Adrenergic receptor stimulation of the mitogen-activated protein kinase cascade and cardiac hypertrophy.

Phenylephrine and noradrenaline (alpha-adrenergic agonism) or isoprenaline (beta-adrenergic agonism) stimulated protein synthesis rates, increased the activity of the atrial natriuretic factor gene promoter and activated mitogen-activated protein kinase (MAPK). The EC50 for MAPK activation by noradrenaline was 2-4 microM and that for isoprenaline was 0.2-0.3 microM. Maximal activation of MAPK by isoprenaline was inhibited by the beta-adrenergic antagonist, propranolol, whereas the activation by noradrenaline was inhibited by the alpha1-adrenergic antagonist, prazosin. FPLC on a Mono-Q column separated two peaks of MAPK (p42MAPK and p44MAPK) and two peaks of MAPK-activating activity (MEK) activated by isoprenaline or noradrenaline. Prolonged phorbol ester exposure partially down-regulated the activation of MAPK by noradrenaline but not by isoprenaline. This implies a role for protein kinase C in MAPK activation by noradrenaline but not isoprenaline. A role for cyclic AMP in activation of the MAPK pathway was eliminated when other agonists that elevate cyclic AMP in the cardiac myocyte did not activate MAPK. In contrast, MAPK was activated by exposure to ionomycin, Bay K8644 or thapsigargin that elevate intracellular Ca2+. Furthermore, depletion of extracellular Ca2+ concentrations with bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid (BAPTA) or blocking of the L-type Ca2+ channel with nifepidine or verapamil inhibited the response to isoprenaline without inhibiting the responses to noradrenaline. We conclude that alpha- and beta-adrenergic agonists can activate the MEK/MAPK pathway in the heart by different signalling pathways. Elevation of intracellular Ca2+ rather than cyclic AMP appears important in the activation of MAPK by isoprenaline in the cardiac myocyte.

Stimulation of phosphatidylinositol hydrolysis, protein kinase C translocation, and mitogen-activated protein kinase activity by bradykinin in rat ventricular myocytes: dissociation from the hypertrophic response.

In ventricular myocytes cultured from neonatal rat hearts, bradykinin (BK), kallidin or BK(1-8) [(Des-Arg9)BK] stimulated PtdinsP2 hydrolysis by 3-4-fold. EC50 values were 6 nM (BK), 2 nM (kallidin), and 14 microM [BK(1-8)]. BK or kallidin stimulated the rapid (less than 30 s) translocation of more than 80% of the novel protein kinase C (PKC) isoforms nPKC-delta and nPKC-epsilon from the soluble to the particulate fraction. EC50 values for nPKC-delta translocation by BK or kallidin were 10 and 2 nM respectively. EC50 values for nPKC-epsilon translocation by BK or kallidin were 2 and 0.6 nM respectively. EC50 values for the translocation of nPKC-delta and nPKC-epsilon by BK(1-8) were more than 5 microM. The classical PKC, cPKC-alpha, and the atypical PKC, nPKC-zeta, did not translocate. BK caused activation and phosphorylation of p42-mitogen-activated protein kinase (MAPK) (maximal at 3-5 min, 30-35% of p42-MAPK phosphorylated). p44-MAPK was similarly activated. EC50 values for p42/p44-MAPK activation by BK were less than 1 nM whereas values for BK(1-8) were more than 10 microM. The order of potency [BK approximately equal to kallidin > BK (1-8)] for the stimulation of PtdInsP2 hydrolysis, nPKC-delta and nPKC-epsilon translocation, and p42/p44-MAPK activities suggests involvement of the B2 BK receptor subtype. In addition, stimulation of all three processes by BK was inhibited by the B2BK receptor-selective antagonist HOE140 but not by the B1-selective antagonist Leu8BK(1-8). Exposure of cells to phorbol 12-myristate 13-acetate for 24 h inhibited subsequent activation of p42/p44-MAPK by BK suggesting participation of nPKC (and possibly cPKC) isoforms in the activation process. Thus, like hypertrophic agents such as endothelin-1 (ET-1) and phenylephrine (PE), BK activates PtdInsP2 hydrolysis, translocates nPKC-delta, and nPKC-epsilon, and activates p42/p44-MAPK. However, in comparison with ET-1 and PE, BK was only weakly hypertrophic as assessed by cell morphology and patterns of gene expression. This difference could not be attributed to dissimilarities between the duration of activation of p42/p44-MAPK by BK or ET-1. Thus activation of these signalling pathways alone may be insufficient to induce a powerful hypertrophic response.

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 multiple mitogen-activated protein kinase sub-families by oxidative stress and phosphorylation of the small heat shock protein, HSP25/27, in neonatal ventricular myocytes.

We investigated the activation of three subfamilies of mitogen-activated protein kinases (MAPKs), namely the stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs), the extracellularly responsive kinases (ERKs) and p38-MAPK, by oxidative stress as exemplified by H2O2 in primary cultures of neonatal rat ventricular myocytes. The 46 and 54 kDa species of SAPKs/JNKs were activated 5- and 10-fold, respectively, by 0.1 mM H2O2 (the maximally effective concentration). Maximal activation occurred at 15-30 min, but was still detectable after 2 h. Both ERK1 and ERK2 were activated 16-fold by 0.1 mM H2O2 with a similar time course to the SAPKs/JNKs, and this was comparable with their activation by 1 microM PMA, the most powerful activator of ERKs that we have so far identified in these cells. The activation of ERKs by H2O2 was inhibited by PD98059, which inhibits the activation of MAPK (or ERK) kinases, and by the protein kinase C (PKC) inhibitor, GF109203X. ERK activation was also inhibited by down-regulation of PMA-sensitive PKC isoforms. p38-MAPK was activated by 0.1 mM H2O2 as shown by an increase in its phosphorylation. However, maximal phosphorylation (activation) was more rapid (<5 min) than for the SAPKs/JNKs or the ERKs. We studied the downstream consequences of p38-MAPK activation by examining activation of MAPK-activated protein kinase 2 (MAPKAPK2) and phosphorylation of the MAPKAPK2 substrate, the small heat shock protein HSP25/27. As with p38-MAPK, MAPKAPK2 was rapidly activated (maximal within 5 min) by 0.1 mM H2O2. This activation was abolished by 10 microM SB203580, a selective inhibitor of certain p38-MAPK isoforms. The phosphorylation of HSP25/27 rapidly followed activation of MAPKAPK2 and was also inhibited by SB203580. Phosphorylation of HSP25/27 was associated with a decrease in its aggregation state. These data indicate that oxidative stress is a powerful activator of all three MAPK subfamilies in neonatal rat ventricular myocytes. Activation of all three MAPKs has been associated with the development of the hypertrophic phenotype. However, stimulation of p38-MAPK and the consequent phosphorylation of HSP25/27 may also be important in cardioprotection.

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.

Regulation of protein kinase B and 4E-BP1 by oxidative stress in cardiac myocytes.

Stimulation of phosphatidylinositol 3'-kinase (PI3K) and protein kinase B (PKB) is implicated in the regulation of protein synthesis in various cells. One mechanism involves PI3K/PKB-dependent phosphorylation of 4E-BP1, which dissociates from eIF4E, allowing initiation of translation from the 7-methylGTP cap of mRNAs. We examined the effects of insulin and H(2)O(2) on this pathway in neonatal cardiac myocytes. Cardiac myocyte protein synthesis was increased by insulin, but was inhibited by H(2)O(2). PI3K inhibitors attenuated basal levels of protein synthesis and inhibited the insulin-induced increase in protein synthesis. Insulin or H(2)O(2) increased the phosphorylation (activation) of PKB through PI3K, but, whereas insulin induced a sustained response, the response to H(2)O(2) was transient. 4E-BP1 was phosphorylated in unstimulated cells, and 4E-BP1 phosphorylation was increased by insulin. H(2)O(2) stimulated dephosphorylation of 4E-BP1 by increasing protein phosphatase (PP1/PP2A) activity. This increased the association of 4E-BP1 with eIF4E, consistent with H(2)O(2) inhibition of protein synthesis. The effects of H(2)O(2) were sufficient to override the stimulation of protein synthesis and 4E-BP1 phosphorylation induced by insulin. These results indicate that PI3K and PKB are important regulators of protein synthesis in cardiac myocytes, but other factors, including phosphatase activity, modulate the overall response.

Phenylephrine promotes phosphorylation of Bad in cardiac myocytes through the extracellular signal-regulated kinases 1/2 and protein kinase A.

Studies in non-cardiomyocytic cells have shown that phosphorylation of the Bcl-2 family protein Bad on Ser-112, Ser-136 and Ser-155 decreases its pro-apoptotic activity. Both phenylephrine (100 microM) and the cell membrane-permeating cAMP analog, 8-(4-chlorophenylthio)-cAMP (100 microM), protected against 2-deoxy-D-glucose-induced apoptosis in neonatal rat cardiac myocytes as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). In cardiac myocytes, phenylephrine primarily stimulates the alpha-adrenoceptor, but, at high concentrations (100 microM), it also increases the activity of the cAMP-dependent protein kinase, protein kinase A (PKA) through the beta-adrenoceptor. Phenylephrine (100 microM) promoted rapid phosphorylation of Bad(Ser-112) and Bad(Ser-155), though we were unable to detect phosphorylation of Bad(Ser-136). Phosphorylation of Bad(Ser-112) was antagonized by either prazosin or propranolol, indicating that this phosphorylation required stimulation of both alpha(1)- and beta-adrenoceptors. Phosphorylation of Bad(Ser-155) was antagonized only by propranolol and was thus mediated through the beta-adrenoceptor. Inhibitor studies and partial purification of candidate kinases by fast protein liquid chromatography showed that the p90 ribosomal S6 kinases, p90RSK2/3 [which are activated by the extracellular signal-regulated kinases 1 and 2 (ERK1/2)] directly phosphorylated Bad(Ser-112), whereas the PKA catalytic subunit directly phosphorylated Bad(Ser-155). However, efficient phosphorylation of Bad(Ser-112) also required PKA activity. These data suggest that, although p90RSK2/3 phosphorylate Bad(Ser-112) directly, phosphorylation of this site is enhanced by phosphorylation of Bad(Ser-155). These phosphorylations potentially diminish the pro-apoptotic activity of Bad and contribute to the cytoprotective effects of phenylephrine and 8-(4-chlorophenylthio)-cAMP.

Peptide growth factors signal differentially through protein kinase C to extracellular signal-regulated kinases in neonatal cardiomyocytes

The extracellular signal-regulated kinases 1/2 (ERK1/2) are activated in cardiomyocytes by Gq protein-coupled receptors and are associated with induction of hypertrophy. Here, we demonstrate that, in primary cardiomyocyte cultures, ERK1/2 were also significantly activated by platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or fibroblast growth factor (FGF), but insulin, insulin-like growth factor 1 (IGF-1) and nerve growth factor (NGF) had relatively minor effects. PDGF, EGF or FGF increased cardiomyocyte size via ERK1/2, whereas insulin, IGF-1 or NGF had no effect suggesting minimum thresholds/durations of ERK1/2 signaling are required for the morphological changes associated with hypertrophy. Peptide growth factors are widely accepted to activate phospholipase C gamma1 (PLCgamma1) and protein kinase C (PKC). In cardiomyocytes, only PDGF stimulated tyrosine phosphorylation of PLCgamma1 and nPKCdelta. Furthermore, activation of ERK1/2 by PDGF, but not EGF, required PKC activity. In contrast, EGF substantially increased Ras.GTP with rapid activation of c-Raf, whereas stimulation of Ras.GTP loading by PDGF was minimal and activation of c-Raf was delayed. Our data provide clear evidence for differential coupling of PDGF and EGF receptors to the ERK1/2 cascade, and indicate that a minimum threshold/duration of ERK1/2 signaling is required for the development of cardiomyocyte hypertrophy.

Carbon monoxide protects against oxidant-induced apoptosis via inhibition of Kv2.1

Oxidative stress induces neuronal apoptosis and is implicated in cerebral ischemia, head trauma, and age-related neurodegenerative diseases. An early step in this process is the loss of intracellular K(+) via K(+) channels, and evidence indicates that K(v)2.1 is of particular importance in this regard, being rapidly inserted into the plasma membrane in response to apoptotic stimuli. An additional feature of neuronal oxidative stress is the up-regulation of the inducible enzyme heme oxygenase-1 (HO-1), which catabolizes heme to generate biliverdin, Fe(2+), and carbon monoxide (CO). CO provides neuronal protection against stresses such as stroke and excitotoxicity, although the underlying mechanisms are not yet elucidated. Here, we demonstrate that CO reversibly inhibits K(v)2.1. Channel inhibition by CO involves reactive oxygen species and protein kinase G activity. Overexpression of K(v)2.1 in HEK293 cells increases their vulnerability to oxidant-induced apoptosis, and this is reversed by CO. In hippocampal neurons, CO selectively inhibits K(v)2.1, reverses the dramatic oxidant-induced increase in K(+) current density, and provides marked protection against oxidant-induced apoptosis. Our results provide a novel mechanism to account for the neuroprotective effects of CO against oxidative apoptosis, which has potential for therapeutic exploitation to provide neuronal protection in situations of oxidative stress.

Carbon monoxide inhibits L-type Ca2+ channels via redox modulation of key cysteine residues by mitochondrial reactive oxygen species

Conditions of stress, such as myocardial infarction, stimulate up-regulation of heme oxygenase (HO-1) to provide cardioprotection. Here, we show that CO, a product of heme catabolism by HO-1, directly inhibits native rat cardiomyocyte L-type Ca2+ currents and the recombinant alpha1C subunit of the human cardiac L-type Ca2+ channel. CO (applied via a recognized CO donor molecule or as the dissolved gas) caused reversible, voltage-independent channel inhibition, which was dependent on the presence of a spliced insert in the cytoplasmic C-terminal region of the channel. Sequential molecular dissection and point mutagenesis identified three key cysteine residues within the proximal 31 amino acids of the splice insert required for CO sensitivity. CO-mediated inhibition was independent of nitric oxide and protein kinase G but was prevented by antioxidants and the reducing agent, dithiothreitol. Inhibition of NADPH oxidase and xanthine oxidase did not affect the inhibitory actions of CO. Instead, inhibitors of complex III (but not complex I) of the mitochondrial electron transport chain and a mitochondrially targeted antioxidant (Mito Q) fully prevented the effects of CO. Our data indicate that the cardioprotective effects of HO-1 activity may be attributable to an inhibitory action of CO on cardiac L-type Ca2+ channels. Inhibition arises from the ability of CO to promote generation of reactive oxygen species from complex III of mitochondria. This in turn leads to redox modulation of any or all of three critical cysteine residues in the channel's cytoplasmic C-terminal tail, resulting in channel inhibition.

Modulation of hTREK-1 by carbon monoxide

TREK-1 is a background K channel important in the regulation of neuronal excitability. Here, we demonstrate that recombinant human TREK-1 is activated by low concentrations of carbon monoxide (CO) and nitric oxide (NO), applied via their respective donor molecules. Related channels hTASK-1 and hTASK-3 were unaffected by CO. Effects of both CO and NO were prevented by preincubation of cells with the protein kinase G inhibitor, Rp-8-Br-PET-cGMPS. The effects of CO were independent of NO formation. At higher concentrations, both NO and CO were inhibitory. As both NO and CO are important neuronal gasotransmitters and TREK is crucial in regulating neuronal excitability, our results provide a novel means by which these gases may modulate neuronal activity.