Abstract 1078: β-Adrenergic Stimulation of Ca/Calmodulin-Dependent Protein Kinase II (CaMKII) Overexpressing Myocytes Increases Sarcoplasmic Reticulum (SR) Calcium Leak Causing KN-93 Sensitive Arrhythmias

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Daniel P Wadsack ◽  
Michael Kohlhaas ◽  
Adam G Rokita ◽  
Stefan Neef ◽  
Lars S Maier
Keyword(s):  
Control Level ◽  
Adrenergic Stimulation ◽  
Dependent Protein Kinase ◽  
Inotropic Effects ◽  
Positive Inotropic Effects ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
Ca Homeostasis ◽  
Sr Calcium Leak ◽  
Inotropic Stimulation

CaMKII is associated with hypertrophy, heart failure and alters intracellular Ca homeostasis. An increased SR Ca leak due to phosphorylation of SR Ca release channels by CaMKII leads to decreased SR Ca content and impaired contractility. This loss of Ca from the SR may also contribute to arrhythmias. We investigated whether β-adrenergic stimulation with isoproterenol (ISO) normalizes SR Ca content and whether inhibiting CaMKII reduces arrhythmias. CaMKII-overexpressing rabbit and mouse myocytes were investigated. Cell shortening, Ca fluorescence (fluo-3) and the incidence of arrhythmias were assessed. An arrhythmia-score differentiated between: early-spike-arrhythmias (ESA), late-spike-arrhythmias (LSA) and permanent arrhythmias (PA). ISO (37°C) had significantly different effects on myocytes with acute (24 h, rabbit, n=34) or chronic (22 w, mouse, n=34) CaMKII overexpression vs corresponding control myocytes (LacZ, n=21 or WT n=34). CaMKII overexpression lead to an ISO concentration-dependent (10 −10 -10 −5 mol/L) inotropic but compared to WT (or LacZ, respectively) impaired shortening and Ca transients (two-way ANOVA, P <0.05). A similar difference between CaMKII-overexpressing (n=17) and WT (n=19) myocytes was also seen during a shortening-frequency protocol (stepwise increase from 0.1– 4 Hz, two-way ANOVA, P <0.05). Arrhythmias spontaneously occurred in CaMKII-overexpressing mouse myocytes. With β-inotropic stimulation (10 −6 mol/L ISO) arrhythmias were increased 6.4-fold. Appearance of ESA and PA could be significantly reduced by KN-93 (1 μmol/L). At a basal stimulation rate of 1 Hz and 10 −7 mol/L ISO, PA could be dramatically reduced by half from control-level 21.43% (KN-92, inactive derivative, n=42) down to 10.87% (KN-93, n=46) arrhythmic events. ESA could be reduced almost 4-fold from 16.67% (KN-92) to 4.35% in the presence of KN-93. We conclude from these data that increasing ISO concentrations exerts positive inotropic effects but cannot normalize altered Ca handling in CaMKII-overexpressing myocytes. This may be due to an increased SR Ca leak under these conditions thus contributing to the arrhythmias observed. CaMKII inhibition clearly can reduce arrhythmias in the presence of β-adrenergic stimulation with ISO.

scholarly journals Effects of increased systolic Ca2+ and phospholamban phosphorylation during β-adrenergic stimulation on Ca2+ transient kinetics in cardiac myocytes

2011 ◽  
Vol 301 (4) ◽  
pp. H1570-H1578 ◽  
Author(s):  
Steve R. Roof ◽  
Thomas R. Shannon ◽  
Paul M. L. Janssen ◽  
Mark T. Ziolo
Keyword(s):  
Adrenergic Stimulation ◽  
Transient Kinetics ◽  
Dependent Protein Kinase ◽  
Time To Peak ◽  
Decline Rate ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
Camkii Activation ◽  
Phospholamban Phosphorylation ◽  
Concentration Levels

Previous studies demonstrated higher systolic intracellular Ca2+ concentration ([Ca2+]i) amplitudes result in faster [Ca2+]i decline rates, as does β-adrenergic (β-AR) stimulation. The purpose of this study is to determine the major factor responsible for the faster [Ca2+]i decline rate with β-AR stimulation, the increased systolic Ca2+ concentration levels, or phosphorylation of phospholamban. Mouse myocytes were perfused under basal conditions [1 mM extracellular Ca2+ concentration ([Ca2+]o)], followed by high extracellular Ca2+ (3 mM [Ca2+]o), washout with 1 mM [Ca2+]o, followed by 1 μM isoproterenol (ISO) with 1 mM [Ca2+]o. ISO increased Ser16 phosphorylation compared with 3 mM [Ca2+]o, whereas Thr17 phosphorylation was similar. Ca2+ transient (CaT) (fluo 4) data were obtained from matched CaT amplitudes with 3 mM [Ca2+]o and ISO. [Ca2+]i decline was significantly faster with ISO compared with 3 mM [Ca2+]o. Interestingly, the faster decline with ISO was only seen during the first 50% of the decline. CaT time to peak was significantly faster with ISO compared with 3 mM [Ca2+]o. A Ca2+/calmodulin-dependent protein kinase (CAMKII) inhibitor (KN-93) did not affect the CaT decline rates with 3 mM [Ca2+]o or ISO but normalized ISO's time to peak with 3 mM [Ca2+]o. Thus, during β-AR stimulation, the major factor for the faster CaT decline is due to Ser16 phosphorylation, and faster time to peak is due to CAMKII activation.

Cardiac myocyte calcium transport in phospholamban knockout mouse: relaxation and endogenous CaMKII effects

1998 ◽  
Vol 274 (4) ◽  
pp. H1335-H1347 ◽  
Author(s):  
Li Li ◽  
Guoxiang Chu ◽  
Evangelia G. Kranias ◽  
Donald M. Bers
Keyword(s):  
Cardiac Myocyte ◽  
Ventricular Myocytes ◽  
Flux Analysis ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Ca Transport ◽  
Pump Activity ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
Exchange Activity

Increases in heart rate are accompanied by acceleration of relaxation. This effect is apparent at the single myocyte level and depends on sarcoplasmic reticulum (SR) Ca transport and Ca/calmodulin dependent protein kinase [CaMKII; see R. A. Bassani, A. Mattiazzi, and D. M. Bers. Am. J. Physiol. 268 ( Heart Circ. Physiol. 37): H703–H712, 1995]. Because phosphorylation of phospholamban (PLB) by CaMKII can stimulate SR Ca transport, it is a plausible candidate mechanism. We examined this issue using ventricular myocytes isolated from wild-type (WT) mice and those in which the PLB gene was ablated by gene targeting (PLB-KO). During steady-state (SS) stimulation, twitch relaxation and intracellular Ca concentration ([Ca]i) decline were significantly faster than after a rest in both WT and PLB-KO myocytes. Furthermore, the CaMKII inhibitor KN-93 (1 μM) abolished the stimulation-dependent acceleration of twitch [Ca]i decline in PLB-KO. This indicates that neither PLB nor its phosphorylation are required for the CaMKII-dependent acceleration of the SS twitch [Ca]i decline and relaxation. Other quantitative aspects of Ca transport in WT and PLB-KO myocytes were also examined. As expected, the time constant (τ) of [Ca]i decline during the SS twitch is much faster in PLB-KO than in WT myocytes (112 ± 6 vs. 188 ± 14 ms, P < 0.0001). There was also an increase in SS SR Ca load, based on the change of [Ca]i during rapid caffeine-induced contractures (CafC) with Na/Ca exchange blocked (565 ± 74 nM for WT, 1118 ± 133 nM for PLB-KO, P < 0.01). Accounting for cytosolic Ca buffering, this implies a 37% increase in SR Ca content. The τ for [Ca]idecline of the CafC with Na present indicated slower extrusion by Na/Ca exchange in the PLB-KO mouse (2.2 ± 0.2 s in WT vs. 3.2 ± 0.2 s in PLB-KO, P < 0.01), although exchanger protein expression was unchanged. Integrated Ca flux analysis in WT and PLB-KO myocytes, respectively, shows that 90 and 96% of Ca during twitch relaxation is removed by the SR Ca-ATPase, 9 and 3.4% by Na/Ca exchange, and 0.5 and 0.1% by slow mechanisms (mitochondria Ca uniporter and sarcolemmal Ca-ATPase). We conclude that the PLB-KO myocytes retain a CaMKII-dependent acceleration of SS twitch [Ca]i decline. The PLB-KO (vs. WT) myocytes also have higher SR Ca pump activity, higher SR Ca load, and reduced Na/Ca exchange activity.

Effects of isoproterenol on cylic-AMP metabolism in rat ventral prostate

1976 ◽  
Vol 54 (3) ◽  
pp. 327-335 ◽  
Author(s):  
B. K. Tsang ◽  
R. L. Singhal
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Kinase Activity ◽  
Activity Ratio ◽  
Ventral Prostate ◽  
Adrenergic Stimulation ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Stimulation Of

β-Adrenergic stimulation of the ventral prostate cyclic-AMP system was investigated by examining the influence of isoproterenol on endogenous cyclic-AMP levels as well as on the activities of adenylate cyclase (EC 4.6.1.1) and cyclic-AMP-dependent and independent protein kinases (EC 2.7.1.37). Administration of isoproterenol (1 mg/kg, ip) resulted in rapid elevation of adenylate cyclase activity (119%) and cyclic-AMP levels (593%). The observed isoproterenol-stimulated changes in cyclic-AMP metabolism of the ventral prostate were time-dependent and maximal stimulation was seen 5 min after treatment with this β-adrenergic agonist. The increases in prostatic adenylate cyclase and cyclic-AMP also were related to the dose of isoproterenol administered and maximal enhancement of these parameters was seen with 1 mg/kg dose of the agonist. Whereas pretreatment of rats with propranolol (3 mg/kg, ip) partially reversed these alterations, administration of an α-adrenergic antagonist, phentolamine, even at a dose of 5 mg/kg, failed to elicit any appreciable effect. Stimulation of prostatic soluble protein kinase by isoproterenol was associated with a decrease (33%) in the activity of the cyclic-AMP-dependent protein kinase with a concomitant increase (25%) in that of the independent enzyme. Whereas the ability of the enzyme to bind cyclic-[3H] AMP in vitro was decreased (54%) following isoproterenol treatment, the protein kinase activity ratio (−cyclic-AMP/+cyclic-AMP) was significantly elevated from 0.51 ± 0.05 to 0.95 ± 0.08. Although propranolol alone had little or no effect on these parameters, it inhibited partially the isoproterenol-induced alterations in cyclic-AMP-dependent protein kinase and the cyclic-AMP binding capacity. Treatment with propranolol also blocked the increases in the kinase activity ratio and in the activity of cyclic-AMP-independent enzyme seen with isoproterenol. Data suggest that the concentration of ventral prostate cyclic-AMP as well as the activities of adenylate cyclase and cyclic-AMP-dependent and independent form of protein kinases are subject to modulation by β-adrenergic stimulation.

Mechanisms for gonadotropin-releasing hormone potentiation of growth hormone rebound following norepinephrine inhibition in goldfish pituitary cells

2007 ◽  
Vol 292 (1) ◽  
pp. E203-E214 ◽  
Author(s):  
Anderson O. L. Wong ◽  
Maggie C. Y. Chuk ◽  
Hiu Chi Chan ◽  
Eric K. Y. Lee
Keyword(s):  
Growth Hormone ◽  
Adrenergic Receptors ◽  
Gonadotropin Releasing Hormone ◽  
Pituitary Cells ◽  
Adrenergic Stimulation ◽  
Dependent Protein Kinase ◽  
Releasing Hormone ◽  
Gh Secretion ◽  
Dependent Protein ◽  
Subsequent Activation

In the goldfish, norepinephrine (NE) inhibits growth hormone (GH) secretion through activation of pituitary α2-adrenergic receptors. Interestingly, a GH rebound is observed after NE withdrawal, which can be markedly enhanced by prior exposure to gonadotropin-releasing hormone (GnRH). Here we examined the mechanisms responsible for GnRH potentiation of this “postinhibition” GH rebound. In goldfish pituitary cells, α2-adrenergic stimulation suppressed both basal and GnRH-induced GH mRNA expression, suggesting that a rise in GH synthesis induced by GnRH did not contribute to its potentiating effect. Using a column perifusion approach, GnRH given during NE treatment consistently enhanced the GH rebound following NE withdrawal. This potentiating effect was mimicked by activation of PKC and adenylate cyclase (AC) but not by induction of Ca2+ entry through voltage-sensitive Ca2+ channels (VSCC). Furthermore, GnRH-potentiated GH rebound could be alleviated by inactivation of PKC, removal of extracellular Ca2+, blockade of VSCC, and inhibition of Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII). Inactivation of AC and PKA, however, was not effective in this regard. These results, as a whole, suggest that GnRH potentiation of GH rebound following NE inhibition is mediated by PKC coupled to Ca2+ entry through VSCC and subsequent activation of CaMKII. Apparently, the Ca2+-dependent cascades are involved in GH secretion during the rebound phase but are not essential for the initiation of GnRH potentiation. Since GnRH has been previously shown to have no effects on cAMP synthesis in goldfish pituitary cells, the involvement of cAMP-dependent mechanisms in GnRH potentiation is rather unlikely.

Regulation of calcium-activated chloride channels in smooth muscle cells: a complex picture is emerging

2005 ◽  
Vol 83 (7) ◽  
pp. 541-556 ◽  
Author(s):  
Normand Leblanc ◽  
Jonathan Ledoux ◽  
Sohag Saleh ◽  
Amy Sanguinetti ◽  
Jeff Angermann ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Chloride Channels ◽  
Muscle Cells ◽  
Cell Types ◽  
Dependent Protein Kinase ◽  
Intracellular Ca ◽  
Dependent Protein

Calcium-activated chloride channels (ClCa) are ligand-gated anion channels as they have been shown to be activated by a rise in intracellular Ca2+ concentration in various cell types including cardiac, skeletal and vascular smooth muscle cells, endothelial and epithelial cells, as well as neurons. Because ClCa channels are normally closed at resting, free intracellular Ca2+ concentration (~100 nmol/L) in most cell types, they have generally been considered excitatory in nature, providing a triggering mechanism during signal transduction for membrane excitability, osmotic balance, transepithelial chloride movements, or fluid secretion. Unfortunately, the genes responsible for encoding this class of ion channels is still unknown. This review centers primarily on recent findings on the properties of these channels in smooth muscle cells. The first section discusses the functional significance and biophysical and pharmacological properties of ClCa channels in smooth muscle cells, and ends with a description of 2 candidate gene families (i.e., CLCA and Bestrophin) that are postulated to encode for these channels in various cell types. The second section provides a summary of recent findings demonstrating the regulation of native ClCa channels in vascular smooth muscle cells by calmodulin-dependent protein kinase II and calcineurin and how their fine tuning by these enzymes may influence vascular tone. Key words: calcium-activated chloride channels, vascular smooth muscle cells, ion channels, calmodulin-dependent protein kinase II, calcineurin

Anti-adrenergic effects of nitric oxide donor SIN-1 in rat cardiac myocytes

2001 ◽  
Vol 281 (1) ◽  
pp. C342-C349 ◽  
Author(s):  
Miroslav O. Stojanovic ◽  
Mark T. Ziolo ◽  
Gordon M. Wahler ◽  
Beata M. Wolska
Keyword(s):  
Nitric Oxide ◽  
Troponin I ◽  
Intracellular Concentration ◽  
Nitric Oxide Donor ◽  
Adrenergic Stimulation ◽  
Inotropic Effects ◽  
Positive Inotropic Effects ◽  
Rat Myocytes ◽  
Rat Cardiac Myocytes ◽  
Adrenergic Effects

We studied how the nitric oxide (NO · ) donor 3-morpholinosydnonimine (SIN-1) alters the response to β-adrenergic stimulation in cardiac rat myocytes. We found that SIN-1 decreases the positive inotropic effect of isoproterenol (Iso) and decreases the extent of both cell shortening and Ca2+ transient. These effects of SIN-1 were associated with an increased intracellular concentration of cGMP, a decreased intracellular concentration of cAMP, and a reduction in the levels of phosphorylation of phospholamban (PLB) and troponin I (TnI). The guanylyl cyclase inhibitor 1 H-8-bromo-1,2,4-oxadiazolo (3,4-d)benz(b)(1,4)oxazin-1-one (ODQ) was not able to prevent the SIN-1-induced reduction of phosphorylation levels of PLB and TnI. However, the effects of SIN-1 were abolished in the presence of superoxide dismutase (SOD) or SOD and catalase. These data suggest that, in the presence of Iso, NO-related congeners, rather than NO · , are responsible for SIN-1 effects. Our results provide new insights into the mechanism by which SIN-1 alters the positive inotropic effects of β-adrenergic stimulation.

scholarly journals Nitric Oxide Modulates Endothelin 1-Induced Ca2+ Mobilization and Cytoskeletal F-Actin Filaments in Human Cerebromicrovascular Endothelial Cells

1999 ◽  
Vol 19 (2) ◽  
pp. 133-138 ◽  
Author(s):  
Ye Chen ◽  
Richard M. McCarron ◽  
Joliet Bembry ◽  
Christl Ruetzler ◽  
Nabil Azzam ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Actin Filaments ◽  
Protein Kinase G ◽  
Microvascular Endothelium ◽  
Dependent Protein Kinase ◽  
Endothelin 1 ◽  
Mediated Effects ◽  
Intracellular Ca ◽  
Dependent Protein

A functional interrelation between nitric oxide (NO), the endothelial-derived vasodilating factor, and endothelin 1 (ET-1), the potent vasoconstrictive peptide, was investigated in microvascular endothelium of human brain. Nor-1 dose-dependently decreased the ET-1–stimulated mobilization of Ca2+. This response was mimicked with cGMP and abrogated by inhibitors of guanylyl cyclase or cGMP-dependent protein kinase G. These findings indicate that NO and ET-1 interactions involved in modulation of intracellular Ca2+ are mediated by cGMP/protein kinase G. In addition, Nor-1–mediated effects were associated with rearrangements of cytoskeleton F-actin filaments. The results suggest mechanisms by which NO–ET-1 interactions may contribute to regulation of microvascular function.

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