5 to 2.5 mM shifted the reversal potential of the AA-induced current by 10 mV rather than the approximately 42 mV predicted for a highly potassium-selective channel. 3. In cells kept in sodium solution plus 12.5 mM potassium and treated with 20 microM nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase pathway of AA metabolism, AA activated only inward currents following hyperpolarizing voltage steps. In this condition, the shift in reversal potential of the AA-induced current was 40 mV when extracellular potassium concentration was changed fivefold. Consequently, in cells treated with NDGA, AA appeared to activate only an inwardly rectifying potassium current. 4. Decreasing the extracellular chloride concentration by approximately 90% did not alter the reversal potential of the AA-activated current when the extracellular sodium concentration was kept constant and the external potassium concentration was 2.5 mM. In the low-chloride solution, AA potentiated both inward and outward current amplitudes. These results suggested that AA did not activate a chloride current in these cells. 5. In a sodium-deficient, N-methyl-D-glucamine (NMG)-containing solution, AA only activated currents for voltage steps to potentials more negative than the holding potential. In the NMG-substituted solution, changing the extracellular potassium concentration fivefold shifted the reversal potential of the AA-induced current by 40 mV. Therefore, in the NMG solution, AA primarily activated an inwardly rectifying potassium current. 6. Exchanging the control solution containing AA to an external solution containing AA and barium (barium blocks the inwardly rectifying potassium current) shifted the current-voltage relationship to more positive voltages such that the extrapolated reversal potential was approximately 0 mV. In other experiments, using the barium-containing solution, the reversal potential for the AA-induced current was -3.3 +/- 2.4 (SE) mV. 7. In conclusion, the results of the present study indicate that at least two membrane currents are activated in the presence of AA: an inwardly rectifying potassium current and an NDGA-sensitive, sodium-dependent current that has a reversal potential more positive than the potassium equilibrium potential. We suggest the second current component is due to the activation of a nonselective cationic conductance.

Arachidonic acid may mediate the galanin-induced hyperpolarization in parasympathetic neurons from Necturus maculosus.

The effects of arachidonic acid (AA) and compounds that inhibit intracellular signalling pathways on membrane potential and galanin-induced hyperpolarizations were investigated in parasympathetic neurons from Necturus maculosus. Treatment for 10-90 min with 10-20 microM 4-bromophenacylbromide or 10 microM cyclosporin A caused a progressive decrease in the amplitude of galanin-induced hyperpolarizations without any change in resting membrane potential. The galanin-induced hyperpolarization was not altered following a 10-120 min treatment with the protein kinase inhibitor H-7. These results indicated that phospholipase A2 activation, but not protein kinase activation, may be required for the galanin-induced hyperpolarization. Arachidonic acid (20-100 microM) caused a concentration-dependent membrane hyperpolarization of the parasympathetic neurons and a decrease in the amplitude of the galanin-induced hyperpolarization. These data indicate that phospholipase A2-catalyzed liberation of AA may be involved in the galanin-induced membrane hyperpolarization observed in mudpuppy parasympathetic neurons.