Abstract MP236: PI3Kgamma-PP2A Axis In Regulation Of SERCA Function

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Maradumane L Mohan ◽  
Conner P Witherow ◽  
Robert Papay ◽  
Yu Sun ◽  
Kate Stenson ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Protein Phosphatase ◽  
Significant Interaction ◽  
Cardiac Contractility ◽  
Ventricular Rate ◽  
Cardiac Stress ◽  
Adult Cardiomyocytes ◽  
Genetic Deletion ◽  
Phosphoinositide 3 Kinase ◽  
Camp Levels

Genetic deletion of Phosphoinositide 3-kinase (PI3Kγ) in mice (PI3Kγ -/- ) results in increased cAMP levels and enhanced ventricular rate/contractility. We investigated whether PI3Kγ plays a role in cardiac contractility by altering intracellular calcium recycling. Caffeine treatment of adult cardiomyocytes from PI3Kγ -/- mice showed significantly reduced calcium reuptake by sarcoendoplasmic reticulum (SR) indicating that PI3Kγ locally regulates SR function. This resulted in elevated levels of intracellular calcium for prolonged period following caffeine. Our findings show that delayed re-uptake of calcium was caused by changes in phosphorylation of phospholamban (PLN), a major regulator of SR calcium reuptake. PI3Kγ -/- cardiomyocytes showed significantly reduced PLN phosphorylation due to increase in PLN-associated protein phosphatase (PP) activity as reflected by decreased demethylated-PP2A. Abrogation of PLN phosphorylation in the PI3Kγ -/- cardiomyocytes shows that the loss in the steady-state phosphorylation of PLN leads to increased inhibition of SERCA. This inhibition is reflected by the slow reuptake of calcium by the SR in the PI3Kγ -/- cardiomyocytes. Concomitantly, significant interaction was observed between SERCA and PLN in the PI3Kγ -/- hearts compared to the controls. Consistently, the altered calcium regulation in the cardiomyocytes of PI3Kγ -/- can be restored by inhibition of PP by okadaic acid. Unexpectedly, cardiomyocyte-specific overexpression of kinase-dead PI3Kγ (PI3Kγ inact ) in the global PI3Kγ -/- cardiomyocytes normalized caffeine induced calcium reuptake, restored PLN phosphorylation, and decreased PLN-associated PP activity reflected by increased demethylated-PP2A. These studies bring-to-fore an unrecognized kinase-independent regulation of PLN by PI3Kγ through PP2A with implications in deleterious cardiac remodeling as PI3Kγ is significantly upregulated following cardiac stress.

scholarly journals Kinase independent function of PI3Kγ modulates calcium re-uptake by regulating phospholamban function

2021 ◽  
Author(s):  
Maradumane L Mohan ◽  
Conner P Witherow ◽  
Robert S Papay ◽  
Yu Sun ◽  
Kate Stenson ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Protein Phosphatase ◽  
Cardiac Contractility ◽  
Ventricular Rate ◽  
Independent Function ◽  
Cardiac Stress ◽  
Adult Cardiomyocytes ◽  
Genetic Deletion ◽  
Phosphoinositide 3 Kinase ◽  
Camp Levels

Rationale Genetic deletion of Phosphoinositide 3-kinase (PI3Kγ) in mice (PI3Kγ-/-) results in increased cAMP levels and enhanced ventricular rate/contractility. Whether PI3Kγ plays a role in cardiac contractility by altering intracellular calcium recycling is not known. Objective To understand the mechanism of PI3Kγ mediated regulation of cardiac contractility. Methods and Results Caffeine treatment of adult cardiomyocytes from PI3Kγ-/- mice showed significantly reduced calcium reuptake by sarcoendoplasmic reticulum (SR) indicating that PI3Kγ locally regulates SR function. This resulted in elevated levels of intracellular calcium for prolonged period following caffeine. Our findings show that delayed re-uptake of calcium was caused by changes in phosphorylation of phospholamban (PLN), a major regulator of SR calcium reuptake. PI3Kγ-/- cardiomyocytes show significantly reduced PLN phosphorylation due to increase in PLN-associated protein phosphatase (PP) activity as reflected by decreased demethylated-PP2A. Consistently, the altered calcium regulation in the cardiomyocytes of PI3Kγ-/- can be restored by inhibition of PP by okadaic acid. Unexpectedly, cardiomyocyate-specific overexpression of kinase-dead PI3Kγ (PI3Kγinact) in the global PI3Kγ-/- cardiomyocytes normalized caffeine induced calcium reuptake, restored PLN phosphorylation, and decreased PLN-associated PP activity reflected by increased demethylated-PP2A. Conclusions These studies bring-to-fore an unrecognized regulation of PLN by PI3Kγ through PP2A with implications in deleterious cardiac remodeling as PI3Kγ is significantly upregulated following cardiac stress.

Abstract MP135: Cardiac Sarcoplasmic Reticulum Calcium Cycling is Regulated by Kinase Independent Function of Pi3kgamma

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Maradumane L Mohan ◽  
Conner P Witherow ◽  
Robert S Papay ◽  
Sathyamangla V Naga Prasad
Keyword(s):  
Intracellular Calcium ◽  
Calcium Release ◽  
Cardiac Contractility ◽  
Underlying Mechanism ◽  
Calcium Handling ◽  
Ventricular Contractility ◽  
Independent Function ◽  
Calcium Cycling ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Phosphoinositide 3 Kinase

Genetic deletion of Phosphoinositide 3-kinase (PI3Kγ) in mice (PI3Kγ -/- ) results in increased cAMP levels and enhanced ventricular contractility. We investigated whether the lack of PI3Kγ plays a role in cardiac contractility by altering intracellular calcium recycling. Isolated cardiomyocytes from PI3Kγ -/- mice showed significantly reduced calcium reuptake by sarcoendoplasmic reticulum (SR) following caffeine induced calcium release indicating that PI3Kγ locally regulates the function of SR. The intracellular calcium remained at elevated levels in the cardiomyocytes of PI3Kγ -/- for a prolonged period after caffeine treatment. This could be due to changes in phosphorylation of SERCA2, Ryanodine receptor (RyR 2 ) or phospholamban (PLN). In fact, when we looked at phosphorylation of PLN in cardiac lysates, a major regulator of cardiac contractility and relaxation, PI3Kγ -/- mice showed significantly reduced PLN phosphorylation compared to littermate controls. Previous studies from our laboratory suggested that absence of PI3Kγ leads to increase in protein phosphatase (PP) activity which could be possible reason for rapid dephosphorylation of PLN, resulting in inhibition of SERCA2 pump. We observed increased SR associated PP activity and PLN associated PP activity in PI3Kγ -/- mice. We also observed increased association of PP-1 and PP2A with PLN in the absence of PI3Kγ. The altered calcium handling in the cardiomyocytes of PI3Kγ -/- mice could be restored to the level of WT controls by okadaic acid mediated inhibition of PP, suggesting that PI3Kγ plays a role in regulating PP activity associated with SR. To test whether PI3Kγ activity is required for PLN dephosphorylation and SR calcium cycling, we used mice with cardiac specific overexpression of kinase dead PI3Kγ (PI3Kγ inact ) in global PI3Kγ -/- mice (PI3Kγ inact /PI3Kγ -/- ). PI3Kγ inact /PI3Kγ -/- mice showed restored PLN phosphorylation, improved caffeine induced calcium reuptake, decreased SR and PLN associated PP activity. These studies show a novel regulation of PP and SR calcium regulation by kinase independent function of PI3Kγ. The underlying mechanism of PP regulation by PI3Kγ will be presented.

Substrate specificity and acute regulation of the tumour suppressor phosphatase, PTEN

2007 ◽  
Vol 74 ◽  
pp. 69-80 ◽  
Author(s):  
C. Peter Downes ◽  
Nevin Perera ◽  
Sarah Ross ◽  
Nick R. Leslie
Keyword(s):  
Protein Phosphatase ◽  
Current Model ◽  
Tumour Suppressor ◽  
Protein Substrates ◽  
Dual Specificity ◽  
Survival And Growth ◽  
Phosphoinositide 3 Kinase ◽  
Phosphatase And Tensin Homologue ◽  
A Current ◽  
Inhibit Cell Proliferation

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumour suppressor that functions as a PtdIns(3,4,5)P3 3-phosphatase to inhibit cell proliferation, survival and growth by antagonizing PI3K (phosphoinositide 3-kinase)-dependent signalling. Recent work has begun to focus attention on potential biological functions of the protein phosphatase activity of PTEN and on the possibility that some of its functions are phosphatase-independent. We discuss here the structural and regulatory mechanisms that account for the remarkable specificity of PTEN with respect to its PtdIns substrates and how it avoids the soluble headgroups of PtdIns that occur commonly in cells. Secondly we discuss the concept of PTEN as a constitutively active enzyme that is subject to negative regulation both physiologically and pathologically. Thirdly, we review the evidence that PTEN functions as a dual specificity phosphatase with discrete lipid and protein substrates. Lastly we present a current model of how PTEN may participate in the control of cell migration.

Abstract 212: Inhibition of Protein Phosphatase 2A (PP2A) Leads to Beta-adrenergic Receptor Dysfunction With Cardiac Stress Following Pressure-overload Hypertrophy

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Arunachal Chatterjee ◽  
Neelakantan Vasudevan ◽  
Maradumane Mohan ◽  
Elizabeth Martelli ◽  
John George ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Adenylyl Cyclase ◽  
Cardiac Dysfunction ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Cyclase Activity ◽  
Adenylyl Cyclase Activity ◽  
Cardiac Stress ◽  
Phosphatase 2A

Beta-Adrenergic receptors (bARs) play a key role in regulating cardiac function. Loss of surface receptors and desensitization (impaired G-protein coupling) of bARs are hallmarks of a failing heart. Desensitization occurs by phosphorylation of bARs. The bARs are resensitized by protein phosphatase 2A (PP2A) mediated dephosphorylation in the endosomes before recycling to the plasma membrane. While mechanisms of desensitization are well understood, little is known about mechanisms regulating resensitization. Our previous work has shown that PI3Kg phosphorylates an endogenous inhibitor of PP2A (I2PP2A) on serine 9 & 93, which then robustly binds to PP2A inhibiting bAR resensitization. Since it is not known whether resensitization is altered in response to cardiac stress or whether altered bAR resensitization contributes to cardiac hypertrophy and failure, we generated transgenic mice with cardiomyocyte specific overexpression of wild type I2PP2A (WT I2PP2A Tg), I2PP2A phospho-mimetic mutants S9, 93D and mutants with constitutively dephosphorylated S9, 93A state. To test whether resensitization is critical in the development of bAR dysfunction during cardiac hypertrophy, WT I2PP2A Tg mice were subjected to transverse aortic constriction (TAC) for 8 weeks. Echocardiographic analysis post-TAC showed that WT I2PP2A Tg mice had accelerated cardiac dysfunction compared to their littermate controls [HW (mg)/BW(g): Sham: WT - 4.83, WT I2PP2A Tg - 4.82, TAC: WT- 6.47, WT I2PP2A Tg - 7.61; %EF: Sham: WT - 83.53, WT I2PP2A Tg - 74.72, TAC: WT - 70.47, WT I2PP2A Tg - 49.62]. To directly test whether resensitization mechanisms are altered, plasma membranes and endosomes were isolated and in vitro Adenylyl Cyclase activity assessed. Our studies show that compared to littermate controls, WT I2PP2A Tg had altered in vitro adenylyl cyclase activity showing that resensitization mechanisms in the endosomes may in part, contribute to cardiac dysfunction. Mechanistic underpinnings of the resensitization pathways using the I2PP2A S9, 93A and S9, 93D will be presented showing that bAR resensitization a process considered passive is altered in conditions of cardiac stress that in part may contribute to bAR dysfunction leading to cardiac hypertrophy and heart failure.

Involvement of the β-adrenergic system in the cardiac chronic form of experimental Trypanosoma cruzi infection

Parasitology ◽  
2009 ◽  
Vol 136 (8) ◽  
pp. 905-918 ◽  
Author(s):  
M. S. LO PRESTI ◽  
H. W. RIVAROLA ◽  
A. R. FERNÁNDEZ ◽  
J. E. ENDERS ◽  
G. LEVIN ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Late Stage ◽  
Early Stage ◽  
Cardiac Contractility ◽  
Receptor Interaction ◽  
Adrenergic System ◽  
Receptor Affinity ◽  
Chronic Form ◽  
Β Receptor ◽  
Camp Levels

SUMMARYChanges in the cardiac β-adrenergic system in early stages of Trypanosoma cruzi infection have been described. Here, we studied an early (135 days post-infection–p.i.) and a late stage (365 days p.i.) of the cardiac chronic form of the experimental infection (Tulahuen or SGO-Z12 strains), determining plasma epinephrine and norepinephrine levels, β-receptor density, affinity and function, cardiac cAMP concentration and phosphodiesterase activity, cardiac contractility, and the presence of β-receptor autoantibodies. Tulahuen-infected mice presented lower epinephrine and norepinephrine levels; lower β-receptor affinity and density; a diminished norepinephrine response and higher cAMP levels in the early stage, and a basal contractility similar to non-infected controls in the early and augmented in the late stage. The Tulahuen strain induced autoantibodies with weak β-receptor interaction. SGO-Z12-infected mice presented lower norepinephrine levels and epinephrine levels that diminished with the evolution of the infection; lower β-receptor affinity and an increased density; unchanged epinephrine and norepinephrine response in the early and a diminished response in the late stage; higher cAMP levels and unchanged basal contractility. The SGO-Z12 isolate induced β-receptor autoantibodies with strong interaction with the β-receptors. None of the antibodies, however, acted a as β-receptor agonist. The present results demonstrate that this system is seriously compromised in the cardiac chronic stage of T. cruzi infection.

Stimulatory phosphorylation of cAMP-specific PDE4D5 by contractile agonists is mediated by PKC-dependent inactivation of protein phosphatase 2A

2008 ◽  
Vol 294 (1) ◽  
pp. G327-G335 ◽  
Author(s):  
Karnam S. Murthy ◽  
Wimolpak Sriwai
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Dependent Inactivation ◽  
Rhythmic Cycles ◽  
Phosphatase 2A ◽  
Contraction And Relaxation ◽  
Camp Levels

Smooth muscle of the gut undergoes rhythmic cycles of contraction and relaxation. Various constituents in the pathways that mediate muscle contraction could act to cross-regulate cAMP or cGMP levels and terminate subsequent relaxation. We have previously shown that cAMP levels are regulated by PKA-mediated phosphorylation of cAMP-specific phosphodiesterase 3A (PDE3A) and PDE4D5; the latter is the only PDE4D isoform expressed in smooth muscle. In the present study we have elucidated a mechanism whereby cholecystokinin (CCK) and, presumably, other contractile agonists capable of activating PKC can cross-regulate cAMP levels. Forskolin stimulated PDE4D5 phosphorylation and PDE4D5 activity. CCK significantly increased forskolin-stimulated PDE4D5 phosphorylation and activity and attenuated forskolin-stimulated cAMP levels. The effect of CCK on forskolin-induced PDE4D5 phosphorylation and activity and on cAMP levels was blocked by the inhibitors of PLC or PKC and in cultured muscle cells by the expression of Gαq minigene. The effects of CCK on PDE4D5 phosphorylation, PDE4D5 activity, and cAMP levels were mimicked by low (1 nM) concentrations of okadaic acid, but not by a low (10 nM) concentration of tautomycin, suggesting involvement of PP2A. Purified catalytic subunit of PP2A but not PP1 dephosphorylated PDE4D5 in vitro. Coimmunoprecipitation studies demonstrated association of PDE4D5 with PP2A and the association was decreased by the activation of PKC. In conclusion, cAMP levels are cross-regulated by contractile agonists via a mechanism that involves PLC-β-dependent, PKC-mediated inhibition of PP2A activity that leads to increase in PDE4D5 phosphorylation and activity and inhibition of cAMP levels.

Mechanism of action of γ-aminobutyric acid on frog melanotrophs

1995 ◽  
Vol 14 (1) ◽  
pp. 1-12 ◽  
Author(s):  
L Desrues ◽  
H Vaudry ◽  
M Lamacz ◽  
M C Tonon
Keyword(s):  
Intracellular Calcium ◽  
Calcium Concentration ◽  
Inhibitory Effect ◽  
Aminobutyric Acid ◽  
Calcium Stores ◽  
Camp Production ◽  
Biphasic Effect ◽  
Camp Levels ◽  
Camp Formation ◽  
Stimulation Of

ABSTRACT We have previously demonstrated that γ-aminobutyric acid (GABA) is a potent regulator of secretory and electrical activity in melanotrophs of the frog pituitary. The aim of the present study was to investigate the intracellular events which mediate the response of melanotrophs to GABA. We first observed that GABA (1–100 μm inhibited both basal and forskolin-stimulated cyclic AMP (cAMP) formation. The inhibitory effect of GABA on cAMP levels was mimicked by the GABAB receptor agonist baclofen (100 μm) and totally abolished by a 4-h pretreatment with pertussis toxin (01 μg/ml). In contrast, the specific GABAA agonist 3-aminopropane sulphonic acid (3APS) did not affect cAMP production. Both GABA and 3APS (100 μm each) induced a biphasic effect on α-MSH release from perifused frog neurointermediate lobes, i.e. a transient stimulation followed by an inhibition of α-MSH secretion. Administration of forskolin (10 μm) prolonged the stimulatory phase and attenuated the inhibitory phase evoked by GABA and 3APS, indicating that cAMP modulates the response of melanotrophs to GABAA agonists. Ejection of 3APS (1 μm) in the vicinity of cultured melanotrophs caused a massive increase in intracellular calcium concentration ([Ca2+]i). The stimulatory effect of 3APS on [Ca2+]i was abolished when the cells were incubated in a chloride-free medium. The formation of inositol trisphosphate was not affected by 3APS, suggesting that the increase in [Ca2+]i cannot be ascribed to mobilization of intracellular calcium stores. ω-Conotoxin did not alter the secretory response of frog neurointermediate lobes to 3APS, while nifedipine blocked the stimulation of α-MSH secretion induced by 3APS. In conclusion, the present data indicate that, in frog pituitary melanotrophs, (i) the stimulatory phase evoked by GABAA agonists can be accounted for by an influx of calcium through L-type calcium channels, (ii) the inhibitory effect evoked by GABAB agonists can be ascribed to inhibition of adenylate cyclase activity and (iii) cAMP attenuates the inhibitory phase evoked by GABAA agonists. Taken together, these data suggest that activation of GABAB receptors may modulate GABAA receptor function.

scholarly journals Visualization of β-adrenergic receptor dynamics and differential localization in cardiomyocytes

2021 ◽  
Vol 118 (23) ◽  
pp. e2101119118
Author(s):  
Marc Bathe-Peters ◽  
Philipp Gmach ◽  
Horst-Holger Boltz ◽  
Jürgen Einsiedel ◽  
Michael Gotthardt ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Adrenergic Receptor ◽  
Cardiac Contractility ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Receptor Subtypes ◽  
Specific Cell ◽  
Adult Cardiomyocytes ◽  
Outer Plasma Membrane

A key question in receptor signaling is how specificity is realized, particularly when different receptors trigger the same biochemical pathway(s). A notable case is the two β‐adrenergic receptor (β‐AR) subtypes, β1 and β2, in cardiomyocytes. They are both coupled to stimulatory Gs proteins, mediate an increase in cyclic adenosine monophosphate (cAMP), and stimulate cardiac contractility; however, other effects, such as changes in gene transcription leading to cardiac hypertrophy, are prominent only for β1‐AR but not for β2-AR. Here, we employ highly sensitive fluorescence spectroscopy approaches, in combination with a fluorescent β‐AR antagonist, to determine the presence and dynamics of the endogenous receptors on the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. These techniques allow us to visualize that the β2‐AR is confined to and diffuses within the T-tubular network, as opposed to the β1‐AR, which is found to diffuse both on the outer plasma membrane as well as on the T-tubules. Upon overexpression of the β2‐AR, this compartmentalization is lost, and the receptors are also seen on the cell surface. Such receptor segregation depends on the development of the T-tubular network in adult cardiomyocytes since both the cardiomyoblast cell line H9c2 and the cardiomyocyte-differentiated human-induced pluripotent stem cells express the β2‐AR on the outer plasma membrane. These data support the notion that specific cell surface targeting of receptor subtypes can be the basis for distinct signaling and functional effects.

P2Y12 ADP receptor-dependent tyrosine phosphorylation of proteins of 27 and 31 kDa in thrombin-stimulated human platelets

2005 ◽  
Vol 93 (05) ◽  
pp. 880-888 ◽  
Author(s):  
Knut Fälker ◽  
Danica Lange ◽  
Peter Presek
Keyword(s):  
Adenylyl Cyclase ◽  
Tyrosine Phosphorylation ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
P2y12 Receptor ◽  
Dependent Manner ◽  
Adp Receptor ◽  
Txa2 Receptor Antagonist ◽  
Phosphoinositide 3 Kinase ◽  
Camp Levels

SummaryIn thrombin-stimulated human platelets several proteins undergo rapid and transient changes in tyrosine phosphorylation. We demonstrate that a set of proteins of 27, 29, 31, 34, and 39 kDa is affected by released ADP and P2Y12 receptor signaling during platelet activation. AR-C69931MX, an antagonist of the Gi2-coupled P2Y12 ADP receptor, inhibits initial tyrosine phosphorylation of p27 and p31 and prevents subsequent dephosphorylation of p29, p34, and p39. Antagonists of the Gq-coupled P2Y1 ADP receptor have no effect. Precluding integrin αIIbβ3 outside-in signaling with RGDS or S1197 does not affect the increase in tyrosine phosphorylation of the set of proteins but inhibits their subsequent dephosphorylation. Besides the ADP analogue 2-MeS-ADP, other platelet agonists such as collagen and the TXA2-mimetic U46619 also induce p27 and p31 tyrosine phosphorylation in a P2Y12 receptor-dependent manner. Tyrosine phosphorylation of p27 and p31 in response to collagen, but not thrombin, is prevented by aspirin and the TXA2 receptor antagonist SQ29548, indicating that the effect of collagen strongly relies on TXA2 signaling. Furthermore, epinephrine, acting via inhibitory Gz-coupled α2A-adrenoceptors, bypasses the inhibitory effect of AR-C69931MX on thrombin-induced p27 and p31 tyrosine phosphorylation. Finally, we demonstrate that tyrosine phosphorylation of p27 and p31 downstream of P2Y12 receptors is due to the inhibition of adenylyl cyclase but not phosphoinositide 3-kinase (PI 3-K) activation. Elevating cAMP levels with PGI2 or forskolin precludes thrombin-induced p27 and p31 tyrosine phosphorylation. Moreover, direct inhibition of adenylyl cyclase by SQ22536 reverses the effect of AR-C69931MX. Our data indicate that the observed changes in tyrosine phosphorylation are the result of both primary Gq signaling, initiating the release of ADP, as well as subsequent P2Y12 receptor-mediated Gi coupling.

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