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.

scholarly journals Stretch-activated whole cell currents in adult rat cardiac myocytes

2000 ◽  
Vol 278 (2) ◽  
pp. H548-H557 ◽  
Author(s):  
Tao Zeng ◽  
Glenna C. L. Bett ◽  
Frederick Sachs
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Reversal Potential ◽  
Cell Voltage ◽  
Cellular Level ◽  
Whole Cell ◽  
Adult Rat ◽  
Longitudinal Stretch ◽  
Single Channel Currents ◽  
Channel Currents

Mechanoelectric transduction can initiate cardiac arrhythmias. To examine the origins of this effect at the cellular level, we made whole cell voltage-clamp recordings from acutely isolated rat ventricular myocytes under controlled strain. Longitudinal stretch elicited noninactivating inward cationic currents that increased the action potential duration. These stretch-activated currents could be blocked by 100 μM Gd3+ but not by octanol. The current-voltage relationship was nearly linear, with a reversal potential of approximately −6 mV in normal Tyrode solution. Current density varied with sarcomere length (SL) according to I (pA/pF) = 8.3 − 5.0SL (μm). Repeated attempts to record single channel currents from stretch-activated ion channels failed, in accord with the absence of such data from the literature. The inability to record single channel currents may be a result of channels being located on internal membranes such as the T tubules or, possibly, inactivation of the channels by the mechanics of patch formation.

Modulation of ICa-L by α1-adrenergic stimulation in rat ventricular myocytes

2005 ◽  
Vol 83 (11) ◽  
pp. 1015-1024 ◽  
Author(s):  
Shetuan Zhang ◽  
Jijin Lin ◽  
Yuji Hirano ◽  
Masayasu Hiraoka
Keyword(s):  
Protein Kinase ◽  
Patch Clamp ◽  
Kinase Inhibitor ◽  
Ventricular Myocytes ◽  
Adrenergic Stimulation ◽  
Pipette Solution ◽  
Rat Ventricular Myocytes ◽  
Perforated Patch ◽  
Intracellular Ca ◽  
Perforated Patch Clamp

We found when L-type calcium current (ICa-L) was recorded with the perforated patch-clamp method in rat ventricular myocytes that bath application of phenylephrine (with propranolol) evoked a biphasic response characterized by an initial transient suppression followed by a sustained potentiation. The transient suppression occurred 30–60 s after phenylephrine perfusion and reached peak inhibition at approximately 2 min. The biphasic modulation of ICa-L was also elicited by methoxamine, and the effects of phenylephrine were blocked by prazosin, indicating that the responses were mediated through α1-adrenoceptors. Pretreatment of cells with H7 (100 µmol/L), a broad-spectrum protein kinase inhibitor that inhibits both protein kinase C and A, eliminated potentiation but did not affect transient suppression. The transient suppression occurred concurrently with the acceleration of the fast component of ICa-L inactivation. Depletion of intracellular Ca2+ stores by ryanodine plus caffeine or thapsigargin eliminated the transient suppression. When ICa-L was recorded with whole-cell patch-clamp and with 0.05 mmol/L EGTA in the pipette solution to allow intracellular Ca2+ to fluctuate, phenylephrine evoked a transient suppression as in the perforated patch recordings. Heparin, a specific blocker of IP3 (inositol 1,4,5-trisphosphate) receptors, eliminated the phenylephrine-induced transient suppression of ICa-L when added to the pipette solution. Intensive chelation of intracellular Ca2+ by 5 mmol/L BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) in the pipette solution also eliminated the phenylephrine-induced transient suppression of ICa-L. We conclude that transient increase in the concentration of intracellular calcium ([Ca2+]i) caused by Ca2+ release from intracellular stores underlies the transient suppression of ICa-L, whereas the potentiation of ICa-L is a result of activation of protein kinases.Key words: Ca2+ mobilization, IP3, Ca2+-induced inactivation of Ca2+ current, perforated patch-clamp.

Perforated-patch recording does not enhance effect of 3-isobutyl-1-methylxanthine on cardiac calcium current

1994 ◽  
Vol 266 (6) ◽  
pp. C1619-C1627 ◽  
Author(s):  
A. Kawamura ◽  
G. M. Wahler
Keyword(s):  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Rapid Decline ◽  
Response Curves ◽  
Whole Cell ◽  
Perforated Patch ◽  
Cell Responses ◽  
Whole Cell Recording ◽  
Cell Recording ◽  
Beta Adrenergic Agonist

Conventional whole cell voltage-clamp recording results in washout of the cardiac Ca2+ current (ICa) response to the beta-adrenergic agonist isoproterenol (Iso), for reasons which are not clear. When dose-response curves for the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) were compared using perforated-patch vs. conventional whole cell recording in guinea pig ventricular myocytes, the conventional whole cell IBMX responses were unexpectedly larger than the perforated-patch responses. Furthermore, during conventional whole cell recording the response to repeated application of Iso declined rapidly, whereas the IBMX response initially increased and then declined. When pipette [Ca2+] was increased to 10(-7) M, conventional whole cell responses to 300 microM IBMX and 10(-9) M Iso were identical to perforated-patch responses. Thus loss of the Iso response during conventional whole cell recording seems to not be solely due to a washout of some constituent of the adenosine 3',5'-cyclic monophosphate pathway. We suggest that unphysiological intracellular [Ca2+] enhances the relative PDE activity and that this contributes to the rapid decline of the Iso response and the initial enhancement of the IBMX response.

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.

Effects of sarcoplasmic reticulum Ca2+ load on the gain function of Ca2+ release by Ca2+ current in cardiac cells

1995 ◽  
Vol 268 (2) ◽  
pp. H916-H920 ◽  
Author(s):  
A. M. Janczewski ◽  
H. A. Spurgeon ◽  
M. D. Stern ◽  
E. G. Lakatta
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Voltage Clamp ◽  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Cardiac Cells ◽  
Gain Function ◽  
Whole Cell ◽  
Rat Ventricular Myocytes

We studied the effects of variable sarcoplasmic reticulum (SR) Ca2+ loading on changes in the gain index of Ca2+ release from the SR, measured as the ratio of the amount of Ca2+ released to the magnitude of the Ca2+ current (ICa) integrated for the initial 20 ms of the depolarization, in whole cell voltage-clamped rat ventricular myocytes dialyzed with the Ca2+ indicator indo 1 salt at 23 degrees C. Changes in ICa were measured directly, and changes in the SR Ca2+ release were indexed by changes in the amplitudes and rates of rise of cytosolic Ca2+ (Ca2+i) transients. The SR Ca2+ load was graded by the duration of conditioning voltage-clamp steps and verified by caffeine-dependent Ca2+i transients. A train of abbreviated (from 100 to 20 ms) voltage-clamp depolarizations, which triggers SR Ca2+ release but fails to replenish the SR with Ca2+, diminished the SR Ca2+ load by 56 +/- 5%, did not alter peak ICa but reduced the amplitudes of the ICa-dependent Ca2+i transients by 52 +/- 3%, and decreased the gain index by 60 +/- 3% (SE; n = 5 or 6). Changes in the amplitudes of Ca2+i transients elicited by ICa and changes in the gain index were linearly correlated (r2 = 0.83 and 0.79, respectively; P < 0.001 for each) with changes in amplitudes of Ca2+i transients elicited by caffeine pulses applied in lieu of the respective voltage-clamp pulses.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Altered Na/Ca exchange distribution in ventricular myocytes from failing hearts

2016 ◽  
Vol 310 (2) ◽  
pp. H262-H268 ◽  
Author(s):  
Hanne C. Gadeberg ◽  
Simon M. Bryant ◽  
Andrew F. James ◽  
Clive H. Orchard
Keyword(s):  
Heart Failure ◽  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Coronary Artery Ligation ◽  
Sham Operation ◽  
Ca Current ◽  
Artery Ligation ◽  
Whole Cell ◽  
Rat Hearts ◽  
T Tubules

In mammalian cardiac ventricular myocytes, Ca efflux via Na/Ca exchange (NCX) occurs predominantly at T tubules. Heart failure is associated with disrupted t-tubular structure, but its effect on t-tubular function is less clear. We therefore investigated t-tubular NCX activity in ventricular myocytes isolated from rat hearts ∼18 wk after coronary artery ligation (CAL) or corresponding sham operation (Sham). NCX current ( INCX) and l-type Ca current ( ICa) were recorded using the whole cell, voltage-clamp technique in intact and detubulated (DT) myocytes; intracellular free Ca concentration ([Ca]i) was monitored simultaneously using fluo-4. INCX was activated and measured during application of caffeine to release Ca from sarcoplasmic reticulum (SR). Whole cell INCX was not significantly different in Sham and CAL myocytes and occurred predominantly in the T tubules in Sham myocytes. CAL was associated with redistribution of INCX and ICa away from the T tubules to the cell surface and an increase in t-tubular INCX/ ICa density from 0.12 in Sham to 0.30 in CAL myocytes. The decrease in t-tubular INCX in CAL myocytes was accompanied by an increase in the fraction of Ca sequestered by SR. However, SR Ca content was not significantly different in Sham, Sham DT, and CAL myocytes but was significantly increased by DT of CAL myocytes. In Sham myocytes, there was hysteresis between INCX and [Ca]i, which was absent in DT Sham but present in CAL and DT CAL myocytes. These data suggest altered distribution of NCX in CAL myocytes.

Sign in / Sign up

Export Citation Format

Share Document