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.

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.

Protein kinase Cε and the antiadrenergic action of adenosine in rat ventricular myocytes

2004 ◽  
Vol 287 (4) ◽  
pp. H1721-H1729 ◽  
Author(s):  
Koji Miyazaki ◽  
Satoshi Komatsu ◽  
Mitsuo Ikebe ◽  
Richard A. Fenton ◽  
James G. Dobson
Keyword(s):  
Electrical Stimulation ◽  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Adrenergic Agonist ◽  
Inhibitory Peptide ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Tubular System ◽  
Stimulation Of

Adenosine-induced antiadrenergic effects in the heart are mediated by adenosine A1 receptors (A1R). The role of PKCε in the antiadrenergic action of adenosine was explored with adult rat ventricular myocytes in which PKCε was overexpressed. Myocytes were transfected with a pEGFP-N1 vector in the presence or absence of a PKCε construct and compared with normal myocytes. The extent of myocyte shortening elicited by electrical stimulation of quiescent normal and transfected myocytes was recorded with video imaging. PKCε was found localized primarily in transverse tubules. The A1R agonist chlorocyclopentyladenosine (CCPA) at 1 μM rendered an enhanced localization of PKCε in the t-tubular system. The β-adrenergic agonist isoproterenol (Iso; 0.4 μM) elicited a 29–36% increase in myocyte shortening in all three groups. Although CCPA significantly reduced the Iso-produced increase in shortening in all three groups, the reduction caused by CCPA was greatest with PKCε overexpression. The CCPA reduction of the Iso-elicited shortening was eliminated in the presence of a PKCε inhibitory peptide. These results suggest that the translocation of PKCε to the t-tubular system plays an important role in A1R-mediated antiadrenergic actions in the heart.

Subcellular distribution of sodium pump isoform subunits in mammalian cardiac myocytes

1996 ◽  
Vol 270 (4) ◽  
pp. C1221-C1227 ◽  
Author(s):  
A. A. McDonough ◽  
Y. Zhang ◽  
V. Shin ◽  
J. S. Frank
Keyword(s):  
Guinea Pig ◽  
Driving Force ◽  
Human Heart ◽  
Sodium Pump ◽  
Immunogold Labeling ◽  
Guinea Pig Heart ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Na Pump ◽  
T Tubules

The cardiac Na+ pump (Na+ -K+ -ATPase) provides the driving force for the Na+/Ca2+ exchanger, a determinant of intracellular Ca2+ stores. Three Na+ pump alpha-catalytic subunit isoforms are expressed in human heart, alpha1 and alpha2 are expressed in rat heart, and only alpha1 is expressed in guinea pig heart. The objective of this study was to determine whether there are isoform-specific patterns of expression in the transverse tubules (T tubules) vs. the peripheral sarcolemma. In adult rat cardiomyocytes, anti-alpha1-specific antibodies labeled the T tubules more intensely than the peripheral sarcolemma, in which labeling was patchy, the same pattern reported for distribution of the Na+/Ca2+ exchanger (J. S. Frank, G. Mottino, D. Reid, R. S. Molday, and K. D. Philipson, J. Cell Biol. 117: 337-345, 1992), whereas anti-alpha2- and anti-beta1-antibodies uniformly labeled T tubules and peripheral sarcolemma. In guinea pig cardiomyocytes, an anti-alpha-antibody against an extracellular epitope evenly labeled the peripheral sarcolemma and T tubules, and immunogold labeling demonstrated coincidence of alpha-subunits and intramembranous particles in sarcolemma. In summary, Na+ pumps are located in both peripheral sarcolemma and T tubules of cardiomyocytes expressing either multiple or single Na+ pump isoforms.

Spontaneous and propagated calcium release in isolated cardiac myocytes viewed by confocal microscopy

1992 ◽  
Vol 262 (3) ◽  
pp. C731-C742 ◽  
Author(s):  
D. A. Williams ◽  
L. M. Delbridge ◽  
S. H. Cody ◽  
P. J. Harris ◽  
T. O. Morgan
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Calcium Release ◽  
Laser Scanning Confocal Microscopy ◽  
Cardiac Cells ◽  
Rat Cardiomyocytes ◽  
Scanning Confocal Microscopy ◽  
Time Period ◽  
Isolated Cardiac Myocytes ◽  
Cell Depolarization

Laser scanning confocal microscopy of the Ca(2+)-sensitive fluorophore fluo-3 has been used to investigate spontaneous and propagated calcium release at high temporal and spatial resolution in enzymatically dispersed rat cardiomyocytes. Waves of fluorescence which propagated throughout the cytosol were evident in spontaneously contracting cardiac cells containing fluo-3, but not in cells containing Ca(2+)-insensitive fluorophores [2',7'-bis (carboxyethyl)-5,6-carboxyfluorescein, SNARF-1, rhodamine-123, or tetramethylrhodamine-labeled dextran]. These waves represent localized areas of elevated [Ca2+] [975 +/- 13 (SE) nM, range 800-1,500 nM; n = 16 cells]. Ca2+ waves were initiated by the spontaneous release of Ca2+ from the sarcoplasmic reticulum (SR) and propagated through cells at rates of 50-150 microns/s. Ca2+ waves were usually initiated at the cell ends, but multiple and variable initiation foci were observed in some cells. Where waves intersected within a single cell there was extinction of wave propagation, confirming the SR as the direct source of Ca2+ and revealing a refractory period in SR Ca2+ release. In some cells high-frequency Ca2+ waves lead to synchronized elevation of [Ca2+] throughout the entire cytosol and within the time period associated with cell depolarization. These observations support the hypothesis that some cardiac arrhythmias are initiated by spontaneous and propagated Ca2+ release and involve subsequent depolarization, global elevation of intracellular [Ca2+], and cell contraction.

Contraction-independent effects of catecholamines on glucose transport in isolated rat cardiomyocytes

1996 ◽  
Vol 270 (4) ◽  
pp. C1204-C1210 ◽  
Author(s):  
Y. Fischer ◽  
J. Thomas ◽  
G. D. Holman ◽  
H. Rose ◽  
H. Kammermeier
Keyword(s):  
Cell Surface ◽  
Glucose Transport ◽  
Basal Cells ◽  
Adrenergic Agonist ◽  
Rat Cardiomyocytes ◽  
Maximal Stimulation ◽  
Two Phases ◽  
Independent Effects ◽  
Beta Adrenergic Agonist ◽  
Isolated Rat

The effects of catecholamines on glucose transport were studied in noncontracting isolated rat cardiomyocytes. alpha-Adrenergic treatment (phenylephrine, or norepinephrine + propranolol) led to an approximately fourfold stimulation of glucose transport in basal cells (no insulin). The effect of phenylephrine was suppressed by the alpha 2-antagonist yohimbine or the beta-antagonist propranolol. The beta-adrenergic agonist isoproterenol partially counteracted the action of phenylephrine (but not that of insulin). Phenylephrine increased glucose transport in two phases with apparent half times of 3.2 and 13.0 min, respectively. Correspondingly, different EC50 values were found after 10 and 45 min on phenylephrine addition (5.0 +/- 1.9 vs. 31.6 +/- 9.6 microM, respectively). Maximal stimulation by phenylephrine was at least partially additive to that of insulin and of other stimulators of glucose transport (e.g., H2O2, vanadate, lithium). Phenylephrine significantly increased the level of cell surface glucose carriers GLUT-1 (1.54-fold) and GLUT-4 (1.78-fold), as assessed by using the specific photolabel 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]- 1,3-bis(D-mannos-4-yloxy)propyl-2-amine. In conclusion, catecholamines stimulate cardiomyocyte glucose transport through alpha 1-adrenergic receptors independently or downstream of a contraction-evoked stimulus. This effect is at least partially explained by a recruitment of glucose transporters to the cell surface. The mechanism(s) and/or signals involved differ from those triggered by insulin and insulinomimetic agents.

scholarly journals Identification and Characterization of Novel Perivascular Adventitial Cells in the Whole Mount Mesenteric Branch Artery Using Immunofluorescent Staining and Scanning Confocal Microscopy Imaging

2012 ◽  
Vol 2012 ◽  
pp. 1-6
Author(s):  
Chandra Somasundaram ◽  
Rahul K. Nath ◽  
Richard D. Bukoski ◽  
Debra I. Diz
Keyword(s):  
Confocal Microscopy ◽  
Neurite Growth ◽  
Immunofluorescent Staining ◽  
Neural Cells ◽  
Microscopy Imaging ◽  
Stress Injury ◽  
Scanning Confocal Microscopy ◽  
Adult Rat ◽  
Calcium Sensing ◽  
Whole Mount

A novel perivascular adventitial cell termed, adventitial neuronal somata (ANNIES) expressing the neural cell adhesion molecule (NCAM) and the vasodilator neuropeptide, calcitonin gene-related peptide (CGRP), exists in the adult rat mesenteric branch artery (MBA) in situ. In addition, we have previously shown that ANNIES coexpress CGRP and NCAM. We now show that ANNIES express the neurite growth marker, growth associated protein-43(Gap-43), palladin, and the calcium sensing receptor (CaSR), that senses changes in extracellular Ca(2+) and participates in vasodilator mechanisms. Thus, a previously characterized vasodilator, calcium sensing autocrine/paracrine system, exists in the perivascular adventitia associated with neural-vascular interface. Images of the whole mount MBA segments were analyzed under scanning confocal microscopy. Confocal analysis showed that the Gap-43, CaSR, and palladin were present in ANNIES about 37 ± 4%, 94 ± 6%, and 80 ± 10% respectively, comparable to CGRP (100%). Immunoblots from MBA confirmed the presence of Gap-43 (48 kD), NCAM (120 and 140 kD), and palladin (90–92 and 140 kD). In summary, CGRP, and NCAM-containing neural cells in the perivascular adventitia also express palladin and CaSR, and coexpress Gap-43 which may participate in response to stress/injury and vasodilator mechanisms as part of a perivascular sensory neural network.

scholarly journals Unexpected anti-hypertrophic responses to low-level stimulation of protease-activated receptors in adult rat cardiomyocytes

2014 ◽  
Vol 387 (10) ◽  
pp. 1001-1007
Author(s):  
Anke C. Fender ◽  
Goran Pavic ◽  
Grant R. Drummond ◽  
Gregory J. Dusting ◽  
Rebecca H. Ritchie
Keyword(s):  
Protease Activated Receptors ◽  
Rat Cardiomyocytes ◽  
Low Level ◽  
Adult Rat ◽  
Stimulation Of

Synergistic interaction of glucose and neurohumoral agonists to stimulate islet phosphoinositide hydrolysis

1995 ◽  
Vol 269 (3) ◽  
pp. E575-E582 ◽  
Author(s):  
G. G. Kelley ◽  
K. C. Zawalich ◽  
W. S. Zawalich
Keyword(s):  
Insulin Secretion ◽  
Phospholipase C ◽  
Insulin Release ◽  
Synergistic Interaction ◽  
Highly Sensitive ◽  
Isolated Rat Islets ◽  
Pi Hydrolysis ◽  
First Time ◽  
Isolated Rat ◽  
Stimulation Of

The interaction between neurohumoral agonists and glucose to stimulate phosphoinositide (PI)-specific phospholipase C (PLC) and insulin release was examined. In freshly isolated rat islets, maximal glucose (40 mM), cholecystokinin (CCK; 300 nM), or carbachol (CCh; 1 mM) stimulated PI hydrolysis 6.5-, 9.8-, and 5.7-fold, respectively, above basal. The combination of glucose and CCK or of glucose and CCh, but not of CCK and CCh, synergistically increased PI hydrolysis 23.2- and 21.6-fold, respectively, indicating that these secretagogues activate PLC by distinct pathways and that there is an interaction between them. This synergy was maximal at physiological concentrations of stimulatory glucose (8-10 mM) and was paralleled by a marked synergistic stimulation of insulin secretion. The enhanced PI response was partially Ca2+ dependent and may involve the activation of distinct isozymes of PLC, which we identify in islets. These studies demonstrate for the first time a unique and highly sensitive synergistic interaction between glucose and neurohumoral agonists to stimulate PI hydrolysis, and they suggest that enhanced PI hydrolysis is important in the potentiation of glucose- and neurohumoral-stimulated insulin secretion.

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