Phorbol ester-mediated neurotensin secretion is dependent on the PKC-α and -δ isoforms

2002 ◽  
Vol 283 (5) ◽  
pp. G1197-G1206 ◽  
Author(s):  
Jing Li ◽  
Mark R. Hellmich ◽  
George H. Greeley ◽  
Courtney M. Townsend ◽  
B. Mark Evers
Keyword(s):  
Critical Role ◽  
Specific Inhibitor ◽  
Immunofluorescent Staining ◽  
Nanomolar Concentration ◽  
Pkc Isoforms ◽  
Pkc Signaling ◽  
Gf 109203X ◽  
Pkc Inhibitors ◽  
Bon Cells

Neurotensin (NT) plays an important role in gastrointestinal secretion, motility, and growth. The mechanisms regulating NT secretion are not entirely known. Our purpose was to define the role of the PKC signaling pathway in secretion of NT from BON cells, a human pancreatic carcinoid cell line that produces and secretes NT peptide. We demonstrated expression of all 11 PKC isoforms at varying levels in untreated BON cells. Expression of PKC-α, -β2, -δ, and -μ isoforms was most pronounced. Immunofluorescent staining showed PKC-α and -μ expression throughout the cytoplasm and in the membrane. Also, significant fluorescence of PKC-δ was noted in the nucleus and cytoplasm. Treatment with PMA induced translocation of PKC-α, -δ, and -μ from cytosol to membrane. Activation of PKC-α, -δ, and -μ was further confirmed by kinase assays. Addition of PKC-α inhibitor Gö-6976 at a nanomolar concentration, other PKC inhibitors Gö-6983 and GF-109203X, or PKC-δ-specific inhibitor rottlerin significantly inhibited PMA-mediated NT release. Overexpression of either PKC-α or -δ increased PMA-mediated NT secretion compared with control cells. We demonstrated that PMA-mediated NT secretion in BON cells is associated with translocation and activation of PKC-α, -δ, and -μ. Furthermore, inhibition of PKC-α and -δ blocked PMA-stimulated NT secretion, suggesting a critical role for these isoforms in NT release.

scholarly journals Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7

Reproduction ◽  
2014 ◽  
Vol 148 (2) ◽  
pp. 221-235 ◽  
Author(s):  
Juan M Gallardo Bolaños ◽  
Carolina M Balao da Silva ◽  
Patricia Martín Muñoz ◽  
Antolín Morillo Rodríguez ◽  
María Plaza Dávila ◽  
...  
Keyword(s):  
Critical Role ◽  
Membrane Integrity ◽  
Specific Inhibitor ◽  
Akt Inhibitor ◽  
Akt Phosphorylation ◽  
Mitochondrial Superoxide ◽  
Phosphorylated Akt ◽  
Sperm Survival

AKT, also referred to as protein kinase B (PKB or RAC), plays a critical role in controlling cell survival and apoptosis. To gain insights into the mechanisms regulating sperm survival after ejaculation, the role of AKT was investigated in stallion spermatozoa using a specific inhibitor and a phosphoflow approach. Stallion spermatozoa were washed and incubated in Biggers–Whitten–Whittingham medium, supplemented with 1% polyvinyl alcohol (PVA) in the presence of 0 (vehicle), 10, 20 or 30 μM SH5, an AKT inhibitor. SH5 treatment reduced the percentage of sperm displaying AKT phosphorylation, with inhibition reaching a maximum after 1 h of incubation. This decrease in phosphorylation was attributable to either dephosphorylation or suppression of the active phosphorylation pathway. Stallion spermatozoa spontaneously dephosphorylated during in vitro incubation, resulting in a lack of a difference in AKT phosphorylation between the SH5-treated sperm and the control after 4 h of incubation. AKT inhibition decreased the proportion of motile spermatozoa (total and progressive) and the sperm velocity. Similarly, AKT inhibition reduced membrane integrity, leading to increased membrane permeability and reduced the mitochondrial membrane potential concomitantly with activation of caspases 3 and 7. However, the percentage of spermatozoa exhibiting oxidative stress, the production of mitochondrial superoxide radicals, DNA oxidation and DNA fragmentation were not affected by AKT inhibition. It is concluded that AKT maintains the membrane integrity of ejaculated stallion spermatozoa, presumably by inhibiting caspases 3 and 7, which prevents the progression of spermatozoa to an incomplete form of apoptosis.Free Spanish abstractA Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/148/2/221/suppl/DC1.

scholarly journals S100A8/A9 modulates inflammatory collateral tissue damage during intraperitoneal origin systemic candidiasis

2020 ◽  
Author(s):  
Madhu Shankar ◽  
Nathalie Uwamahoro ◽  
Sandra Holmberg ◽  
Maria Joanna Niemiec ◽  
Johannes Roth ◽  
...  
Keyword(s):  
Tissue Damage ◽  
Critical Role ◽  
Specific Inhibitor ◽  
Surgical Intensive Care ◽  
Inflammatory Protein ◽  
Systemic Level ◽  
Combating Infection ◽  
Deficient Mice

AbstractPeritonitis is a leading cause of severe sepsis in surgical intensive care units, as over 70% of patients diagnosed with peritonitis develop septic shock. A critical role of the immune system is to return to homeostasis after combating infection. S100A8/A9 (calprotectin) is an antimicrobial, pro-inflammatory protein complex often used as a biomarker for diagnosis of disease activities in many inflammatory disorders. Here we describe the role of S100A8/A9 on inflammatory collateral tissue damage (ICTD).We performed an in vivo Candida albicans disseminated peritonitis mouse model using WT and S100A9-deficient mice and stimulated primary macrophages with recombinant S100A8/A9 in the presence or absence of the compound paquinimod, a specific inhibitor of S100A9. In addition, the effects on ICTD and fungal clearance were investigated. S100A9-deficient mice developed less ICTD than wildtype mice. Restoration of S100A8/A9 in S100A9 knockout mice resulted in increased ICTD and fungal clearance comparable to wildtype levels. Treatment with paquinimod abolished ICTD.The data indicated that S100A8/A9 controls ICTD levels and host antimicrobial modulation at a systemic level during intra-abdominal candidiasis (IAC).

1,25-Dihydroxyvitamin D3 and TPA activate phospholipase D in Caco-2 cells: role of PKC-α

1999 ◽  
Vol 276 (4) ◽  
pp. G993-G1004 ◽  
Author(s):  
Sharad Khare ◽  
Marc Bissonnette ◽  
Beth Scaglione-Sewell ◽  
Ramesh K. Wali ◽  
Michael D. Sitrin ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Dihydroxyvitamin D3 ◽  
Dihydroxyvitamin D ◽  
1,25 Dihydroxyvitamin D ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Gf 109203X ◽  
Pkc Β ◽  
Stimulation Of

1,25-Dihydroxyvitamin D3[1,25(OH)2D3] and 12- O-tetradecanoylphorbol 13-acetate (TPA) both activated phospholipase D (PLD) in Caco-2 cells. GF-109203x, an inhibitor of protein kinase C (PKC) isoforms, inhibited this activation by both of these agonists. 1,25(OH)2D3activated PKC-α, but not PKC-β1, -βII, -δ, or -ζ, whereas TPA activated PKC-α, -β1, and -δ. Chronic treatment with TPA (1 μM, 24 h) significantly reduced the expression of PKC-α, -βI, and -δ and markedly reduced the ability of 1,25(OH)2D3or TPA to acutely stimulate PLD. Removal of Ca2+ from the medium, as well as preincubation of cells with Gö-6976, an inhibitor of Ca2+-dependent PKC isoforms, significantly reduced the stimulation of PLD by 1,25(OH)2D3or TPA. Treatment with 12-deoxyphorbol-13-phenylacetate-20-acetate, which specifically activates PKC-βI and -βII, however, failed to stimulate PLD. In addition, the activation of PLD by 1,25(OH)2D3or TPA was markedly reduced or accentuated in stably transfected cells with inhibited or amplified PKC-α expression, respectively. Taken together, these observations indicate that PKC-α is intimately involved in the stimulation of PLD in Caco-2 cells by 1,25(OH)2D3or TPA.

scholarly journals Nuclear signalling by Rac GTPase: essential role of phospholipase A2

1997 ◽  
Vol 326 (2) ◽  
pp. 333-337 ◽  
Author(s):  
Byung-Chul KIM ◽  
Jae-Hong KIM
Keyword(s):  
Phospholipase A2 ◽  
Critical Role ◽  
Specific Inhibitor ◽  
Major Product ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Inhibitory Protein ◽  
Rac Gtpase

Rac, one member of Rho family GTPases, stimulates c-fos serum response element (SRE)–luciferase reporter gene in Rat-2 fibroblast cells. By transient transfection analysis, we demonstrated that the activation of phospholipase A2 (PLA2) and the subsequent production of arachidonic acid (AA) are essential for Rac-induced c-fos SRE activation, implying a critical role for PLA2 in the Rac-signalling pathway to the nucleus. Either pretreatment with mepacrine, a specific inhibitor of PLA2, or co-transfection with the expression plasmid of lipocortin-1, a proposed inhibitory protein of PLA2, selectively abolished RacV12-induced SRE activation. Further, we demonstrated that subsequent metabolism of AA, a major product of Rac-activated PLA2, by lipoxygenase (LO) is essential for Rac-induced c-fos SRE activation. In agreement with the role of the PLA2–AA–LO cascade as a potential mediator of Rac signalling to the nucleus, the addition of exogenous AA stimulated c-fos SRE-luciferase activity in an LO-dependent manner. Together, our results demonstrate that ‘Rac-activated PLA2 and subsequent AA metabolism by LO’ constitute a novel and specific pathway in Rac GTPase-induced c-fos SRE activation.

Aberrant Megakaryocyte Antigen Expression on Myelodysplastic Erythroid Cells: Role of Protein Kinase C?.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2625-2625
Author(s):  
Rachel Elliott ◽  
Lorna Pearn ◽  
Ala Al-Sabah ◽  
Steven Knapper ◽  
Alan K. Burnett ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Antigen Expression ◽  
Model System ◽  
Morphological Evidence ◽  
Erythroid Cells ◽  
Pkc Isoforms ◽  
Pkc Inhibitors ◽  
Vitro Model

Abstract Erythroid lineage dysplasia is one of the most frequent findings in Myelodysplastic Syndrome (MDS) and in clinical terms contributes significantly to the morbidity in affected patients. There is currently no satisfactory treatment available for the majority of these patients and many rely on regular red cell transfusions for support. Previous work using an in vitro model system based on human CD34+ progenitors indicated that the developmental abnormalities affecting the erythroid cells may in part arise from inappropriate activation of protein kinase C (PKC). Evidence suggests that PKC acts as a lineage discriminator for bipotential erythro-megakaryocytic progenitor cells where low PKC activity promotes erythroid commitment and high PKC activity promotes megakaryocytic commitment. In the in vitro model system aberrant activation of PKC in erythroid cells led to developmental disruption and inappropriate megakaryocytic antigen expression such as GPIIb (CD41). Restoration of normal development was possible by inhibition of PKC. In this study we investigate the extent to which aberrant megakaryocyte antigen expression occurs in MDS, and whether PKC inhibition can normalise this and the associated erythroid lineage dysplasia. Bone marrow was obtained from 27 MDS patients and compared with 16 normal bone marrows. Fresh bone marrow mononuclear cells from both groups were analysed by 4- colour flow cytometry to establish the frequency of aberrant CD41 expression on nucleated erythroid (GlyA+ CD36+) cells. Cells bearing adherent platelets were excluded based on co-expression of CD42b. We have established that aberrant expression of CD41 occurred in 56% of the MDS patient marrows compared with normal bone marrow erythroid cells (n=27 and 16 respectively). These patients also showed clear morphological evidence of erythro-megakaryocytic lineage dysplasia. To determine the effect of PKC inhibition on this aberrant CD41 expression, some of these cases were examined in bulk liquid culture in the presence of PKC inhibitors. We assessed three PKC inhibitors in this way: GF109203X, a proprietary staurosporine derivative and Tamoxifen. In 42% of the cases examined in this way there was a phenotypic response to the highly selective PKC inhibitor GF109203X in terms of a reduction in CD41 expression. There was also morphological evidence of erythroid differentiation. The other two agents showed no effect on these parameters. The expression profile of individual PKC isoforms in the erythroid (GlyA+) cells was examined by western blotting, to determine if as in the model system the developmental abnormalites seen in the MDS marrow resulted from hyperactivation of PKC. We were able to evaluate 13 MDS and 6 normal controls in this way. The expression of 3 PKC isoforms (α, ε, ι) was determined as well as the level of phosphorylated PKC and the level of PDK1 kinase (which phosphorylates PKC). More than half of the patients examined demonstrated elevated levels of PKC expression. Aberrations in phosphorylation of PKC were less common. Patients with the highest expression of CD41 also had the highest expression of PKCα. These data suggest a role of inappropriate PKC activity in the erythroid lineage dysplasia and subsequent developmental failure seen in MDS, and, that PKC inhibitors may be valuable therapeutic agents in a subset of these patients.

scholarly journals Protein Kinase C Downregulation upon Rapamycin Treatment Attenuates Neuroinflammation and Mitochondrial Disease

2020 ◽  
Vol 4 (Supplement_1) ◽  
pp. 889-890
Author(s):  
Anthony Grillo ◽  
Alessandro Bitto ◽  
Matt Kaeberlein
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Critical Role ◽  
Specific Inhibitor ◽  
Mitochondrial Diseases ◽  
Leigh Syndrome ◽  
Premature Death ◽  
Kinase C ◽  
Pkc Β ◽  
Pkc Inhibitors

Abstract Mitochondrial dysfunction causes many poorly understood diseases, such as Leigh Syndrome, that are often caused by dysfunctions in proteins involved in the electron transport chain. My lab previously reported mTOR is pathologically involved in the neurodegenerative phenotype and premature death of mice missing the Complex I subunit Ndufs4 (Ndufs4-/- mice). We discovered treatment with rapamycin extends lifespan, reduces neuroinflammation, and attenuates the neurodegenerative phenotype in these mice, although the mechanisms remain unclear. Rapamycin-treated Ndufs4-/- mice exhibited decreased activation of the mTORC1 pathway. It also deactivated the mTORC2 pathway. We observed that phosphorylation of the canonical protein kinase C (PKC) isoforms (PKC-α, -β, and -γ) decreased more than any other kinases, leading us to hypothesize its deactivation contributes to the observed lifespan extension. To test this, we treated Ndufs4-/- mice with three different PKC inhibitors: the pan-PKC inhibitors GO6983 and GF109203X, and the PKC-β specific inhibitor ruboxistaurin. Similar to rapamycin, all three drugs were able to significantly delay the onset of neurological symptoms (i.e. clasping) and increase survival. We also observed that PKC-β inhibition reduced skin inflammation to suppress the hair loss phenotype displayed by Ndufs4-/- mice at weaning. We further discovered PKC-β inhibition reduces neuroinflammation by deactivating the NF-kB inflammatory pathway. These results suggest that mTORC2 may play a critical role in the etiology of mitochondrial diseases such as Leigh Syndrome.

Role of protein kinase C-δ in the age-dependent secretagogue action of bile acids in mammalian colon

2007 ◽  
Vol 293 (6) ◽  
pp. C1851-C1861 ◽  
Author(s):  
Jainuch Kanchanapoo ◽  
Mei Ao ◽  
Roli Prasad ◽  
Christopher Moore ◽  
Cynthia Kay ◽  
...  
Keyword(s):  
Bile Acids ◽  
Short Circuit ◽  
Specific Inhibitor ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Specific Effects ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Do So

The role of specific PKC isoforms in the regulation of epithelial Cl− secretion by Ca2+-dependent secretagogues remains controversial. In the developing rabbit distal colon, the bile acid taurodeoxycholate (TDC) acts via intracellular calcium to stimulate Cl− transport in adult, but not in young, animals, whereas the PKC activator phorbol dibutyrate (PDB) stimulates Cl− transport at all ages. We tested the hypothesis that specific PKC isoforms account for the age-specific effects of TDC. The effects of conventional (cPKC) and novel (nPKC) PKC-specific inhibitors on TDC- and PDB-stimulated Cl− transport in adult and weanling colonocytes were assessed by using 6-methoxy-quinolyl acetoethyl ester. In adult colonocytes, the cPKC inhibitor Gö-6976 inhibited PDB action but not TDC action, whereas the cPKC and nPKC inhibitor Gö-6850 blocked both TDC and PDB actions. Additionally, rottlerin and the PKC-δ-specific inhibitor peptide (δV1-1) inhibited TDC- and PDB-stimulated Cl− transport in adult colonocytes. Rottlerin also decreased TDC-stimulated short-circuit current in intact colonic epithelia. Only Gö-6976, but neither rottlerin nor δV1-1, inhibited PDB-stimulated transport in weanling colonocytes. Colonic lysates express PKC-α, -λ, and -ι protein equally at all ages, but they do not express PKC-γ or -θ at any age. Expression of PKC-β and PKC-ε protein was newborn>adult>weanling, whereas PKC-δ was expressed in adult but not in weanling or newborn colonocytes. TDC (1.6-fold) and PDB (2.0-fold) stimulated PKC-δ enzymatic activity in adult colonocytes but failed to do so in weanling colonocytes. PKC-δ mRNA expression showed age dependence. Thus PKC-δ appears critical for the action of TDC in the adult colon, and its low expression in young animals may account for their inability to secrete in response to bile acids.

scholarly journals Cyclin A2-Cyclin-Dependent Kinase 2 Cooperates with the PLK1-SCFβ-TrCP1-EMI1-Anaphase-Promoting Complex/Cyclosome Axis To Promote Genome Reduplication in the Absence of Mitosis

2009 ◽  
Vol 29 (24) ◽  
pp. 6500-6514 ◽  
Author(s):  
Hoi Tang Ma ◽  
Yiu Huen Tsang ◽  
Miriam Marxer ◽  
Randy Y. C. Poon
Keyword(s):  
Genome Stability ◽  
Critical Role ◽  
Ectopic Expression ◽  
Specific Inhibitor ◽  
Genome Replication ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Cyclin E2 ◽  
Cyclin A2

ABSTRACT Limiting genome replication to once per cell cycle is vital for maintaining genome stability. Inhibition of cyclin-dependent kinase 1 (CDK1) with the specific inhibitor RO3306 is sufficient to trigger multiple rounds of genome reduplication. We demonstrated that although anaphase-promoting complex/cyclosome (APC/C) remained inactive during the initial G2 arrest, it was activated upon prolonged inhibition of CDK1. Using cellular biosensors and live-cell imaging, we provide direct evidence that genome reduplication was associated with oscillation of APC/C activity and nuclear-cytoplasmic shuttling of CDC6 even in the absence of mitosis at the single-cell level. Genome reduplication was abolished by ectopic expression of EMI1 or depletion of CDC20 or CDH1, suggesting the critical role of the EMI1-APC/C axis. In support of this, degradation of EMI1 itself and genome reduplication were delayed after downregulation of PLK1 and β-TrCP1. In the absence of CDK1 activity, activation of APC/C and genome reduplication was dependent on cyclin A2 and CDK2. Genome reduplication was then promoted by a combination of APC/C-dependent destruction of geminin (thus releasing CDT1), accumulation of cyclin E2-CDK2, and CDC6. Collectively, these results underscore the crucial role of cyclin A2-CDK2 in regulating the PLK1-SCFβ-TrCP1-EMI1-APC/C axis and CDC6 to trigger genome reduplication after the activity of CDK1 is suppressed.

Classical PKCs Regulate ADP-Induced Thromboxane Generation by Modulating Tyrosine Phosphorylation On Novel PKC Isoform Delta Through Shptp-1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1064-1064
Author(s):  
Dheeraj Bhavanasi ◽  
Carol T Dangelmaier ◽  
Jin Jianguo ◽  
Soochong Kim ◽  
Satya P. Kunapuli
Keyword(s):  
Tyrosine Phosphorylation ◽  
Conflicts Of Interest ◽  
Tyrosine Phosphatase ◽  
Src Family Kinases ◽  
Dependent Manner ◽  
Functional Responses ◽  
Pkc Isoforms ◽  
Adp Receptors ◽  
Gf 109203X

Abstract Abstract 1064 Introduction: Adenosine Di-phosphate (ADP) is stored in dense granules of platelets and is released upon platelet activation acting as a feedback activator by binding to G-protein coupled P2Y1 and P2Y12 receptors. ADP stimulation causes platelets to change shape, aggregate, release dense and a-granule contents and synthesize thromboxane A2 that can further act as a feedback activator potentiating platelet responses by binding to thromboxane receptor (TP). Protein kinase C is a serine threonine specific kinase that regulates multiple platelet functional responses. Specific PKC isoforms regulating platelet responses downstream of ADP receptors are not completely known. Aim: The aim of the current study is to elucidate the role of PKC isoforms in regulating ADP-induced platelet functional responses in platelets. Methods: We sought to delineate the mechanism of ADP-induced platelet responses by performing platelet aggregation (aggregometry), ATP secretion (luciferin-luciferase reaction) and thromboxane generation (ELISA kit measuring TxB2) in human or murine platelets by pre-incubating the platelets with control (DMSO) or inhibitors wherever mentioned. We also evaluated the role of PKCd to ADP-induced platelet responses by using murine platelets lacking PKCd. Background and Results: Murugappan et al have shown that PKCd was not activated downstream of ADP receptors based on the inability of ADP to cause threonine 507 phosphorylation on PKCd in platelets. However, studies from other labs have shown that PKCd can be activated when it is phosphorylated on its tyrosine residues. In the current study we show that, upon stimulation with 2MeSADP, PKCd is phosphorylated on tyrosine residue 311 in a time-dependent manner in platelets (Fig A). Also, ADP-induced thromboxane generation (Fig B) and ADP-induced thromboxane-mediated dense granule secretion were significantly inhibited in PKCd knockout murine platelets compared to those of wild type platelets. Similarly, thromboxane generation downstream of ADP receptors in human platelets pre-incubated with a PKCd inhibitor is significantly inhibited compared to control indicating a role for PKCd in mediating ADP-induced responses in platelets. Bynagari et al have shown that ADP-induced thromboxane generation is potentiated in the presence of the pan-PKC inhibitor, GF 109203X and the isoform regulating this effect is PKCe. We observed that pre-incubation of PKCe knockout murine platelets with GF 109203X further potentiated ADP-induced thromboxane generation suggesting that there are other PKC isoforms negatively regulating ADP-induced thromboxane generation. We show that this potentiating effect of thromboxane generation with GF 109203X in WT or PKCe KO murine platelets correlate with an increase in the phosphorylation of Y311 on PKCd (Fig C) suggesting that ADP-induced thromboxane generation is regulated through PKCd Y311 phosphorylation. Tyrosine phosphorylation on PKCd is mediated by Src family kinases (SFKs) as the phosphorylation is abolished with PP2, a SFK inhibitor and is only partially inhibited in Fyn knockout murine platelets suggesting that other SFKs also mediate this tyrosine phosphorylation. Surprisingly, pre-incubation of platelets with LY-333531, a classical PKC isoform (a/b) inhibitor potentiated PKCd Y311 phosphorylation (Fig D) as well as thromboxane generation (Fig E) downstream of ADP receptors suggesting a role for classical PKCs. Also, platelets pre-incubated with LY-333531 showed a decrease in the phosphorylation of SHPTP-1 (Fig F), a tyrosine phosphatase, rendering it active. The active SHPTP-1 phosphatase may dephosphorylate and activate SFKs, which can now phosphorylate PKCd on Y311 in platelets. Conclusions: In the current study, we report for the first time that the novel PKC isoform d is tyrosine phosphorylated downstream of ADP receptors through which it mediates ADP-induced thromboxane generation. We also show a novel role for classical PKC isoforms a/b in regulating tyrosine phosphorylation on novel isoform, PKCd possibly through the tyrosine phosphatase SHPTP-1 and Src family kinases in platelets. Disclosures: No relevant conflicts of interest to declare.

Essential activation of PKC-δ in opioid-initiated cardioprotection

2001 ◽  
Vol 280 (3) ◽  
pp. H1346-H1353 ◽  
Author(s):  
Ryan M. Fryer ◽  
Yigang Wang ◽  
Anna K. Hsu ◽  
Garrett J. Gross
Keyword(s):  
Opioid Receptor ◽  
Coronary Artery Occlusion ◽  
Artery Occlusion ◽  
Immunofluorescent Staining ◽  
Opioid Antagonist ◽  
Area At Risk ◽  
Receptor Stimulation ◽  
Gf 109203X ◽  
Δ Opioid Receptor

Stimulation of the δ1-opioid receptor confers cardioprotection to the ischemic myocardium. We examined the role of protein kinase C (PKC) after δ-opioid receptor stimulation with TAN-67 ord-Ala2-d-Leu5-enkephalin (DADLE) in a rat model of myocardial infarction induced by a 30-min coronary artery occlusion and 2-h reperfusion. Infarct size (IS) was determined by tetrazolium staining and expressed as a percentage of the area at risk (IS/AAR). Control animals, subjected to ischemia and reperfusion, had an IS/AAR of 59.9 ± 1.8. DADLE and TAN-67 administered before ischemia significantly reduced IS/AAR (36.9 ± 3.9 and 36.7 ± 4.7, respectively). The δ1-selective opioid antagonist 7-benzylidenenaltrexone (BNTX) abolished TAN-67-induced cardioprotection (54.4 ± 1.3). Treatment with the PKC antagonist chelerythrine completely abolished DADLE- (61.8 ± 3.2) and TAN-67-induced cardioprotection (55.4 ± 4.0). Similarly, the PKC antagonist GF 109203X completely abolished TAN-67-induced cardioprotection (54.6 ± 6.6). Immunofluorescent staining with antibodies directed against specific PKC isoforms was performed in myocardial biopsies obtained after 15 min of treatment with saline, chelerythrine, BNTX, or TAN-67 and chelerythrine or BNTX in the presence of TAN-67. TAN-67 induced the translocation of PKC-α to the sarcolemma, PKC-β1 to the nucleus, PKC-δ to the mitochondria, and PKC-ε to the intercalated disk and mitochondria. PKC translocation was abolished by chelerythrine and BNTX in TAN-67-treated rats. To more closely examine the role of these isoforms in cardioprotection, we utilized the PKC-δ selective antagonist rottlerin. Rottlerin abolished opioid-induced cardioprotection (48.9 ± 4.8) and PKC-δ translocation without affecting the translocation of PKC-α, -β1, or -ε. These results suggest that PKC-δ is a key second messenger in the cardioprotective effects of δ1-opioid receptor stimulation in rats.

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