Activation of p21-activated protein kinase alpha (alpha PAK) by hyperosmotic shock in neonatal ventricular myocytes.

The p21-activated protein kinases (PAKs) may participate in signalling from Cdc42/Rac1 to the stress-regulated MAPKs (SAPKs/JNKs and p38-/HOG-1-related-MAPKs). We characterized the expression and regulation of alpha PAK in cultured ventricular myocytes. alpha PAK was specifically immunoprecipitated from myocyte extracts. High basal alpha PAK activity was detected in unstimulated myocytes. Its activity was increased rapidly (<30 s) by hyperosmotic shock in the presence of okadaic acid, and was maximal by 3 min (187 +/- 7% relative to unstimulated cells). Endothelin-1 and interleukin-1beta, which also activate SAPKs/JNKs, did not increase alpha PAK activity and presumably act through different PAK isoforms or other mechanisms.

Stimulation of "stress-regulated" mitogen-activated protein kinases (stress-activated protein kinases/c-Jun N-terminal kinases and p38-mitogen-activated protein kinases) in perfused rat hearts by oxidative and other stresses.

"Stress-regulated" mitogen-activated protein kinases (SR-MAPKs) comprise the stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinases (JNKs) and the p38-MAPKs. In the perfused heart, ischemia/reperfusion activates SR-MAPKs. Although the agent(s) directly responsible is unclear, reactive oxygen species are generated during ischemia/reperfusion. We have assessed the ability of oxidative stress (as exemplified by H2O2) to activate SR-MAPKs in the perfused heart and compared it with the effect of ischemia/reperfusion. H2O2 activated both SAPKs/JNKs and p38-MAPK. Maximal activation by H2O2 in both cases was observed at 0.5 mM. Whereas activation of p38-MAPK by H2O2 was comparable to that of ischemia and ischemia/reperfusion, activation of the SAPKs/JNKs was less than that of ischemia/reperfusion. As with ischemia/reperfusion, there was minimal activation of the ERK MAPK subfamily by H2O2. MAPK-activated protein kinase 2 (MAPKAPK2), a downstream substrate of p38-MAPKs, was activated by H2O2 to a similar extent as with ischemia or ischemia/reperfusion. In all instances, activation of MAPKAPK2 in perfused hearts was inhibited by SB203580, an inhibitor of p38-MAPKs. Perfusion of hearts at high aortic pressure (20 kilopascals) also activated the SR-MAPKs and MAPKAPK2. Free radical trapping agents (dimethyl sulfoxide and N-t-butyl-alpha-phenyl nitrone) inhibited the activation of SR-MAPKs and MAPKAPK2 by ischemia/reperfusion. These data are consistent with a role for reactive oxygen species in the activation of SR-MAPKs during ischemia/reperfusion.

Activation of mitogen-activated protein kinases (p38-MAPKs, SAPKs/JNKs and ERKs) by the G-protein-coupled receptor agonist phenylephrine in the perfused rat heart.

We investigated the ability of phenylephrine (PE), an alpha-adrenergic agonist and promoter of hypertrophic growth in the ventricular myocyte, to activate the three best-characterized mitogen-activated protein kinase (MAPK) subfamilies, namely p38-MAPKs, SAPKs/JNKs (i.e. stress-activated protein kinases/c-Jun N-terminal kinases) and ERKs (extracellularly responsive kinases), in perfused contracting rat hearts. Perfusion of hearts with 100 microM PE caused a rapid (maximal at 10 min) 12-fold activation of two p38-MAPK isoforms, as measured by subsequent phosphorylation of a p38-MAPK substrate, recombinant MAPK-activated protein kinase 2 (MAPKAPK2). This activation coincided with phosphorylation of p38-MAPK. Endogenous MAPKAPK2 was activated 4-5-fold in these perfusions and this was inhibited completely by the p38-MAPK inhibitor, SB203580 (10 microM). Activation of p38-MAPK and MAPKAPK2 was also detected in non-contracting hearts perfused with PE, indicating that the effects were not dependent on the positive inotropic/chronotropic properties of the agonist. Although SAPKs/JNKs were also rapidly activated, the activation (2-3-fold) was less than that of p38-MAPK. The ERKs were activated by perfusion with PE and the activation was at least 50% of that seen with 1 microM PMA, the most powerful activator of the ERKs yet identified in cardiac myocytes. These results indicate that, in addition to the ERKs, two MAPK subfamilies, whose activation is more usually associated with cellular stresses, are activated by the Gq/11-protein-coupled receptor (Gq/11PCR) agonist, PE, in whole hearts. These data indicate that Gq/11PCR agonists activate multiple MAPK signalling pathways in the heart, all of which may contribute to the overall response (e.g. the development of the hypertrophic phenotype).

Regulation of mitogen-activated protein kinase cascades in the heart.

Using primary cultures of neonatal rat ventricular myocytes and isolated adult rat hearts as models, we have characterized extensively the regulation of MAPKs in the heart. The ERKs are activated primarily by GPCR agonists acting through PKC. These agonists can also activate the JNKs although the mechanism is unclear. Cellular stresses stimulate strong activation of the JNKs, but also cause some stimulation of ERKs. Activation of p38-MAPK has so far only been demonstrated in intact adult hearts subjected to stresses and probably leads to activation of MAPKAPK2. Both cellular stresses and GPCR agonists induce phosphorylation of c-Jun, but only the latter causes upregulation of c-Jun protein.

Endothelin-1 promotes phosphorylation of CREB transcription factor in primary cultures of neonatal rat cardiac myocytes: implications for the regulation of c-jun expression.

Cardiac myocyte hypertrophy is associated with an increase in expression of immediate early genes (e.g. c-jun) via activation of pre-existing transcription factors. The activity of CREB transcription factor is regulated through phosphorylation of Ser-133 by one of several protein kinases (e.g. protein kinase A (PKA), p90 ribosomal S6 kinases (RSKs) and the related kinase, MSK1). A cell-permeable form of cAMP, hypertrophic agonists (endothelin-1 (ET-1), phenylephrine (PE)) and hyperosmotic shock all promoted phosphorylation of CREB(Ser-133) in rat neonatal cardiac myocytes. The response to endothelin-1 required the extracellular signal-regulated kinase cascade which stimulates both RSKs and MSK1. Phosphorylation of CREB(Ser-133) in response to ET-1 was not associated with any increase in DNA binding to a consensus cAMP-response element (CRE). The rat c-jun promoter contains elements which may bind either c-Jun/ATF2 or CREB/ATF1 dimers. Using extracts from rat cardiac myocytes, we identified at least two complexes which bind to the most proximal of these elements, one of which contained CREB and the other c-Jun. Thus, phosphorylation and activation of CREB in cardiac myocytes may be effected by a range of different stimuli to influence the expression of immediate early genes such as c-jun.

The neurotoxicity of 5-S-cysteinyldopamine is mediated by the early activation of ERK1/2 followed by the subsequent activation of ASK1/JNK1/2 pro-apoptotic signalling

Parkinson's disease is characterized by the progressive and selective loss of dopaminergic neurons in the substantia nigra. It has been postulated that endogenously formed CysDA (5-S-cysteinyldopamine) and its metabolites may be, in part, responsible for this selective neuronal loss, although the mechanisms by which they contribute to such neurotoxicity are not understood. Exposure of neurons in culture to CysDA caused cell injury, apparent 12-48 h post-exposure. A portion of the neuronal death induced by CysDA was preceded by a rapid uptake and intracellular oxidation of CysDA, leading to an acute and transient activation of ERK2 (extracellular-signal-regulated kinase 2) and caspase 8. The oxidation of CysDA also induced the activation of apoptosis signal-regulating kinase 1 via its de-phosphorylation at Ser967, the phosphorylation of JNK (c-Jun N-terminal kinase) and c-Jun (Ser73) as well as the activation of p38, caspase 3, caspase 8, caspase 7 and caspase 9. Concurrently, the inhibition of complex I by the dihydrobenzothiazine DHBT-1 [7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid], formed from the intracellular oxidation of CysDA, induces complex I inhibition and the subsequent release of cytochrome c which further potentiates pro-apoptotic mechanisms. Our data suggest a novel comprehensive mechanism for CysDA that may hold relevance for the selective neuronal loss observed in Parkinson's disease.

Estrogen receptor-mediated transcription involves the activation of multiple kinase pathways in neuroblastoma cells

While many physiological effects of estrogens (E) are due to regulation of gene transcription by liganded estrogen receptors (ERs), several effects are also mediated, at least in part, by rapid non-genomic actions of E. Though the relative importance of rapid versus genomic effects in the central nervous system is controversial, we showed previously that membrane-limited effects of E, initiated by an estradiol bovine serum albumin conjugate (E2-BSA), could potentiate transcriptional effects of 17beta-estradiol from an estrogen response element (ERE)-reporter in neuroblastoma cells. Here, using specific inhibitors and activators in a pharmacological approach, we show that activation of phosphatidylinositol-3-phosphate kinase (PI3K) and mitogen activated protein kinase (MAPK) pathways, dependent on a Galphaq coupled receptor signaling are important in this transcriptional potentiation. We further demonstrate, using ERalpha phospho-deficient mutants, that E2-BSA mediated phosphorylation of ERalpha is one mechanism to potentiate transcription from an ERE reporter construct. This study provides a possible mechanism by which signaling from the membrane is coupled to transcription in the nucleus, providing an integrated view of hormone signaling in the brain.

Activation of the G-protein coupled receptor 30 (GPR30) has different effects on anxiety in male and female mice

The GPR30, a former orphan GPCR, is a putative membrane estrogen receptor that can activate rapid signaling pathways such as extracellular regulated kinase (ERK) in a variety of cells and may contribute to estrogen's effects in the central nervous system. The distribution of GPR30 in the limbic system predicts a role for this receptor in the regulation of learning and memory and anxiety by estrogens. Though acute G-1 treatment is reported to be anxiogenic in ovariectomised female mice and in gonadally intact male mice, the effect of GPR30 activation is unknown in gonadectomised male mice. In this study, we show that an acute administration of G-1 to gonadectomised male mice, but not female mice, was anxiolytic on an elevated plus maze task, without affecting locomotor activity. In addition, though G-1 treatment did not regulate ERK, it was associated with increased estrogen receptor (ER)alpha phosphorylation in the ventral, but not dorsal, hippocampus of males. In the female, G-1 increased the ERK activation solely in the dorsal hippocampus, independent of state anxiety. This is the first study to report an anxiolytic effect of GPR30 activation in male mice, in a rapid time frame that is commensurate with non-genomic signaling by estrogen.