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.

Stimulation of adult rat ventricular myocyte protein synthesis and phosphoinositide hydrolysis by the endothelins.

The effects of endothelin-1 (ET-1) on protein synthesis and phosphoinositide (PI) hydrolysis were investigated in ventricular myocytes isolated by collagenase digestion of adult rat hearts. The maximum stimulation of protein synthesis by ET-1 was about 35% and the EC50 value was about 0.3 nM. The stimulation was exerted at the translational stage since it was insensitive to inhibition by actinomycin D. The maximum stimulation of PI hydrolysis by ET-1 as measured by the formation of [3H]inositol phosphates was about 11-fold and the EC50 value was about 0.7 nM. The ET-1 analogue sarafotoxin-6b stimulated protein synthesis by a maximum of 27% and stimulated PI hydrolysis about 8- to 9-fold. The EC50 values were 1.6 nM and 0.6 nM, respectively. Other endothelins stimulated protein synthesis and PI hydrolysis in the following order of potency: ET-1 approximately ET-2 > ET-3. This order of potency suggests that the stimulation of both protein synthesis and PI hydrolysis is mediated through the ETA receptor. Although both angiotensin II and [Arg]vasopressin stimulated PI hydrolysis significantly, the stimulation was less than 60%, i.e., much less than the stimulation by ET-1 and its analogues. Neither insulin nor substance P stimulated PI hydrolysis. Stimulation of protein synthesis by ET-1 and its analogues correlated strongly with the stimulation of PI hydrolysis and we suggest that the stimulation of protein synthesis may be dependent on the stimulation of PI hydrolysis. We hypothesize that the mechanism may involve a protein kinase C-mediated increase in intracellular pH.

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.

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.

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.

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.

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, phorbol esters and phenylephrine stimulate MAP kinase activities in ventricular cardiomyocytes.

ET-1 stimulated MBP kinase activity in cultured cardiomyocytes. Maximal activation (3.5-fold) was at 5 min. EC50 was 0.2 nM. PMA or PE also increased MBP kinase (4- or 2.5-fold, respectively). Pre-treatment with PMA down-regulated the subsequent response to ET-1 or PMA. ET-1- or PMA-stimulated MBP kinase was resolved into 2 major (peaks II and IV) and 2 minor peaks by FPLC on Mono Q. Peaks II and IV were inactivated by either LAR or PP2A. Renatured MBP kinase activities following SDS-PAGE in MBP-containing gels and immunoblot analysis showed that peak II was a p42 MAP kinase and peak IV was a p44 MAP kinase.

Interaction of cimetidine with human serum albumin.

Ultrafiltration studies have detected the existence of a weak interaction between cimetidine and human serum albumin, a finding supported by corresponding studies with this xenobiotic and bovine serum albumin. Furthermore, the binding characteristics of the interaction with human serum albumin (4 sites, K = 630 M-1) more than suffice to account for the proportion of protein-bound drug in the serum of patients subjected to cimetidine therapy. Thus, although alpha 1-acid glycoprotein is usually regarded as the specific transporter of basic drugs, the present evidence implicates albumin as the likely binding protein for cimetidine in serum.

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.

cAMP and protein synthesis in isolated adult rat heart preparations.

The involvement of adenosine 3',5'-cyclic monophosphate (cAMP) in the stimulation of ventricular protein synthesis by aortic hypertension or adrenergic agonists in the adult rat heart was investigated. In either the retrogradely or anterogradely perfused heart, aortic hypertension increased protein synthesis rates by up to 19%. However, no changes in cAMP concentrations or in cAMP-dependent protein kinase activity ratios could be detected either at early (< 5 min) or late (90 min) time points. Although isoproterenol, 3-isobutyl-1-methylxanthine, or forskolin raised cAMP concentrations (by up to 4.5-fold) and cAMP-dependent protein kinase ratios (by up to 4-fold), protein synthesis rates were not increased; however, under some perfusion conditions, glucagon did stimulate protein synthesis by 25%. Epinephrine stimulated protein synthesis by up to 32%, an effect that was not prevented by propranolol. Phenylephrine also stimulated protein synthesis, an effect that was prevented by prazosin but was unaffected by yohimbine. These findings implicate the alpha 1-adrenoceptor in the regulation of cardiac protein synthesis. Because changes in adenine nucleotide concentrations were similar in hearts perfused with epinephrine or with the agents that raised cAMP, it is unlikely that adenine nucleotide depletion is responsible for the failure to observe effects of the latter group of agents on protein synthesis. Although isoproterenol or forskolin raised cAMP concentrations in isolated ventricular cardiomyocytes where ATP depletion was minimal, neither stimulated protein synthesis. alpha 1-Adrenergic agonists stimulate phosphoinositide hydrolysis in the heart (Brown, J. H., I. L. Buxton, and L. L. Brunton. Circ. Res. 57:532-537, 1985). Aortic hypertension doubled the rate of phosphoinositide hydrolysis in the perfused heart. We suggest that the phosphoinositide-linked signal transduction pathway is more likely to be involved in stimulation of cardiac protein synthesis by hypertension or adrenergic agonism than the adenylyl cyclase/cAMP-linked pathway.

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.

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.

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.

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.

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.

Characterization of protein kinase C isotype expression in adult rat heart. Protein kinase C-epsilon is a major isotype present, and it is activated by phorbol esters, epinephrine, and endothelin.

The pattern of protein kinase C (PKC) isotype expression in whole extracts of dispersed, freshly isolated adult rat ventricular myocytes and adult rat heart ventricle was examined by immunoblot analysis using antisera specific for PKC-alpha, -beta 1, -gamma, -delta, -epsilon, -zeta, or -eta isotypes. This analysis revealed significant levels of expression of the Ca(2+)-independent isotype PKC-epsilon, which was detected as band of 97-kd molecular mass. PKC-zeta was detected principally as a 66-kd band that probably represented a proteolytic product of the holoenzyme. PKC-eta was detected only in whole ventricle as a doublet at 75 and 81 kd and was therefore probably present in nonmyocytic cells. PKC-alpha, -beta 1, -gamma, and -delta could not be detected. Because of our inability to detect PKC-alpha, -beta 1, -gamma, and -delta in whole extracts, PKC isotypes were partially purified from whole heart by DEAE Sepharose chromatography. PKC-alpha, -beta 1, -gamma, and -delta could still not be detected in the appropriate fractions. All PKC isotypes were detectable in appropriate positive control extracts (brain or certain cultured cell lines). In unstimulated isolated cardiomyocytes, the majority (80-95%) of the PKC-epsilon immunoreactivity was present in the soluble fraction of the extract. On exposure of the cardiomyocytes to 1 microM phorbol 12-myristate 13-acetate (PMA), PKC-epsilon undergoes a rapid (< 30 seconds), sustained (at least 60 minutes), and virtually complete association with the Triton X-100-soluble membrane fraction. There was an associated loss of PKC-epsilon from the soluble fraction. The EC50 for PMA of the translocation event was 15-37 nM. Exposure of cardiomyocytes to 1 microM 4 beta-phorbol 12,13-didecanoate or 1 microM phorbol 12,13-dibutyrate also resulted in translocation of PKC-epsilon to the membrane fraction, whereas exposure to 1 microM 4 alpha-phorbol 12,13-didecanoate was without effect. PKC-epsilon also translocated on exposure of cardiomyocytes to 50 microM epinephrine or 100 nM endothelin-1. However, in both cases, the extent of translocation was significantly less than that after exposure to PMA. We conclude that interventions that lead to hypertrophy of cardiomyocytes (phorbol esters, epinephrine, and endothelin-1) activate PKC-epsilon.

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.

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.

Characterization of stimulation of phosphoinositide hydrolysis by alpha 1-adrenergic agonists in adult rat hearts.

The coupling of the pharmacologically defined alpha 1A- and alpha 1B-adrenoceptors to the hydrolysis of phospho[3H]inositides (PI) was investigated in ventricular myocytes freshly isolated from adult rat hearts. The alpha 1-adrenoceptor population in the heart was characterized by competitive binding experiments using [3H]prazosin and the alpha 1A-adrenoceptor-selective antagonist 5-methyl urapidil. It was heterogeneous with approximately 25% being pharmacologically of the alpha 1A-adrenoceptor subtype and 75% being of the alpha 1B-adrenoceptor subtype. Epinephrine, norepinephrine, or phenylephrine stimulated PI hydrolysis in the presence or absence of propranolol. The greatest stimulation (7-fold) was with epinephrine. The half-maximum effective concentrations for agonists were approximately 0.5-3.5 and 0.2 microM in the absence and presence of propranolol, respectively. The inhibition by 5-methyl urapidil of the stimulation of PI hydrolysis by a fixed concentration of epinephrine fitted a two-site competition curve. The distribution between high-affinity (25%) and low-affinity (75%) sites suggested that both the alpha 1A- and alpha 1B-adrenoceptors were coupled to PI hydrolysis in proportion to their relative abundance. Equally, the stimulation of PI hydrolysis by epinephrine in the presence of a fixed concentration of 5-methyl urapidil was biphasic. In addition, chloroethylclonidine, an irreversible inhibitor of the alpha 1B-adrenoceptor, inhibited the epinephrine stimulation of PI hydrolysis by 35%. We conclude that the pharmacologically defined alpha 1A- and alpha 1B-adrenoceptor subtypes are both coupled to PI hydrolysis in the ventricular myocyte.