scholarly journals FXYD1 phosphorylation in vitro and in adult rat cardiac myocytes: threonine 69 is a novel substrate for protein kinase C

2009 ◽  
Vol 296 (6) ◽  
pp. C1346-C1355 ◽  
Author(s):  
William Fuller ◽  
Jacqueline Howie ◽  
Linda M. McLatchie ◽  
Roberta J. Weber ◽  
C. James Hastie ◽  
...  
Keyword(s):  
Sodium Pump ◽  
Ventricular Myocytes ◽  
Phosphorylation Site ◽  
Cell Voltage ◽  
Kinase Activation ◽  
Kinase Substrate ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Rat Cardiac Myocytes

FXYD1 (phospholemman), the primary sarcolemmal kinase substrate in the heart, is a regulator of the cardiac sodium pump. We investigated phosphorylation of FXYD1 peptides by purified kinases using HPLC, mass spectrometry, and Edman sequencing, and FXYD1 phosphorylation in cultured adult rat ventricular myocytes treated with PKA and PKC agonists by phosphospecific immunoblotting. PKA phosphorylates serines 63 and 68 (S63 and S68) and PKC phosphorylates S63, S68, and a new site, threonine 69 (T69). In unstimulated myocytes, FXYD1 is ∼30% phosphorylated at S63 and S68, but barely phosphorylated at T69. S63 and S68 are rapidly dephosphorylated following acute inhibition of PKC in unstimulated cells. Receptor-mediated PKC activation causes sustained phosphorylation of S63 and S68, but transient phosphorylation of T69. To characterize the effect of T69 phosphorylation on sodium pump function, we measured pump currents using whole cell voltage clamping of cultured adult rat ventricular myocytes with 50 mM sodium in the patch pipette. Activation of PKA or PKC increased pump currents (from 2.1 ± 0.2 pA/pF in unstimulated cells to 2.9 ± 0.1 pA/pF for PKA and 3.4 ± 0.2 pA/pF for PKC). Following kinase activation, phosphorylated FXYD1 was coimmunoprecipitated with sodium pump α1-subunit. We conclude that T69 is a previously undescribed phosphorylation site in FXYD1. Acute T69 phosphorylation elicits stimulation of the sodium pump additional to that induced by S63 and S68 phosphorylation.

Lipopolysaccharide induces apoptosis in adult rat ventricular myocytes via cardiac AT1 receptors

2002 ◽  
Vol 283 (2) ◽  
pp. H461-H467 ◽  
Author(s):  
Hai Ling Li ◽  
Jun Suzuki ◽  
Evelyn Bayna ◽  
Fu-Min Zhang ◽  
Erminia Dalle Molle ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Ventricular Myocytes ◽  
Annexin V ◽  
Left Ventricular ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Cardiac Apoptosis

Lipopolysaccharide (LPS) from gram-negative bacteria circulates in acute, subacute, and chronic conditions. It was hypothesized that LPS directly induces cardiac apoptosis. In adult rat ventricular myocytes (isolated with depyrogenated digestive enzymes to minimize tolerance), LPS (10 ng/ml) decreased the ratio of Bcl-2 to Bax at 12 h; increased caspase-3 activity at 16 h; and increased annexin V, propidium iodide, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining at 24 h. Apoptosis was blocked by the caspase inhibitor benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone (Z-VAD-fmk), captopril, and angiotensin II type 1 receptor (AT1) inhibitor (losartan), but not by inhibitors of AT2 receptors (PD-123319), tumor necrosis factor-α (TNFRII:Fc), or nitric oxide ( N G-monomethyl-l-arginine). Angiotensin II (100 nmol/l) induced apoptosis similar to LPS without additive effects. LPS in vivo (1 mg/kg iv) increased apoptosis in left ventricular myocytes for 1–3 days, which dissipated after 1–2 wk. Losartan (23 mg · kg−1 · day−1 in drinking water for 3 days) blocked LPS-induced in vivo apoptosis. In conclusion, low levels of LPS induce cardiac apoptosis in vitro and in vivo by activating AT1 receptors in myocytes.

scholarly journals Reinduction of T-Type Calcium Channels by Endothelin-1 in Failing Hearts In Vivo and in Adult Rat Ventricular Myocytes In Vitro

Circulation ◽  
2003 ◽  
Vol 108 (20) ◽  
pp. 2530-2535 ◽  
Author(s):  
Toshiaki Izumi ◽  
Yasuki Kihara ◽  
Nobuaki Sarai ◽  
Takeshi Yoneda ◽  
Yoshitaka Iwanaga ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Ventricular Myocytes ◽  
Rat Ventricular Myocytes ◽  
Endothelin 1 ◽  
Adult Rat

Early redistribution of plasma membrane phosphatidylserine during apoptosis of adult rat ventricular myocytes in vitro

1999 ◽  
Vol 94 (3) ◽  
pp. 171-179 ◽  
Author(s):  
C. R�cker-Martin ◽  
M. H�naff ◽  
S.N. Hatem ◽  
E. Delpy ◽  
J.-J. Mercadier
Keyword(s):  
Plasma Membrane ◽  
Ventricular Myocytes ◽  
Rat Ventricular Myocytes ◽  
Adult Rat

α1-Adrenergic activation of L-type Ca current in rat ventricular myocytes: perforated patch-clamp recordings

1998 ◽  
Vol 274 (6) ◽  
pp. H2203-H2207 ◽  
Author(s):  
Shi J. Liu ◽  
Richard H. Kennedy
Keyword(s):  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Adrenergic Stimulation ◽  
Whole Cell ◽  
Rat Ventricular Myocytes ◽  
Perforated Patch ◽  
Adult Rat ◽  
Transient Decrease ◽  
Adrenergic Antagonist ◽  
Patch Configuration

α1-Adrenergic stimulation has little effect on L-type Ca2+channel current ( I Ca,L) in adult cardiac myocytes measured using conventional whole cell voltage-clamp techniques. In this study using perforated-patch techniques, we reevaluated the effect of α1-adrenergic stimulation on I Ca,L in adult rat ventricular myocytes. Action potentials and I Ca,L were examined in the presence of 1 μM nadolol, a β-adrenergic antagonist, in myocytes internally dialyzed with Na+- and K+-free solutions (Cs+ and tetraethylammonium as substitutes). Phenylephrine (PE; 30 μM) increased the action potential duration measured at 25 and 70% of repolarization by 104 and 86%, respectively. In the perforated-patch configuration, PE elicited a transient decrease followed by a ∼60% increase in I Ca,L, whereas only the transient decrease in I Ca,L was observed in myocytes when the conventional whole cell configuration was used. The PE-induced increase in I Ca,L was reversibly blocked by 1 μM prazosin, an α1-adrenergic antagonist. These results suggest that α1-adrenergic stimulation enhances cardiac I Ca,L and that obligatory intracellular mediators for this action are lost during whole cell recordings.

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