The c-Jun N-terminal protein kinase family of mitogen-activated protein kinases (JNK MAPKs).

The c-Jun N-terminal protein kinase mitogen-activated protein kinases (JNK MAPKs) are an evolutionarily-conserved family of serine/threonine protein kinases. First identified in 1990 when intraperitoneal injection of the protein synthesis inhibitor cycloheximide activated a 54 kDa protein kinase, the JNK MAPKs have now taken on a prominent role in signal transduction. This research has revealed a number of levels of complexity. Alternative gene splicing is now recognised to result in ten different JNK MAPK isoforms of 46-55 kDa, and these isoforms differ in their substrate affinities. Furthermore, although originally classified as stress-activated protein kinases (SAPKs), or SAPKs, the JNK MAPKs are also critical mediators of signal transduction in response to stimulation by cytokines and some growth factors. JNK MAPKs have been shown to be critical mediators in dorsal closure in developing Drosophila embryos, and targeted knockout of murine JNK MAPKs has suggested a critical involvement of these kinases in mammalian embryonic development. Recent work has also highlighted their importance in programmed cell death. Thus, the JNK MAPKs may provide a critical target for regulation in both normal and diseased states.

A role for the extracellular signal-regulated kinase and p38 mitogen-activated protein kinases in interleukin-1 beta-stimulated delayed signal tranducer and activator of transcription 3 activation, atrial natriuretic factor expression, and cardiac myocyte morphology.

We have demonstrated that two hypertrophic agents, interleukin-1 beta (IL-1 beta) and leukemic inhibitory factor (LIF), altered cardiac myocyte morphology with striking similarity and prompted us to investigate the common actions of these cytokines. We compared the phosphorylation/activation of signal tranducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase (ERK), p38(MAPK), and c-Jun N-terminal kinase mitogen-activated protein kinases (MAPKs). The phosphorylation of STAT3 by IL-1 beta was delayed (>60 min), whereas the response to LIF was rapid (<10 min) and transient. We confirmed that IL-1 beta potently stimulated all three MAPK subfamilies. In contrast, LIF promoted strong activation of ERKs, marginal activation of p38(MAPK), and no c-Jun N-terminal kinase activation. To test the roles of ERKs and p38(MAPK), myocytes were pretreated with PD98059 and SB203580. Either inhibitor alone prevented STAT3 phosphorylation, implicating ERKs and p38(MAPK) in the delayed STAT3 response to IL-1 beta. The interplay of MAPKs and STAT3 phosphorylation in regulating IL-1 beta-stimulated hypertrophy was investigated by evaluating the effect of MAPK inhibitors on atrial natriuretic factor (ANF) expression and myocyte morphology. The specific inhibition of either ERK or p38(MAPK) attenuated the IL-1 beta- or LIF-stimulated ANF expression by up to 70%. Inhibition was not further increased in the presence of both inhibitors. Furthermore, although individual inhibition of ERK or p38(MAPK) did not affect morphology, co-treatment with both inhibitors abrogated the hypertrophic morphology stimulated by IL-1 beta but not by LIF. Taken together, our data indicate that the activation of ERK and p38(MAPK) is essential in regulating a delayed STAT3 phosphorylation as well as changes in ANF expression and morphology that follow IL-1 beta treatment. Thus, the role of MAPKs in the hypertrophic response can be dictated at least partly by the nature of the hypertrophic agent employed.

Tissue inhibitor of metalloproteinases-4 inhibits but does not support the activation of gelatinase A via efficient inhibition of membrane type 1-matrix metalloproteinase.

The tissue inhibitors of metalloproteinases 1-4 (TIMPs) have discrete regulatory roles in the activation of matrix metalloproteinase (MMP)-2 (gelatinase A), an important basement membrane-degrading MMP pivotal to tumor metastasis and angiogenesis. TIMP-2 binds to both the hemopexin C domain of progelatinase A and the active site of membrane type-1 (MT1) MMP. This trimeric complex presents the cell surface-bound gelatinase A zymogen to a free MT1-MMP molecule for activation. To investigate the role of TIMP-4 in the activation process, we developed a new procedure for the expression and purification of recombinant human TIMP-4 from baby hamster kidney cells. The recombinant TIMP-4 was a potent inhibitor of gelatinase A (apparent K(i) [Ki(app.)] < or = 9 pM; k(on) (association rate constant), 4.57 +/- 0.13 x 10(6) M(-1)s(-1)) and was less dependent upon hemopexin C domain interactions than TIMP-2 in its mode of binding and inhibition. Unlike TIMP-1, TIMP-4 strongly inhibited MT1-MMP (Ki(app.) < or = 100 pM; k(on), 3.49 +/- 0.34 x 10(6) M(-1)s(-1)) and blocked the concanavalin A-induced cellular activation of progelatinase A. In concanavalin A-stimulated homozygous Timp2 -/- fibroblasts or unstimulated MT1-MMP-transfected Timp2 -/- cells, which cannot activate progelatinase A, activation was restored by the addition of 0.3-5 nM TIMP-2 but not by TIMP-4, unequivocally showing the TIMP-2 dependency of MT1-MMP-induced activation of gelatinase A and the fact that TIMP-4 cannot support activation. The dominance of TIMP-2 in the activation process was further supported by the preferential binding of TIMP-2 compared with TIMP-4 to the hemopexin C domain of progelatinase A in inhibitor mixtures and by the ability of TIMP-2 to displace TIMP-4 from the hemopexin C domain. Hence, TIMP-4 regulates gelatinase A activity by efficient inhibition of MT1-MMP-mediated activation and by inhibiting the activated enzyme and, thus, is a tumor resistance factor in the peritumor stroma.

The mechanism of heat shock activation of ERK mitogen-activated protein kinases in the interleukin 3-dependent ProB cell line BaF3.

We have investigated heat shock stimulation of MAPK cascades in an interleukin 3-dependent cell line, BaF3. Following exposure to 42 degrees C, the stress-activated JNK MAPKs were phosphorylated and activated, but p38 MAPKs remained unaffected. Surprisingly, heat shock also activated ERK MAPKs in a potent (>60-fold), delayed (>30 min), and sustained (>/=120 min) manner. These characteristics suggested a novel mechanism of ERK MAPK activation and became the focus of this study. A MEK-specific inhibitor, PD98059, inhibited heat shock ERK MAPK activation by >75%. Surprisingly, a role for Ras in the heat shock response was eliminated by the failure of a dominant-negative Ras(Asn-17) mutant to inhibit ERK MAPK activation and the failure to observe increases in Ras.GTP. Heat shock also failed to stimulate activation of A-, B-, and c-Raf. Instead, a serine/threonine phosphatase inhibitor, okadaic acid, activated ERK MAPK in a similar manner to heat shock. Furthermore, pretreatment with suramin, generally recognized as a broad range inhibitor of growth factor receptors, inhibited both okadaic acid-stimulated and heat shock-stimulated ERK MAPK activity by >40%. Inhibiting ERK MAPK activation during heat shock with PD98059 enhanced losses in cell viability. These results demonstrate Ras- and Raf-independent ERK MAPK activation maintains cell viability following heat shock.

Intact mitochondrial electron transport function is essential for signalling by hydrogen peroxide in cardiac myocytes.

Oxidative stress has been proposed as a mediator of cardiac injury during ischemia and reperfusion. We examined the signalling events initiated by short-term exposure of cardiac myocytes to oxidative stress elicited by hydrogen peroxide. A potent stimulation of tyrosine phosphorylation was observed within 1 to 2 min exposure to 1 m m hydrogen peroxide. Within 5 min, the ERK mitogen-activated protein kinases (ERK MAPKs) were activated. This activation of ERK MAPKs was blocked by N-acetylcysteine (NAC), implicating a role for free radicals in the signalling events. NAC failed to inhibit ERK MAPK activation by the hypertrophic agent, phenylephrine, or hyperosmotic shock. Myxothiazol, an inhibitor of complex III of the mitochondrial electron transport chain, also inhibited ERK MAPK activation by hydrogen peroxide, but not by 12- O -tetradecanoylphorbol-13-acetate (TPA) or hyperosmotic shock. Myxothiazol completely inhibited the increase in tyrosine phosphorylated proteins observed with hydrogen peroxide treatment. A variety of inhibitors which act at different levels of the mitochondrial electron transport chain (rotenone, theonyltrifluoroacetone, antimycin A, cyanide) also inhibited activation of the ERK MAPKs by hydrogen peroxide but not TPA or hyperosmotic shock. These studies suggest a novel mechanism of regulation of the ERK MAPK pathway and oxidative stress signalling by hydrogen peroxide.

Mitochondrial ATP production is necessary for activation of the extracellular-signal-regulated kinases during ischaemia/reperfusion in rat myocyte-derived H9c2 cells.

To search for the stimuli involved in activating the mitogen-activated protein kinases (MAPKs) during ischaemia and reperfusion, we simulated the event in a system in vitro conducive to continuous and non-invasive measurements of several major perturbations that occur at the time: O(2) tension, mitochondrial respiration and energy status. Using H9c2 cells (a clonal line derived from rat heart), we found that activation of the extracellular signal-regulated MAPKs (ERKs) on reoxygenation was abolished if the mitochondria were inhibited prior to and during reoxygenation. Re-introduction of O(2) per se is therefore not sufficient to activate the ERKs. Recovery and maintenance of cellular ATP levels by mitochondrial respiration is necessary, although ATP recovery alone is not sufficient. ERK activation by H(2)O(2), but not phorbol esters, was also sensitive to mitochondrial inhibition. Thus, reoxygenation and H(2)O(2)-mediated oxidative stress share a mechanism of ERK activation that is ATP- or mitochondrion-dependent, and this common feature suggests that the reoxygenation response is mediated by reactive oxygen species. A correlation between ERK activity and ATP levels was also found during the anoxic phase of ischaemia, an effect that was not due to substrate limitation for the kinases. Our results reveal the importance of cellular metabolism in ERK activation, and introduce ATP as a novel participant in the mechanisms underlying the ERK cascade.

Signalling via stress-activated mitogen-activated protein kinases in the cardiovascular system.

A number of physiological, pharmacological and pathological stimuli initiate cardiac hypertrophy. The intracellular signalling events activated by these stimuli are equally complex. Our ability to treat the hypertrophic and failing myocardium effectively will require clarification of which signalling events regulate growth, remodelling and failure. Much recent attention has focused on the regulation of the mitogen-activated protein kinase cascades (MAPKs), with the importance of these cascades in the development of cardiovascular diseases being extensively explored. These signalling pathways may provide one link from the diverse stress and pharmacological extracellular stimuli to the regulation of gene expression, contractile protein regulation and protein function. This review focuses on the recent progress made in the understanding of the regulation and function of MAPKs in the cardiovascular system, with particular emphasis being placed on the events in the cardiac ventricular myocyte.

Differential regulation of parallel mitogen-activated protein kinases in cardiac myocytes revealed by phosphatase inhibition.

Previous studies have suggested that the contribution of inducible phosphatases to ERK MAPK deactivation is both cell-type- and agonist-specific. The aim of this study was to define the role of inducible phosphatases in ERK MAPK regulation in cardiac myocytes. We examined the kinetics of activation/deactivation of ERK MAPKs following the exposure of cardiac myocytes to endothelin-1 or phorbol ester. Deactivation was prevented by inhibition of protein synthesis indicating a contribution of inducible phosphatases. In contrast, okadaic acid failed to prolong ERK MAPK activation, but activated three myelin basic protein kinases (MBPKs, 55, 62, and 87 kDa) and two c-Jun kinases (46 and 55 kDa). Although the identity of the MBPKs is unknown, the c-Jun kinases corresponded to JNK MAPKs. Simultaneous exposure of cardiac myocytes to okadaic acid and osmotic shock potentiated JNK MAPK activation. Thus, inducible phosphatases regulate ERK MAPK deactivation, whereas okadaic acid-sensitive phosphatases regulate JNK MAPKs and three novel MBPKs.

Stimulation of the stress-activated mitogen-activated protein kinase subfamilies in perfused heart. p38/RK mitogen-activated protein kinases and c-Jun N-terminal kinases are activated by ischemia/reperfusion.

It has recently been recognized that cellular stresses activate certain members of the mitogen-activated protein kinase (MAPK) superfamily. One role of these "stress-activated" MAPKs is to increase the transactivating activity of the transcription factors c-Jun, Elk1, and ATF2. These findings may be particularly relevant to hearts that have been exposed to pathological stresses. Using the isolated perfused rat heart, we show that global ischemia does not activate the 42- and 44-kD extracellular signal-regulated (protein) kinase (ERK) subfamily of MAPKs but rather stimulates a 38-kD activator of MAPK-activated protein kinase-2 (MAPKAPK2). This activation is maintained during reperfusion. The molecular characteristics of this protein kinase suggest that it is a member of the p38/reactivating kinase (RK) group of stress-activated MAPKs. In contrast, stress-activated MAPKs of the c-Jun N-terminal kinase (JNK/SAPKs) subfamily are not activated by ischemia alone but are activated by reperfusion following ischemia. Furthermore, transfection of ventricular myocytes with activated protein kinases (MEKK1 and SEK1) that may be involved in the upstream activation of JNK/ SAPKs induces increases in myocyte size and transcriptional changes typical of the hypertrophic response. We speculate that activation of multiple parallel MAPK pathways may be important in the responses of hearts to cellular stresses.

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.

The role of protein kinases in adaptational growth of the heart.

The ventricular myocyte is a terminally-differentiated cell that can no longer undergo cell division. In response to a variety of stimuli, including exposure to endothelin-1, phenylephrine or mechanical stretch, the myocyte increases its size and its complement of organized myofibrils. These adaptational changes during myocyte hypertrophy are accompanied by distinct changes in gene expression. The signalling cascades that initiate these changes are currently under intensive investigation. Many hypertrophic agonists activate protein kinase C (PKC). Transfection of ventricular myocytes with constitutively-active PKC isoforms initiates the changes in gene expression typical of the hypertrophic response. Similarly, the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway can be activated by a variety of hypertrophic agents. Transfection of ventricular myocytes with components of this pathway has demonstrated that MAPK is essential for the changes in gene expression associated with the development of hypertrophy. However a Ras-dependent, but Raf-independent, pathway may regulate the organization of the contractile apparatus. Other protein kinases, such as ribosomal S6 kinases, p90RSK or p70/p85S6K, which are poorly characterized in the ventricular myocyte, may also regulate changes in gene expression. Further research is required to investigate cross-talk between these signal transduction pathways so that the spatial and temporal relationships that integrate the multiple signaling events leading to the adaptational growth of the ventricular myocyte may be understood.

Endothelin-1-dependent signaling pathways in the myocardium.

Endothelin-1 (ET-1) is a locally acting vasoactive peptide that also has profound effects on the contractile properties and growth of the cardiac myocyte. Binding of ET-1 to its transmembrane heptahelical receptors activates G proteins of the G(q) and G(i) classes. Activation of G(q) stimulates hydrolysis of phosphatidylinositol-4,5-bisphosphate, and the diacylglycerol thus formed stimulates protein kinase C. Subsequently, the protein kinase Raf is activated and this leads to activation of the extracellular signal-regulated protein kinase (ERK) subfamily of mitogen-activated protein kinases. Activation of G(i) counteracts ß-adrenoceptor-mediated increases in cAMP concentrations. We have attempted to rationalize the established physiological consequences of ET-1 agonism in the cardiac myocyte (that is, on contraction and growth) in terms of activation of these signaling pathways.

Cellular stresses differentially activate c-Jun N-terminal protein kinases and extracellular signal-regulated protein kinases in cultured ventricular myocytes.

Anisomycin or osmotic stress induced by sorbitol activated c-Jun N-terminal protein kinases (JNKs) in ventricular myocytes cultured from neonatal rat hearts. After 15-30 min, JNK was activated by 10-20-fold. Activation by anisomycin was transient, but that by sorbitol was sustained for at least 4 h. In-gel JNK assays confirmed activation of two renaturable JNKs of 46 and 55 kDa (JNK-46 and JNK-55, respectively). An antibody against human JNK1 immunoprecipitated JNK-46 activity. Endothelin-1, an activator of extracellular signal-regulated protein kinases (ERKs), also transiently activated JNKs by 2-5-fold after 30 min. Phorbol 12-myristate 13-acetate did not activate the JNKs although it activated ERK1 and ERK2, which phosphorylated the c-Jun transactivation domain in vitro. ATP depletion and repletion achieved by incubation in cyanide+deoxyglucose and its subsequent removal from the medium activated the ERKs but failed to activate the JNKs. Sorbitol (but not anisomycin) also stimulated the ERKs. Sorbitol-stimulated JNK activity could be resolved into three peaks by fast protein liquid chromatography on a Mono Q column. The two major peaks contained JNK-46 or JNK-55. These results demonstrate that cellular stresses differentially activate the JNKs and ERKs and that there may be "cross-talk" between these MAPK pathways.

Hypertrophic agonists stimulate the activities of the protein kinases c-Raf and A-Raf in cultured ventricular myocytes.

We detected expression of two Raf isoforms, c-Raf and A-Raf, in neonatal rat heart. Both isoforms phosphorylated, activated, and formed complexes with mitogen-activated protein kinase kinase 1 in vitro. However, these isoforms were differentially activated by hypertrophic stimuli such as peptide growth factors, endothelin-1 (ET1), or 12-O-tetradecanoylphorbol-13-acetate (TPA) that activate the mitogen-activated protein kinase cascade. Exposure of cultured ventricular myocytes to acidic fibroblast growth factor activated c-Raf but not A-Raf. In contrast, TPA produced a sustained activation of A-Raf and only transiently activated c-Raf. ET1 transiently activated both isoforms. TPA and ET1 were the most potent activators of c-Raf and A-Raf. Both utilized protein kinase C-dependent pathways, but stimulation by ET1 was also partially sensitive to pertussis toxin pretreatment. cRaf was inhibited by activation of cAMP-dependent protein kinase although A-Raf was less affected. Fetal calf serum, phenylephrine, and carbachol were less potent activators of c-Raf and A-Raf. These results demonstrate that A-Raf and c-Raf are differentially regulated and that A-Raf may be an important mediator of mitogen-activated protein kinase cascade activation when cAMP is elevated.

The mitogen-activated protein kinase kinase MEK1 stimulates a pattern of gene expression typical of the hypertrophic phenotype in rat ventricular cardiomyocytes.

Adult mammalian ventricular cardiomyocytes are terminally differentiated cells that enlarge adaptively by hypertrophy. In this situation, genes normally expressed in the fetal ventricular cardiomyocyte (e.g. atrial natriuretic factor (ANF), beta-myosin heavy chain (beta-MHC), and skeletal muscle (SkM) alpha-actin) are re-expressed, and there is transient expression of immediate early genes (e.g. c-fos). Using appropriate reporter plasmids, we studied the effects of transfection of the constitutively active or dominant negative mitogen-activated protein kinase kinase MEK1 on ANF, beta-MHC, and SkM alpha-actin promoter activities in cultured ventricular cardiomyocytes. ANF expression was stimulated (maximally 75-fold) by the hypertrophic agonist phenylephrine in a dose-dependent manner (EC50, 10 microM), and this stimulation was inhibited by dominant negative MEK1. Cotransfection of dominant negative MEK1 with a dominant negative mitogen-activated protein kinase (extracellular signal-regulated protein kinase (ERK2)) increased this inhibition. Transfection with constitutively active MEK1 constructs doubled ANF promoter activity. The additional cotransfection of wild-type ERK2 stimulated ANF promoter activity by about 5-fold. Expression of beta-MHC and SkM alpha-actin was also stimulated. Promoter activity regulated by activator protein-1 or c-fos serum response element consensus sequences was also increased. We conclude that the MEK1/ERK2 cascade may play a role in regulating gene expression during hypertrophy.

Intracellular signalling through protein kinases in the heart.

Protein kinases play important roles in intracellular signalling pathways in probably all cells. In the heart, they are involved in the regulation of ion handling, contractility, fuel metabolism and growth. In this review, we discuss the consequences of activation of protein kinases known to be expressed in the heart. We concentrate principally on the following: cyclic AMP-dependent protein kinase, protein kinase C, mitogen-activated protein kinase, Ca2+/calmodulin-dependent protein kinases and pyruvate dehydrogenase kinase.

Expression of protein kinase C isoforms during cardiac ventricular development.

The expression of protein kinase C (PKC) isoforms (PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta) was studied by immunoblotting in whole ventricles of rat hearts during postnatal development (1-26 days) and in the adult. PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta were detected in ventricles of 1-day-old rats, although PKC-alpha and PKC-beta 1 were only barely detectable. All isoforms were rapidly downregulated during development, with abundances relative to total protein declining in the adult to < 25% of 1-day-old values. PKC-beta 1 was not detectable in adult ventricles. The specific activity of PKC was also downregulated. The rat ventricular myocyte becomes amitotic soon after birth but continues to grow, increasing its protein content 40- to 50-fold between the neonate and the 300-g adult. An important question is thus whether the amount of PKC per myocyte is downregulated. With the use of isolated cells, immunoblotting showed that the contents per myocyte of PKC-alpha and PKC-epsilon increased approximately 10-fold between the neonatal and adult stages. In rat ventricles, the rank of association with the particulate fraction was PKC-delta > PKC-epsilon > PKC-zeta. Association of these isoforms with the particulate fraction was less in the adult than in the neonate. In primary cultures of ventricular myocytes prepared from neonatal rat hearts, 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited translocation of PKC-alpha, PKC-delta, and PKC-epsilon from the soluble to the particulate fraction in < 1 min, after which time no further translocation was observed. Prolonged exposure (16 h) of myocytes to 1 microM TPA caused essentially complete downregulation of these isoforms, although downregulation of PKC-epsilon was slower than for PKC-delta. In contrast, PKC-zeta was neither translocated nor downregulated by 1 microM TPA. Immunoblotting of human ventricular samples also revealed downregulation of PKC relative to total protein during fetal/postnatal development.

Activation of the mitogen-activated protein kinase cascade by pertussis toxin-sensitive and -insensitive pathways in cultured ventricular cardiomyocytes.

The involvement of pertussis toxin (PTX)-sensitive and -insensitive pathways in the activation of the mitogen-activated protein kinase (MAPK) cascade was examined in ventricular cardiomyocytes cultured from neonatal rats. A number of agonists that activate heterotrimeric G-protein-coupled receptors stimulated MAPK activity after exposure for 5 min. These included foetal calf serum (FCS), endothelin-1 (these two being the most effective of the agonists examined), phenylephrine, endothelin-3, lysophosphatidic acid, carbachol, isoprenaline and angiotensin II. Activation of MAPK and MAPK kinase (MEK) by carbachol returned to control levels within 30-60 min, whereas activation by FCS was more sustained. FPLC on Mono Q showed that carbachol and FCS activated two peaks of MEK and two peaks of MAPK (p42MAPK and p44MAPK). Pretreatment of cells with PTX for 24 h inhibited the activation of MAPK by carbachol, FCS and lysophosphatidic acid, but not that by endothelin-1, phenylephrine or isoprenaline. Involvement of G-proteins in the activation of the cardiac MAPK cascade was demonstrated by the sustained (PTX-insensitive) activation of MAPK (and MEK) after exposure of cells to AlF4-. AlF4- activated PtdIns hydrolysis, as did endothelin-1, endothelin-3, phenylephrine and FCS. In contrast, the effect of lysophosphatidic acid on PtdIns hydrolysis was small and carbachol was without significant effect even after prolonged exposure. We conclude that PTX-sensitive (i.e. Gi/G(o)-linked) and PTX-insensitive (i.e. Gq/Gs-linked) pathways of MAPK activation exist in neonatal ventricular myocytes. FCS may stimulate the MAPK cascade through both pathways.

Differential activation of protein kinase C isoforms by endothelin-1 and phenylephrine and subsequent stimulation of p42 and p44 mitogen-activated protein kinases in ventricular myocytes cultured from neonatal rat hearts.

The translocation of protein kinase C (PKC) isoforms PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta from soluble to particulate fractions was studied in ventricular cardiomyocytes cultured from neonatal rats. Endothelin-1 (ET-1) caused a rapid ETA receptor-mediated translocation of PKC-delta and PKC-epsilon (complete in 0.5-1 min). By 3-5 min, both isoforms were returning to the soluble fraction, but a greater proportion of PKC-epsilon remained associated with the particulate fraction. The EC50 of translocation for PKC-delta was 11-15 nM ET-1 whereas that for PKC-epsilon was 1.4-1.7 nM. Phenylephrine caused a rapid translocation of PKC-epsilon (EC50 = 0.9 microM) but the proportion lost from the soluble fraction was less than with ET-1. Translocation of PKC-delta was barely detectable with phenylephrine. Neither agonist caused any consistent translocation of PKC-alpha or PKC-zeta. Activation of p42 and p44 mitogen-activated protein kinase (MAPK) by ET-1 or phenylephrine followed more slowly (complete in 3-5 min). Phosphorylation of p42-MAPK occurred simultaneously with its activation. The proportion of the total p42-MAPK pool phosphorylated in response to ET-1 (50%) was greater than with phenylephrine (20%). In addition to activation of MAPK, an unidentified p85 protein kinase was activated by ET-1 in the soluble fraction whereas an unidentified p58 protein kinase was activated in the particulate fraction.

Regulation of mitogen-activated protein kinase cascade in adult rat heart preparations in vitro.

The regulation of mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) was studied in freshly isolated adult rat heart preparations. In contrast to the situation in ventricular myocytes cultured from neonatal rat hearts, stimulation of MAPK activity by 1 mumol/L phorbol 12-myristate 13-acetate (PMA) was not consistently detectable in crude extracts. After fast protein liquid chromatography, MAPK isoforms p42MAPK and p44MAPK and two peaks of MEK were shown to be activated > 10-fold in perfused hearts or ventricular myocytes exposed to 1 mumol/L PMA for 5 minutes. The identities of MAPK or MEK were confirmed by immunoblotting and, for MAPK, by the "in-gel" myelin basic protein phosphorylation assay. In retrogradely perfused hearts, high coronary perfusion pressure (120 mm Hg for 5 minutes), norepinephrine (50 mumol/L for 5 minutes), or isoproterenol (50 mumol/L for 5 minutes) stimulated MAPK and MEK approximately 2- to 5-fold. In isolated myocytes, endothelin 1 (100 nmol/L for 5 minutes) also stimulated MAPK, but stimulation by norepinephrine or isoproterenol was difficult to detect. Immunoblotting showed that the relative abundances of MAPK and MEK protein in ventricles declined to < 20% of their postpartal abundances after 50 days. This may explain the difficulties encountered in assaying the activity of MAPK in crude extracts from adult hearts. We conclude that potentially hypertrophic agonists and interventions stimulate the MAPK cascade in adult rats and suggest that the MAPK cascade may be an important intracellular signaling pathway in this response.