scholarly journals Purinergic stimulation of rat cardiomyocytes induces tyrosine phosphorylation and membrane association of phospholipase Cγ: a major mechanism for InsP3 generation

1996 ◽  
Vol 318 (2) ◽  
pp. 723-728 ◽  
Author(s):  
Michel PUCEAT ◽  
Guy VASSORT
Keyword(s):  
Tyrosine Kinase ◽  
Time Course ◽  
Kinase Inhibitor ◽  
Extracellular Atp ◽  
Rat Cardiomyocytes ◽  
Major Mechanism ◽  
Adult Rat ◽  
Hydrolysis Of ◽  
Purinergic Stimulation ◽  
Stimulation Of

Phospholipase Cγ (PLCγ) expression and activation by a purinergic agonist were investigated in adult rat cardiomyocytes. PLCγ is expressed in isolated cardiomyocytes. Stimulation of cells with extracellular ATP induces a rapid increase in membrane-associated PLCγ immunoreactivity most probably due to redistribution of the lipase from the cytosol to the membrane. The purine triggers a significant phosphorylation on tyrosine residues of a cytosolic pool of PLCγ with a time course that correlates with that of translocation. Extracellular ATP also increases intracellular Ins(1,4,5)P3 content. All these events (translocation and phosphorylation of PLCγ, InsP3 formation) are blocked by genistein, a tyrosine kinase inhibitor. The purinergic effect on both PLCγ translocation and phosphorylation are Ca-sensitive. We thus propose that the purinergic stimulation activates a non-receptor tyrosine kinase that phosphorylates PLCγ in the presence of an increased Ca level and induces PLCγ redistribution to the membrane. There, PLCγ becomes activated leading to the hydrolysis of phosphatidylinositol diphosphate and in turn Ins(1,4,5)P3 formation. This cascade of events may play a significant role in the induction of arrhythmogenesis by purinergic agonists.

scholarly journals Adenoprotection of the heart involves phospholipase C-induced activation and translocation of PKC-ε to RACK2 in adult rat and mouse

2009 ◽  
Vol 297 (2) ◽  
pp. H718-H725 ◽  
Author(s):  
Richard A. Fenton ◽  
Satoshi Komatsu ◽  
Mitsuo Ikebe ◽  
Lynne G. Shea ◽  
James G. Dobson
Keyword(s):  
Confocal Microscopy ◽  
Phospholipase C ◽  
Adrenergic Agonist ◽  
Rat Cardiomyocytes ◽  
Scanning Confocal Microscopy ◽  
Adult Rat ◽  
Sds Page ◽  
T Tubules ◽  
Isolated Rat ◽  
Stimulation Of

Adenosine protects the heart from adrenergic overstimulation. This adenoprotection includes the direct anti-adrenergic action via adenosine A1 receptors (A1R) on the adrenergic signaling pathway. An indirect A1R-induced attenuation of adrenergic responsiveness involves the translocation of PKC-ε to t-tubules and Z-line of cardiomyocytes. We investigated with sarcomere imaging, immunocytochemistry imaging, and coimmunoprecipitation (co-IP) whether A1R activation of PKC-ε induces the kinase translocation to receptor for activated C kinase 2 (RACK2) in isolated rat and mouse hearts and whether phospholipase C (PLC) is involved. Rat cardiomyocytes were treated with the A1R agonist chlorocyclopentyladenosine (CCPA) and exposed to primary PKC-ε and RACK2 antibodies with secondaries conjugated to Cy3 and Cy5 (indodicarbocyanine), respectively. Scanning confocal microscopy showed that CCPA caused PKC-ε to reversibly colocalize with RACK2 within 3 min. Additionally, rat and mouse hearts were perfused and stimulated with CCPA or phenylisopropyladenosine to activate A1R, or with phorbol 12-myristate 13-acetate to activate PKC. RACK2 was immunoprecipitated from heart extracts and resolved with SDS-PAGE. Western blotting showed that CCPA, phenylisopropyladenosine, and phorbol 12-myristate 13-acetate in the rat heart increased the PKC-ε co-IP with RACK2 by 186, 49, and >1,000%, respectively. The A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine prevented the CCPA-induced co-IP with RACK2. In mouse hearts, CCPA increased the co-IP of PKC-ε with RACK2 by 61%. With rat cardiomyocytes, the β-adrenergic agonist isoproterenol increased sarcomere shortening by 177%. CCPA reduced this response by 47%, an action inhibited by the PLC inhibitor U-73122 and 8-cyclopentyl-1,3-dipropylxanthine. In conclusion, A1R stimulation of the heart is associated with PLC-initiated PKC-ε translocation and association with RACK2.

Activation of pp60(src) is critical for stretch-induced orienting response in fibroblasts

1999 ◽  
Vol 112 (9) ◽  
pp. 1365-1373 ◽  
Author(s):  
X. Sai ◽  
K. Naruse ◽  
M. Sokabe
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Time Course ◽  
Kinase Inhibitor ◽  
Orienting Response ◽  
Cyclic Stretch ◽  
Wild Type ◽  
Herbimycin A ◽  
Molecular Masses

When subjected to uni-axial cyclic stretch (120% in length, 1 Hz), fibroblasts (3Y1) aligned perpendicular to the stretch axis in a couple of hours. Concomitantly with this orienting response, protein tyrosine phosphorylation of cellular proteins (molecular masses of approximately 70 kDa and 120–130 kDa) increased and peaked at 30 minutes. Immuno-precipitation experiments revealed that paxillin, pp125(FAK), and pp130(CAS) were included in the 70 kDa, and 120–130 kDa bands, respectively. Treatment of the cells with herbimycin A, a tyrosine kinase inhibitor, suppressed the stretch induced tyrosine phosphorylation and the orienting response suggesting that certain tyrosine kinases are activated by stretch. We focused on pp60(src), the most abundant tyrosine kinase in fibroblasts. The kinase activity of pp60(src) increased and peaked at 20 minutes after the onset of cyclic stretch. Treatment of the cells with an anti-sense S-oligodeoxynucleotide (S-ODN) against pp60(src), but not the sense S-ODN, inhibited the stretch induced tyrosine phosphorylation and the orienting response. To further confirm the involvement of pp60(src), we performed the same sets of experiments using c-src-transformed 3Y1 (c-src-3Y1) fibroblasts. Cyclic stretch induced a similar orienting response in c-src-3Y1 to that in wild-type 3Y1, but with a significantly faster rate. The time course of the stretch-induced tyrosine phosphorylation was also much faster in c-src-3Y1 than in 3Y1 fibroblasts. These results strongly suggest that cyclic stretch induces the activation of pp60(src) and that pp60(src) is indispensable for the tyrosine phosphorylation of pp130(CAS), pp125(FAK) and paxillin followed by the orienting response in 3Y1 fibroblasts.

Stimulation of tyrosine phosphorylation of distinct proteins in response to antibody-mediated ligation and clustering of alpha 3 and alpha 6 integrins

1995 ◽  
Vol 108 (3) ◽  
pp. 1165-1174
Author(s):  
K. Jewell ◽  
C. Kapron-Bras ◽  
P. Jeevaratnam ◽  
S. Dedhar
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Cell Attachment ◽  
Umbilical Vein ◽  
Integrin Receptors ◽  
Human Prostate Carcinoma ◽  
Beta 1 Integrin ◽  
Umbilical Vein Endothelial Cells ◽  
Stimulation Of

The interaction of cells with components of the extracellular matrix through their integrin receptors results in the stimulation of tyrosine phosphorylation of several proteins, suggesting that these receptors play a key role in signal transduction. Here we report that antibody-mediated ligation and clustering of alpha 3 beta 1 and alpha 6 beta 1/alpha 6 beta 4 integrins resulted in the stimulation of tyrosine phosphorylation of proteins that are specific for each heterodimer. Thus, ligation and clustering of the alpha 3 beta 1 integrin on human prostate carcinoma cells (PC-3) and human umbilical vein endothelial cells (HUVEC) with anti-alpha 3 antibodies resulted in the stimulation of tyrosine phosphorylation of a 55 kDa protein. In contrast, ligation and clustering of the alpha 6 beta 1 integrin on these cells with anti-alpha 6 antibody resulted in the dramatic stimulation of tyrosine phosphorylation of a 90 kDa protein in addition to a 52 kDa protein, and ligation and clustering of alpha 5 beta 1 on HUVEC did not result in the apparent stimulation of tyrosine phosphorylation of any proteins. Clustering with anti-beta 1 antibodies triggered the tyrosine phosphorylation of all of these proteins, whereas ligation and clustering of PC-3 cells with an anti-beta 4 antibody resulted in the tyrosine phosphorylation of a distinct 62 kDa protein. Since the PC-3 cells express both alpha 6 beta 1 and alpha 6 beta 4, these data suggest that these two receptors can transduce distinct signals. All of the phosphorylations could be inhibited by treating the cells with Genistein, a tyrosine kinase inhibitor. Antibody-mediated ligation and clustering of integrins on the two types of cells did not result in the stimulation of tyrosine phosphorylation of pp125 focal adhesion kinase, although this was observed upon cell attachment and spreading on fibronectin, laminin and anti-alpha 3 monoclonal antibody. Collectively, these data demonstrate that cross-linking of different integrin heterodimers can stimulate tyrosine kinase activities, leading to the phosphorylation of distinct proteins, which are also different from those observed when cells are allowed to spread on a matrix.

scholarly journals Tyrosine phosphorylation and acetylcholine receptor cluster formation in cultured Xenopus muscle cells.

1993 ◽  
Vol 120 (1) ◽  
pp. 185-195 ◽  
Author(s):  
L P Baker ◽  
H B Peng
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Acetylcholine Receptor ◽  
Time Course ◽  
Kinase Inhibitor ◽  
Cluster Formation ◽  
Protein S ◽  
Muscle Cells ◽  
Nerve Muscle ◽  
Achr Clustering

Aggregation of the nicotinic acetylcholine receptor (AChR) at sites of nerve-muscle contact is one of the earliest events to occur during the development of the neuromuscular junction. The stimulus presented to the muscle by nerve and the mechanisms underlying postsynaptic differentiation are not known. The purpose of this study was to examine the distribution of phosphotyrosine (PY)-containing proteins in cultured Xenopus muscle cells in response to AChR clustering stimuli. Results demonstrated a distinct accumulation of PY at AChR clusters induced by several stimuli, including nerve, the culture substratum, and polystyrene microbeads. AChR microclusters formed by external cross-linking did not show PY colocalization, implying that the accumulation of PY in response to clustering stimuli was not due to the aggregation of basally phosphorylated AChRs. A semi-quantitative determination of the time course for development of PY labeling at bead contacts revealed early PY accumulation within 15 min of contact before significant AChR aggregation. At later stages (within 15 h), the AChR signal came to approximate the PY signal. We have reported the inhibition of bead-induced AChR clustering in response to beads by a tyrphostin tyrosine kinase inhibitor (RG50864) (Peng, H. B., L. P. Baker, and Q. Chen. 1991. Neuron. 6:237-246). RG50864 also inhibited PY accumulation at bead contacts, providing evidence for tyrosine kinase activation in response to the bead stimulus. These results suggest that tyrosine phosphorylation may play an important role in the generative stages of cluster formation, and may involve protein(s) other than or in addition to AChRs.

scholarly journals Ca+2/Calmodulin-Dependent Protein Kinase Mediates Glucose Toxicity-Induced Cardiomyocyte Contractile Dysfunction

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Rong-Huai Zhang ◽  
Haitao Guo ◽  
Machender R. Kandadi ◽  
Xiao-Ming Wang ◽  
Jun Ren
Keyword(s):  
Kinase Inhibitor ◽  
Maximal Velocity ◽  
Cam Kinase ◽  
Glucose Toxicity ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Cardiac Contractile ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
The Impact

(1) Hyperglycemia leads to cytotoxicity in the heart. Although several theories are postulated for glucose toxicity-induced cardiomyocyte dysfunction, the precise mechanism still remains unclear. (2) This study was designed to evaluate the impact of elevated extracellular Ca2+on glucose toxicity-induced cardiac contractile and intracellular Ca2+anomalies as well as the mechanism(s) involved with a focus on Ca2+/calmodulin (CaM)-dependent kinase. Isolated adult rat cardiomyocytes were maintained in normal (NG, 5.5 mM) or high glucose (HG, 25.5 mM) media for 6-12 hours. Contractile indices were measured including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time-to-PS (TPS), and time-to-90% relengthening (TR90). (3) Cardiomyocytes maintained with HG displayed abnormal mechanical function including reduced PS, ±dL/dt, and prolonged TPS, TR90and intracellular Ca2+clearance. Expression of intracellular Ca2+regulatory proteins including SERCA2a, phospholamban and Na+-Ca2+exchanger were unaffected whereas SERCA activity was inhibited by HG. Interestingly, the HG-induced mechanical anomalies were abolished by elevated extracellular Ca2+(from 1.0 to 2.7 mM). Interestingly, the high extracellular Ca2+-induced beneficial effect against HG was abolished by the CaM kinase inhibitor KN93. (4) These data suggest that elevated extracellular Ca2+protects against glucose toxicity-induced cardiomyocyte contractile defects through a mechanism associated with CaM kinase.

scholarly journals Acute α1-adrenergic stimulation of cardiac protein synthesis may involve increased intracellular pH and protein kinase activity

1991 ◽  
Vol 273 (2) ◽  
pp. 347-353 ◽  
Author(s):  
S J Fuller ◽  
C J Gaitanaki ◽  
R J Hatchett ◽  
P H Sugden
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Intracellular Ph ◽  
Polynomial Regression ◽  
Kinase Inhibitor ◽  
Adenine Nucleotide ◽  
Protein Kinase Activity ◽  
Adrenergic Stimulation ◽  
Rat Cardiomyocytes ◽  
Stimulation Of

In the presence of 5 microM-DL-propranolol and in HCO3(-)-containing buffers, 1 microM-adrenaline acutely stimulated protein synthesis by about 25% in the anterogradely perfused rat heart. This stimulation was opposed by low (1-10 nM) concentrations of prazosin, but not by similar concentrations of yohimbine, suggesting involvement of the alpha 1-adrenoceptor. Under the same conditions, adrenaline raised intracellular pH (pHi) by about 0.1 unit. The increase in pHi induced by adrenaline was prevented by 5 nM-prazosin, but not by 5 nM-yohimbine, again suggesting involvement of the alpha 1-adrenoceptor. Since an increase in pHi stimulates protein synthesis in the heart [Sugden & Fuller (1991) Biochem. J. 273, 339-346], the increase in pHi induced by adrenaline may be involved in its stimulation of protein synthesis. Adrenaline also increased phosphocreatine concentrations. As discussed, the increase in pHi induced by adrenaline may be responsible for this effect. Using second-order polynomial regression analysis, we showed that rates of protein synthesis were significantly correlated (P less than 0.0001) with phosphocreatine concentrations. We discuss two possible reasons for this correlation: (i) increases in pHi stimulate protein synthesis and separately raise phosphocreatine concentrations, or (ii) the increase in protein synthesis rates is a consequence of the raised phosphocreatine concentrations induced by the increase in pHi. Rates of protein synthesis were not significantly correlated with ATP/ADP concentration ratios, nor with any of the following: ATP, ADP, AMP or total adenine nucleotide concentrations. In freshly isolated adult rat cardiomyocytes, the protein kinase inhibitor staurosporine (1 microM) prevented stimulation of protein synthesis by 0.3 microM-adrenaline (and by 1 microM-phorbol 12-myristate 13-acetate or 1 m-unit of insulin/ml). The results are discussed within a mechanistic framework initiated by stimulation of the hydrolysis of membrane phospholipids by alpha 1-adrenergic agonists.

STIMULATION OF SYNTHESIS OF GLYCOPROTEIN ENZYMES IN THE RAT UTERUS BY OESTRADIOL

1968 ◽  
Vol 42 (2) ◽  
pp. 261-NP ◽  
Author(s):  
F. A. DUGAN ◽  
B. RADHAKRISHNAMURTHY ◽  
R. A. RUDMAN ◽  
G. S. BERENSON
Keyword(s):  
Total Activity ◽  
Alkaline Phosphatases ◽  
Rat Uterus ◽  
Oestrogen Treatment ◽  
Immature Rat ◽  
Adult Rat ◽  
Electrophoretic Patterns ◽  
Electrophoretic Mobilities ◽  
Hydrolysis Of ◽  
Stimulation Of

SUMMARY Glycoproteins from immature and immature, oestrogen-stimulated and adult rat uteri were isolated and analysed by chemical and gel electrophoretic methods. Esterase, acid phosphatase, alkaline phosphatase and peroxidase activities were found. Changes in electrophoretic mobilities of certain enzyme bands in polyacrylamide gel were also observed after hydrolysis of the preparations with neuraminidase. These latter observations and chemical analyses provide additional evidence of the carbohydrate nature of the enzymes. The influence of 17β-oestradiol on immature rat uteri caused a significant increase in total protein and sialic acid per uterus compared with controls. Oestrogen treatment also resulted in an increase in the total activity of esterase and acid and alkaline phosphatases per uterus, but there was no increase in specific activities. Observations of electrophoretic patterns of glycoprotein preparations from untreated and oestrogen-stimulated, immature uteri did not show the evolution to a more adult pattern by oestrogen stimulation. These studies show that stimulation with oestrogen increases the synthesis of glycoprotein in the immature rat uterus. Factors which are involved in the more intricate control of glycoprotein biosynthesis need to be elucidated.

Role of tyrosine kinase and p44/42 MAPK in D2-like receptor-mediated stimulation of Na+, K+-ATPase in kidney

2002 ◽  
Vol 282 (4) ◽  
pp. F697-F702 ◽  
Author(s):  
Vihang Narkar ◽  
Tahir Hussain ◽  
Mustafa Lokhandwala
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Mapk Pathway ◽  
Rat Kidney ◽  
Receptor Activation ◽  
Proximal Tubules ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Stimulation Of

Our laboratory has shown that dopamine D2-like receptor activation causes stimulation of Na+, K+-ATPase (NKA) activity in the proximal tubules of the rat kidney. The present study was designed to investigate the cellular signaling mechanisms mediating this response to D2-like receptor activation. We measured the stimulation of NKA activity by bromocriptine (D2-like receptor agonist) in the absence and presence of PD-98059 [p44/42 mitogen-activated protein kinase (MAPK) kinase inhibitor] and genistein (tyrosine kinase inhibitor) in renal proximal tubules. Both agents inhibited bromocriptine-mediated stimulation of NKA, suggesting the involvement of p44/42 MAPK and tyrosine kinase in this response. Additionally, we found that bromocriptine increased the phosphorylation of p44/42 MAPK in the proximal tubules, which was blocked by PD-98059 and genistein. These results show that D2-like receptor activation causes stimulation of NKA activity by means of a tyrosine kinase-p44/42 MAPK pathway in the proximal tubules of the kidney.

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