Selective modulation of PKC isozymes by inflammation in canine colonic circular muscle cells

BACKGROUND & AIMS: Protein kinase C (PKC) is a key signaling molecule in excitation-contraction coupling in several types of smooth muscle cells. We investigated whether the attenuated contraction in inflamed colon cells is caused by alterations in the expression, distribution, and activation of specific PKC isozymes. METHODS: Kinase assays, immunofluorescence imaging, and Western immunoblotting were performed on single circular smooth muscle cells obtained from the normal dog colon as well as from colon with experimental colitis induced by mucosal exposure to ethanol and acetic acid, to determine the distribution, expression, and activation of PKC isozymes. RESULTS: Classical (alpha, beta, and gamma), novel (delta and epsilon), and the atypical PKC (iota, lambda, and zeta) isozymes were detected in colonic circular muscle cells. The expression of PKC alpha, beta, and epsilon isozymes was down-regulated, whereas that of PKC iota and lambda isozymes was up-regulated; other isozymes were not affected by inflammation. Acetylcholine (ACh) treatment translocated only the PKC alpha, beta, and epsilon isozymes from the cytosol to the membrane in normal cells; this translocation was absent in inflamed colon cells. Immunofluorescence imaging confirmed the translocation of PKC alpha from the cytosol to the membrane in response to ACh in normal cells. PKC inhibitors, chelerythrine, and myristoylated peptides to alpha, beta, and epsilon isozymes inhibited the contractile response to ACh in normal, but not in inflamed, cells. PKC iota and lambda did not participate in the contractile response to ACh. CONCLUSIONS: ACh-induced contraction is mediated by PKC alpha, beta, and epsilon isozymes in normal colonic circular muscle cells. Contractile dysfunction in inflamed colon cells is, in part, caused by decreased expression and impaired activation of specific PKC isozymes.     

Responses of cardiac protein kinase C isoforms to distinct pathological stimuli are differentially regulated.

Currently at least 11 protein kinase C (PKC) isoforms have been identified and may play different roles in cell signaling pathways leading to changes in cardiac contractility, the hypertrophic response, and tolerance to myocardial ischemia. The purpose of the present study was to test the hypothesis that responses of individual PKC isoforms to distinct pathological stimuli were differentially regulated in the adult guinea pig heart. Isolated hearts were perfused by the Langendorff method and were exposed to ischemia, hypoxia, H(2)O(2), or angiotensin II. Hypoxia and ischemia induced translocation of PKC isoforms alpha, beta(2), gamma, and zeta, and H(2)O(2) translocated PKC isoforms alpha, beta(2), and zeta. Angiotensin II produced translocation of alpha, beta(2), epsilon, gamma, and zeta isoforms. Inhibition of phospholipase C with tricyclodecan-9-yl-xanthogenate (D609) blocked hypoxia-induced (alpha, beta(2), and zeta) and angiotensin II-induced (alpha, beta(2), gamma, and zeta) translocation of PKC isoforms. Inhibition of tyrosine kinase with genistein blocked translocation of PKC isoforms by hypoxia (beta(2) and zeta) and by angiotensin II (beta(2)). By contrast, neither D609 nor genistein blocked H(2)O(2)-induced translocation of any PKC isoform. We conclude that hypoxia-induced activation of PKC isoforms is mediated through pathways involving phospholipase C and tyrosine kinase, but oxidative stress may activate PKC isoforms independently of Galphaq-phospholipase C coupling and tyrosine kinase signaling. Because oxidative stress may directly activate PKC, and PKC activation appears to be involved in human heart failure, selective inhibition of the PKC isoforms may provide a novel therapeutic strategy for the prevention and treatment of this pathological process.     

Distribution and stimulation by gastrin-releasing peptide of protein kinase C subfamilies in insulin-secreting cells

The role of the different isoforms of protein kinase C (PKC) in modulating insulin secretion is still widely unknown. The aim of our studies was to investigate which isoforms are influenced by gastrin-releasing peptide (GRP), a neuropeptide which has been shown to modulate insulin secretion by activating PKC. Presence of PKC isoforms alpha, beta, gamma, delta, epsilon and zeta was tested by immunoblot analysis in whole pancreatic islets of mouse and rat and in the insulinoma cell line RINm5F. Effects of GRP, the truncated peptide GRP1-16 and KCl were also measured on translocation of PKC isoforms. In pancreatic islets of mouse and rat, the PKC isoforms alpha, beta, gamma, delta, epsilon and zeta could be detected. No PKCgamma activity was present in the pancreatic tumor cell line RINm5F. Incubation of mouse or rat islets or of RINm5F cells with GRP induced translocation of the PKC isoforms alpha, beta and zeta. The N-terminal portion of the peptide GRP1-16 induced partial translocation only of the PKC isoforms alpha, beta and zeta in mouse and rat islets in 4 out of 10 cases, but failed to show any effect on PKC isoforms in RINm5F cells. Depolarization of the islets by KCl did not translocate any tested PKC isoform. However, incubation with GRP followed by depolarization with KCl led to translocation of the PKC isoforms alpha, beta and zeta. It is suggested that PKC alpha, beta and/or zeta may play a role in the modulation of insulin secretion by GRP.     

Diacylglycerol kinase zeta rescues G alpha q-induced heart failure in transgenic mice

BACKGROUND: The G alpha q protein-coupled receptor (GPCR) signaling pathway, which includes diacylglycerol (DAG) and protein kinase C (PKC), plays a critical role in the development of cardiac hypertrophy and heart failure (HF). It has been reported that the expression of a constitutively active mutant of the G protein alpha q subunit in the hearts of transgenic mice (G alpha q-TG) induces cardiac hypertrophy and lethal HF. DAG kinase (DGK) catalyzes DAG and controls its cellular levels, thus acting as a regulator of GPCR signaling. It has been found that transgenic mice with cardiac-specific overexpression of DGK zeta (DGK zeta-TG) inhibit GPCR agonist-induced activation of the DAG-PKC signaling and subsequent cardiac hypertrophy, so this study tested the hypothesis that DGK zeta could rescue G alpha q-TG mice from developing HF. METHODS AND RESULTS: Double transgenic mice (G alpha q/DGK zeta-TG) with cardiac-specific overexpression of both DGK zeta and G alpha q were generated by crossing G alpha q-TG with DGK zeta-TG mice, and the pathophysiological consequences were analyzed. DGK zeta prevented cardiac dysfunction, determined by dilatation of left ventricular (LV) dimensions, reduction of LV fractional shortening, and marked increases in LV end-diastolic pressure in G alpha q-TG mice. Translocation of PKC isoforms, phosphorylation activity of c-jun N-terminal kinase and p38 mitogen-activated protein kinase in G alpha q-TG mice were attenuated by DGK zeta. DGK zeta improved the survival rate of G alpha q-TG mice. CONCLUSIONS: These results demonstrate the first evidence that DGK zeta blocks cardiac dysfunction and progression to lethal HF by activated G alpha q protein without detectable adverse effects in the in-vivo heart and suggest that DGK zeta is a novel therapeutic target for HF.     

Subunit interactions within the T-cell antigen receptor: clues from the study of partial complexes.

The T-cell antigen receptor is a multisubunit complex composed of seven transmembrane chains (alpha beta gamma delta epsilon zeta 2). Subunit interactions within this complex were defined by analyzing the subunit composition of partial complexes. These partial complexes were observed in mutant and tumor T cells that fail to synthesize one or more of the receptor chains or in fibroblasts transfected with genes encoding T-cell antigen receptor chains. In addition, partial complexes were generated by immunoprecipitation with antibodies that cause selective dissociation of T-cell antigen receptor chains. The alpha and beta chains were found to form a disulfide-linked dimer in the absence of any of the other chains. The gamma, delta, and epsilon chains were also efficiently associated in the absence of a complete heterodimer. Complexes composed only of delta epsilon or gamma epsilon could be observed. Both these dimers, as well as the gamma delta epsilon trimer, could form stable complexes with alpha beta, even in the absence of zeta 2. The zeta 2 dimer could bind directly to alpha beta. In the absence of a complete clonotypic heterodimer, zeta 2 was not stably associated with gamma delta epsilon. These observations suggest a model in which alpha beta interacts directly with the gamma delta epsilon trimer and zeta 2, with less-direct interaction between the latter two.     

PKC delta-induced activation of MAPK pathway is required for bFGF-stimulated proliferation of coronary smooth muscle cells

OBJECTIVE: Basic fibroblast growth factor (bFGF)-stimulated proliferation of coronary smooth muscle cells (cSMC) contributes to the pathogenesis of arteriosclerosis and restenosis. However, the molecular mechanisms involved are not fully understood. We have shown previously that protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) are required for the bFGF-stimulated mitogenic process in bovine cSMC. In this study, we determined the PKC isoform(s) involved and investigated their functional role in the bFGF-stimulated signaling and cell cycle progression in human and bovine cSMC. METHODS AND RESULTS: Downregulation of PKC by phorbol 12-myristate 13-acetate (PMA) inhibited bFGF-induced DNA synthesis, the activation of MAPK, and the expression of c-myc, demonstrating the involvement of PMA-sensitive PKC isoforms in growth factor-induced proliferation and the MAPK pathway. The PMA-sensitive classical PKC isoforms alpha, beta, gamma and novel PKC isoforms delta and epsilon were found in human cSMC. Whereas blocking of the classical PKC isoforms had no influence, the suppression of PKC delta by genetic and pharmacological approaches inhibited the bFGF-stimulated c-Raf1-MEK-MAPK-c-myc signaling and DNA synthesis in cSMC. In contrast to PKC epsilon, our results showed that bFGF activated PKC delta by phosphorylation in a time-dependent manner. In addition, inhibition of PKC delta induced a hypophosphorylation of the retinoblastoma protein and suppression of the cyclins D1 and A, demonstrating the importance of PKC delta for bFGF-induced cell cycle progression through the G1 phase in cSMC. CONCLUSIONS: Our results show that PKC delta is required for the bFGF-stimulated c-Raf1-MEK-MAPK-c-myc signaling pathway involved in the proliferation of cSMC. Therefore, it may be an interesting therapeutic target for preventing proliferative vascular disorders.     

Proteasome implication in phorbol ester- and GnRH-induced selective down-regulation of PKC (alpha, epsilon, zeta) in alpha T(3)-1 and L beta T(2) gonadotrope cell lines

We investigated mechanisms underlying selective down-modulation of PKC isoforms (alpha, epsilon, zeta): 1) during 12-O-tetradecanoyl-phorbol-13 acetate (TPA) (10(-7) M) or GnRH (10(-7) M) desensitization conditions (2- to 6-h treatments) in two gonadotrope cell lines (alpha T(3)-1, L beta T(2)) and 2) in primary pituitary cell cultures from male rats during long-term phorbol ester administration. We demonstrated that, as in alpha T(3)-1 cells, in a more differentiated gonadotrope cell line L beta T(2) the GnRH-receptor coupling (PLC, PLA2, PLD) generated second messengers essential for PKCs activation; the characterized isoforms (alpha, beta II, delta, epsilon, zeta) were selectively and differentially down-regulated by TPA (alpha, beta II, delta, epsilon) or GnRH (delta, epsilon). In whole cell lysates, proteasome inhibitors (proteasome inhibitor I and II, Lactacystin, beta-Lactone, Calpain inhibitor I) prevented in both gonadotrope cell lines the TPA-induced depletion of PKC alpha, epsilon, and the GnRH-elicited PKC epsilon down-regulation; they counteracted in mixed pituitary cell cultures as well, the TPA-evoked PKC alpha, epsilon depletion. In contrast, the inhibitors of calpain(s) and lysosomal proteases (Calpeptin, E64d, Calpain inhibitor II, and PD150606), were ineffective. As shown in alpha T(3)-1 subcellular fractions, proteasome abrogation did not affect membrane translocation of TPA- and GnRH- target isoforms (alpha, epsilon) but, preventing their degradation, favored enzyme accumulation to the membrane compartment. Proteolysis processing of PKCs may be dependent upon their phosphorylated state and/or catalytic activity. Inhibition of PKC catalytic activity (GF109203X, G?6976), selectively prevented the TPA-evoked PKC alpha depletion in both mixed pituitary cells and alpha T(3)-1 gonadotropes; in alpha T(3)-1 subcellular fractions, PKC alpha inactivation overcame the TPA-evoked isoenzyme degradation by inducing a pronounced membrane accumulation of the isoform without affecting its membrane relocalization. Thus, the proteasome system by adjusting PKC cellular levels, may represent a regulatory proteolytic pathway implicated in the adaptive mechanisms of the time dependent cell responses.     

Altered cardiac endothelin receptors and protein kinase C in deoxycorticosterone-salt hypertensive rats

The aim of the present study was to assess the status of ET-1 receptor subtypes (ET(A)and ET(B)) in ventricular myocytes and fibroblasts and to determine the role of PKC-dependent pathways in ET-1-stimulated cardiac cells in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Systolic blood pressure and relative heart to body weight were significantly increased in DOCA-salt rats. In unilaterally nephrectomized (Uni-Nx) control rats, more than 90% of cardiomyocyte ET receptors were of the ET(A)subtype, whereas in fibroblasts ET(A)and ET(B)receptors were present in a 1:3 ratio. In DOCA-salt rats, the density of the ET(A)receptor subtype was reduced by 31% in cardiomyocytes and in cardiac fibroblasts only ET(B)receptor density was decreased by 29%. Affinity was unchanged. The relative expression of immunoreactive PKC alpha, gamma and epsilon was significantly increased, whereas PKC delta was not altered in cardiac extracts of DOCA-salt rats. In cardiac fibroblasts from DOCA-salt rats PKC delta was significantly increased and PKC epsilon was not translocated after ET-1 stimulation. The hearts of DOCA-salt hypertensive rats are thus characterized by: (1) decreased density of cardiomyocyte ET(A)receptors and fibroblast ET(B)receptors; (2) cell-specific enhanced expression of some PKC isoenzymes (alpha, gamma, delta and epsilon); and (3) unresponsiveness of PKC epsilon to translocate in the presence of ET-1. Together with alterations of ET-1-induced Ca(2+)handling in cardiac myocytes and fibroblasts, which we previously reported, results from the present study indicate a marked modification of the cardiac ET-1 system of DOCA-salt hypertensive rats.     

Structure of the T-cell antigen receptor: evidence for two CD3 epsilon subunits in the T-cell receptor-CD3 complex.

The T-cell antigen receptor (TCR) consists of heterodimeric glycoproteins (TCR alpha beta or gamma delta) that demonstrate homology with immunoglobulins. Noncovalently associated with the alpha beta (or gamma delta) heterodimer are at least five nonvariant proteins (CD3-gamma, -delta, -epsilon, -zeta, and -eta), which together comprise the TCR-CD3 complex. The stoichiometry of the antigen receptor has been assumed to be either alpha beta gamma delta epsilon zeta zeta or alpha beta gamma delta epsilon zeta eta. In this paper we provide several lines of evidence that support the notion that the mature TCR-CD3 complex on the cell surface contains two CD3-epsilon polypeptide chains. Transfection of two murine T cell-T cell hybridomas with the human DNA encoding CD3-epsilon protein demonstrated that both murine and human CD3-epsilon chains were present within the same TCR-CD3 complex. Analysis of thymocytes isolated from transgenic mice that expressed high copy numbers of the human CD3-epsilon gene showed that the heterologous human CD3-epsilon subunits were coexpressed with murine CD3-epsilon in the same TCR-CD3 complex. Since CD3-epsilon was shown to form disulfide-linked homodimers both in human and murine T cells, the two CD3-epsilon subunits present in the TCR-CD3 complex were in direct contact with one another. The presence of two CD3-epsilon polypeptide chains in close proximity to one another in the TCR-CD3 complex may have important implications for its assembly and its signal transduction mechanisms.     

Norepinephrine increases glucose transport in brown adipocytes via beta3-adrenoceptors through a cAMP, PKA, and PI3-kinase-dependent pathway stimulating conventional and novel PKCs

To identify the signaling pathways that mediate the adrenergic stimulation of glucose uptake in brown adipose tissue, we used mouse brown adipocytes in culture. The endogenous adrenergic neurotransmitter norepinephrine (NE) induced 2-deoxy-D-glucose uptake 3-fold in a concentration-dependent manner (pEC50 approximately 6.5). The uptake was abolished by high doses of propranolol. The NE effect was mimicked by isoprenaline (pEC50 approximately 6.9), BRL 37344 (pEC50 approximately 8.6), CL 316243 (pEC50 approximately 9.7) and CGP 12177 (pEC50 approximately 7.3) and was thus mediated by beta3-adrenergic receptors. The NE-induced effect on 2-deoxy-D-glucose uptake was mediated by adenylyl cyclase and cAMP because responses were inhibited by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine and the protein kinase A inhibitor 4-cyano-3-methylisoquinoline. Cholera toxin and 8-bromoadenosine cAMP were both able to increase 2-deoxy-D-glucose uptake. Involvement of other adrenergic signaling pathways (alpha1-and alpha2-adrenergic receptors) were excluded. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, abolished beta-adrenergic- or 8-bromoadenosine cAMP-stimulated 2-deoxy-D-glucose uptake, demonstrating that a cAMP-dependent PI3K-mediated pathway is positively connected to glucose uptake. Inhibition of the beta-adrenergically stimulated response with protein kinase C (PKC) inhibitors (G? 6983, which inhibits (alpha, beta, gamma), (delta), and (zeta) isoforms and Ro-31-8220, which inhibits (alpha, beta1, beta2, gamma) and (epsilon) but not atypical isoforms) indicated that cAMP-mediated glucose uptake is stimulated via conventional and novel PKCs. These results demonstrate that adrenergic stimulation, through beta3-adrenergic receptors/cAMP/protein kinase A, recruits a PI3K pathway stimulating conventional and novel PKCs, which mediate glucose uptake in brown adipocytes.     

Lysophosphatidic acid stimulates protein kinase C isoforms alpha, beta, epsilon, and zeta in a pertussis toxin sensitive pathway in vascular smooth muscle cells.

The natural phospholipid lysophosphatidic acid (LPA) has been characterized as an important vascular smooth muscle cell (VSMC) mitogen whose effects are mainly mediated by pertussis toxin (PTX)-sensitive guanosine triphosphate (GTP)-binding protein (Gi-protein). Protein kinase C (PKC) isoforms play an important role in intracellular signaling cascades and in growth of VSMC. In the present study we investigated the effect of LPA on activation of PKC isoforms alpha, beta, epsilon, and zeta in VSMC by Western blot of cytosolic and membrane fractions. Furthermore, we examined the role of PKC activation on LPA-induced growth of VSMC using PKC inhibitor 19-27. Stimulation of VSMC by 5 microg/mL LPA for 10 min increased the amount of PKC alpha, beta, epsilon, and zeta in the particulate fraction by 689%, 285%, 424%, and 510%, respectively, and returned to control level after 30 min. Correspondingly, the amount of PKC alpha, beta, epsilon, and zeta in the cytosolic fraction decreased by 32%, 94%, 44%, and 95%, respectively, compared to control. Furthermore, we could show that LPA-induced activation of PKC alpha, beta, epsilon, and zeta isoforms was PTX sensitive. Incubation of VSMC with nonspecific PKC inhibitor 19-27 (10 micromol/L) for 24 h resulted in a 30% inhibition of LPA-induced DNA synthesis as measured by [3H]thymidine incorporation. In conclusion, in VSMC LPA stimulated translocation of PKC isoforms alpha, beta, epsilon, and zeta in a PTX-sensitive manner. Furthermore stimulation of PKC might be critically involved in LPA-induced mitogenesis in VSMC.     

CD3 zeta subunit can substitute for the gamma subunit of Fc epsilon receptor type I in assembly and functional expression of the high-affinity IgE receptor: evidence for interreceptor complementation.

The high-affinity receptor for IgE (Fc epsilon RI) is a four-subunit structure consisting of three distinct polypeptides: the IgE-binding alpha chain, the four-fold membrane-spanning beta chain, and the disulfide-linked gamma-gamma homodimer. cDNAs encoding each subunit have previously been isolated. Here we show that microinjection of Xenopus oocytes with a mixture of in vitro transcribed RNAs encoding each subunit results in expression of IgE receptors at the oocyte surface as detected by binding of IgE or anti-Fc epsilon RI alpha subunit monoclonal antibody to intact oocytes. Surface expression of Fc epsilon RI requires injection of all three subunits (alpha, beta, and gamma) RNAs. In particular, omission of Fc epsilon RI gamma RNA from the mixtures abolishes surface binding of either IgE or anti-Fc epsilon RI alpha monoclonal antibody to microinjected oocytes. However, addition of CD3 zeta RNA to Fc epsilon RI alpha and Fc epsilon RI beta RNAs restores IgE receptor surface expression when this combination is microinjected into oocytes. Metabolic labeling and immunoprecipitation of oocyte microinjected with a mixture of CD3 zeta plus Fc epsilon RI alpha and Fc epsilon RI beta RNAs reveals a noncovalent association between the CD3 zeta-zeta disulfide-linked homodimer and Fc epsilon RI alpha-beta. These results provide direct evidence for the functional relatedness of CD3 zeta and Fc epsilon RI gamma.     

Protein kinase C isozymes epsilon and alpha in murine erythroleukemia cells.

Protein kinase C (PKC) has a role in signal transduction during hexamethylene bisacetamide (HMBA)-induced differentiation of murine erythroleukemia cells (MELC). Separation of MELC PKC isozymes by hydroxylapatite chromatography yields a major peak (III) and a minor peak (II) of PKC activity, previously reported to contain the PKC alpha and beta isozymes, respectively. In the present study, we confirm that peak III activity is PKC alpha but show that peak II contains PKC epsilon and little or no PKC beta. Immunoblot analysis with isozyme-specific anti-alpha and anti-epsilon PKC antibodies detected PKC alpha in peak III and PKC epsilon in peak II. Peak III activity was markedly enhanced (up to 20-fold) by phosphatidylserine, diolein, and Ca2+, whereas addition of these cofactors to the reaction mixture stimulated peak II activity only 2- to 4-fold. RNase protection analysis of MELC RNA showed that PKC alpha and PKC epsilon RNAs were in a ratio of approximately 2:1, but PKC beta RNA was barely detectable. Taken together, these data indicate that MELC contain PKC alpha and PKC epsilon but little or no PKC beta.     

Surface expression of alternative forms of the TCR/CD3 complex.

T-cell antigen receptor (TCR) heterodimers of both the alpha beta and gamma delta types are expressed at the surface of T cells only in association with a complex of invariant chains called CD3. The requirement for individual CD3 components to achieve TCR surface expression was examined by cotransfection of a non-T-cell line with TCR alpha and beta, as well as CD3 delta, epsilon, gamma, and zeta, cDNAs. Both transient and stable transfectants expressing TCR and CD3 epitopes at the cell surface were generated. By transfection of TCR and CD3 components in different combinations, the TCR chains, as well as the CD3 epsilon and zeta chains, were each shown to be essential for reconstituting surface expression. On the other hand, CD3 delta and gamma chains could be used alternatively, providing evidence for two different types of TCR/CD3 complexes.     

Norepinephrine-induced changes in gene expression of phospholipase C in cardiomyocytes

Previously, we have reported that norepinephrine (NE)-mediated cardiac hypertrophy may occur due to stimulation of alpha1-adrenoceptors and phospholipase C (PLC) activity. Since the signal transduction mechanisms involving PLC isozymes in cardiomyocytes are not well established, the present study was conducted to test the hypothesis that stimulation of cardiac PLC activity by NE increases the gene expression for PLC isozymes via a PKC and ERK 1/2-dependent pathway. For this purpose, mRNA levels for PLC beta1, beta3, gamma1, and delta1 isozymes were determined in isolated adult rat cardiomyocytes upon incubation in the absence and presence of NE. The NE-induced increases in PLC isozyme mRNA levels were not only attenuated by prazosin, an inhibitor of alpha1-adrenergic receptor, but also by U73122, an inhibitor of PLC activity. Alterations in NE-induced PLC gene expression by both prazosin and U73122 were associated with inhibition of PLC activity. The inhibition of NE-stimulated PLC gene expression by bisindolylmaleimide, a PKC inhibitor, and PD98059, an ERK1/2 inhibitor, indicated that PKC-MAPK signaling may be involved in this signal transduction pathway. The observed NE-induced changes in gene expression in the presence of different inhibitors were associated with corresponding changes in the protein content. Furthermore, significant increases in mRNA levels and protein contents for all PLC isozymes were found in cardiomyocytes treated with phorbol 12-myristate 13-acetate, a PKC activator. These data indicate that PLC isozymes may regulate their own gene expression through a PKC and ERK 1/2-dependent pathway in a cycle of events associated with the cardiomyocyte hypertrophic response.     

Tissue angiotensin II during progression or ventricular hypertrophy to heart failure in hypertensive rats; differential effects on PKC epsilon and PKC beta

The protein kinase C (PKC) family has been implicated as second messengers in mechanosensitive modulation of cardiac hypertrophy. However, little information is available on the role of expression and activation of specific cardiac PKC isozymes during development of left ventricular hypertrophy (LVH) and failure (LVF). Dahl salt-sensitive rats fed an 8% salt diet developed systemic hypertension and concentric LVH at 11 weeks of age that is followed by left ventricle (LV) dilatation and global hypokinesis at 17 weeks. Among several PKC isozymes expressed in the LV myocardium, only PKC epsilon showed a 94% increase at the LVH stage. At the LVF stage, however, PKC epsilon returned to the control level, whereas PKC beta I and beta II increased by 158% and 155%, respectively. Hearts were studied at each stage using the Langendorff set-up, and a LV balloon was inflated to achieve an equivalent diastolic wall stress. Following mechanical stretch, PKC epsilon was significantly activated in LVH myocardium in which tissue angiotensin II levels were increased by 59%. Pre-treatment with valsartan, an AT(1)-receptor blocker, abolished the stretch-mediated PKC epsilon activation. Mechanical stretch no longer induced PKC epsilon activation in LVF. Chronic administration of valsartan blunted the progression of LVF and inhibited the increase in PKC beta. Mechanosensitive PKC epsilon activation is augmented and therefore may contribute to the development of compensatory hypertrophy. This effect was dependent on activation of tissue angiotensin II. However, this compensatory mechanism becomes inactive in LVF, where PKC beta may participate in the progression to cardiac dysfunction and LV remodeling.     

Expression of GTP-binding proteins and protein kinase C isozymes in platelet-like particles derived from megakaryoblastic leukemia cells (MEG-01)

The expression of the various GTP-binding proteins and protein kinase C (PKC) in the platelet-like particles produced by MEG-01 cells was analyzed by RT-PCR and immunoblotting. We selected 14 human low Mr GTPbinding proteins (LMW-GPs) and nine PKCs expressed in platelets and/or megakaryocytes, and designed specific primer pairs for the proteins. RT-PCR analysis revealed that the particles express the mRNAs of many LMW-GPs such as rap 1A, rap 1B, rap 2B, ral A, rho A, rac 1, rac 2, Cdc 42, rab 1, rab 3B, rab 6, ram/rab 27 and ran . By immunoblotting analysis, Rap1, RhoA, Rac, Cdc42, Rab6 and Rab8 were identified in the particles. As for PKCs, the particles were observed to express the mRNAs of PKC- alpha ,- beta I,- beta -II,- delta , epsilon , eta and theta , but not- gamma and zeta . Using immunoblot analysis, PKC- beta I,- beta II and zeta were shown to exist in the particles, although the contents were lower than those in platelets. Furthermore, the presence of Gi2- alpha , a heterotrimeric G protein that is the major pertussis toxin substrate in human platelets, and beta subunits was observed in the particles. Taken together, the particles possess some similarity to human platelets based on the expression profiles of GTP-binding proteins and PKCs.     

The gamma chain of the high-affinity receptor for IgE is a major functional subunit of the T-cell antigen receptor complex in gamma delta T lymphocytes.

T-cell activation is a consequence of the clonotypic T-cell antigen receptor (TCR) binding to an antigen followed by signal transduction via the invariant subunits of the TCR/CD3 complex. gamma delta TCR cells are a small subset of T cells that populate both the epithelial and lymphoid tissues and have unique antigen specificity and function. However, the composition of invariant chains within the gamma delta TCR/CD3 complex has not been well characterized. Here we report that, unlike the majority of alpha beta T cell, gamma delta T cells isolated from spleen and intestinal epithelial tissue express high levels of the gamma chain of the high-affinity receptor for IgE (Fc epsilon RI gamma) as one invariant subunit of their TCR/CD3 complex. Fc epsilon RI gamma exists as both a homodimer and a heterodimer associated with the TCR zeta chain. Moreover, stimulation of the gamma delta TCR results in rapid tyrosine phosphorylation of Fc epsilon RI gamma. Our results suggest that utilization of distinct receptor signaling components may enable the coupling of antigen stimulation to the activation of different signal transduction pathways in alpha beta and gamma delta T cells.     

Beta(2)-adrenergic and several other G protein-coupled receptors in human atrial membranes activate both G(s) and G(i)

Cardiac G protein-coupled receptors that couple to Galpha(s) and stimulate cAMP formation (eg, beta-adrenergic, histamine, serotonin, and glucagon receptors) play a key role in cardiac inotropy. Recent studies in rodent cardiac myocytes and transfected cells have revealed that one of these receptors, the beta(2)-adrenergic receptor (AR), also couples to the inhibitory G protein Galpha(i) (activation of which inhibits cAMP formation). If beta(2)ARs could be shown to couple to Galpha(i) in the human heart, it would have important ramifications, because levels of Galpha(i) increase with age and in failing human heart. Therefore, we investigated whether beta(2)ARs in the human heart activate Galpha(i). By photoaffinity labeling human atrial membranes with [(32)P]azidoanilido-GTP, followed by immunoprecipitation with antibodies specific for Galpha(i), we found that Galpha(i) is activated by stimulation of beta(2)ARs but not of beta(1)ARs. In addition, we found that other Galpha(s)-coupled receptors also couple to Galpha(i), including histamine, serotonin, and glucagon. When coupling of these receptors to Galpha(i) is disrupted by pertussis toxin, their ability to stimulate adenylyl cyclase is enhanced. These data provide the first evidence that beta(2)AR and many other Galpha(s)-coupled receptors in human atrium also couple to Galpha(i) and that abolishing the coupling of these receptors to Galpha(i) increases the receptor-mediated adenylyl cyclase activity.     

Regulation of CYP11B2 gene expression by protein kinase C

Bisindolylmaleimide, a protein kinase C (PKC) inhibitor, was shown to stimulate the hamster CYP11B2 promoter activity in transfected NCI-H295 cells. In this study we have found that TPA, an activator of PKC, also inhibited the hamster CYP11B2 promoter activity. DAG-dependent PKC alpha and PKC epsilon, and atypical PKC zeta were detected in hamster adrenal zona glomerulosa, whereas the isoforms alpha, epsilon, zeta and theta were found in NCI-H295 cells. CYP11B2 promoter activity was inhibited in cells co-transfected with constitutively active PKC alpha and epsilon mutants, whereas it was increased with the constitutively active PKC zeta mutant. Dominant negative PKC alpha, epsilon and zeta mutants stimulated the promoter activity. G?6976, a specific inhibitor of classical PKCs, enhanced promoter activity, providing further evidence that PKC alpha, the only classical PKC revealed in hamster adrenal and NCI-H295 cells, was involved in the promoter inhibition.