Characterization of stimulation of phosphoinositide hydrolysis by alpha 1-adrenergic agonists in adult rat hearts

1994 ◽  
Vol 267 (3) ◽  
pp. H970-H978 ◽  
Author(s):  
A. Lazou ◽  
S. J. Fuller ◽  
M. A. Bogoyevitch ◽  
K. A. Orfali ◽  
P. H. Sugden
Keyword(s):  
Ventricular Myocytes ◽  
Irreversible Inhibitor ◽  
Adult Rat ◽  
Fixed Concentration ◽  
Rat Hearts ◽  
Site Competition ◽  
Pi Hydrolysis ◽  
Hydrolysis Of ◽  
Stimulation Of

The coupling of the pharmacologically defined alpha 1A- and alpha 1B-adrenoceptors to the hydrolysis of phospho[3H]inositides (PI) was investigated in ventricular myocytes freshly isolated from adult rat hearts. The alpha 1-adrenoceptor population in the heart was characterized by competitive binding experiments using [3H]prazosin and the alpha 1A-adrenoceptor-selective antagonist 5-methyl urapidil. It was heterogeneous with approximately 25% being pharmacologically of the alpha 1A-adrenoceptor subtype and 75% being of the alpha 1B-adrenoceptor subtype. Epinephrine, norepinephrine, or phenylephrine stimulated PI hydrolysis in the presence or absence of propranolol. The greatest stimulation (7-fold) was with epinephrine. The half-maximum effective concentrations for agonists were approximately 0.5-3.5 and 0.2 microM in the absence and presence of propranolol, respectively. The inhibition by 5-methyl urapidil of the stimulation of PI hydrolysis by a fixed concentration of epinephrine fitted a two-site competition curve. The distribution between high-affinity (25%) and low-affinity (75%) sites suggested that both the alpha 1A- and alpha 1B-adrenoceptors were coupled to PI hydrolysis in proportion to their relative abundance. Equally, the stimulation of PI hydrolysis by epinephrine in the presence of a fixed concentration of 5-methyl urapidil was biphasic. In addition, chloroethylclonidine, an irreversible inhibitor of the alpha 1B-adrenoceptor, inhibited the epinephrine stimulation of PI hydrolysis by 35%. We conclude that the pharmacologically defined alpha 1A- and alpha 1B-adrenoceptor subtypes are both coupled to PI hydrolysis in the ventricular myocyte.

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.

Alpha 1b-adrenoceptor-mediated stimulation of Na-K pump current in adult rat ventricular myocytes

1993 ◽  
Vol 264 (4) ◽  
pp. H1315-H1318 ◽  
Author(s):  
A. P. Williamson ◽  
R. H. Kennedy ◽  
E. Seifen ◽  
J. P. Lindemann ◽  
J. R. Stimers
Keyword(s):  
Adrenergic Receptors ◽  
Ventricular Myocytes ◽  
Pump Current ◽  
Peak Effect ◽  
Patch Clamp Technique ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Whole Cell Patch Clamp ◽  
Dose Dependent ◽  
Stimulation Of

The purpose of this study was to determine if myocardial alpha 1a-and/or alpha 1b-adrenoceptors are involved in the increase in Na-K pump current (Ip) elicited by alpha 1-adrenergic agonists. Single rat ventricular myocytes were isolated by enzymatic disaggregation. The whole cell patch-clamp technique was used to examine dose-dependent effects of phenylephrine (PE) on holding current (Ih) and to determine whether observed actions were mediated via alpha 1a-or alpha 1b-adrenergic receptors. To minimize the contribution of transsar-colemmal currents other than Ip to Ih, membrane voltage was held constant -40 mV, and cells were maintained in a Ca-free perfusate containing 1 mM Ba and 0.1 mM Cd. All experiments were conducted in the presence of 3 microM nadolol. PE elicited dose-dependent increases in Ih, with a peak effect of 0.57 +/- 0.03 pA/pF observed at 30 microM. The response to PE was dose dependently inhibited by prazosin and chloroethylclonidine and was totally eliminated by 1 mM ouabain. When used at doses selective for the alpha 1a-subtype, WB4101 failed to significantly antagonize the action of PE. These data suggest that the observed alpha 1-adrenoceptor-mediated increase in Ih in isolated rat ventricular myocytes is the result of an increase in Ip effected via stimulation of alpha 1b-adrenergic receptors.

Characterization of Ca(2+)-release channels in fetal and adult rat hearts

1995 ◽  
Vol 269 (3) ◽  
pp. H778-H782 ◽  
Author(s):  
V. Ramesh ◽  
M. J. Kresch ◽  
A. M. Katz ◽  
D. H. Kim
Keyword(s):  
Fetal Heart ◽  
Postnatal Growth ◽  
Hill Coefficient ◽  
Sprague Dawley ◽  
Release Channel ◽  
Adult Rat ◽  
Biochemical Assays ◽  
Rat Hearts ◽  
Distinguishing Property

The goal of this study was to characterize the Ca(2+)-release channel in whole homogenates of left (LV) and right ventricles (RV) of fetal (22 days in gestation) and adult Sprague-Dawley rat hearts using [3H]ryanodine binding and 45Ca2+ fluxes. Although many features of the Ca(2+)-release channels were similar in fetal and adult hearts, biochemical assays revealed quantitative differences. Similar properties include 1) Ca(2+)-sensitive cooperative ryanodine binding to Ca(2+)-release channel, measured as Ca2+ concentration for half-maximal activation (fetal LV: 0.13 +/- 0.02 microM; adults LV: 0.15 +/- 0.02 microM) and Hill coefficient (fetal LV: 2.5 +/- 0.9; adult LV: 2.7 +/- 0.5), and 2) caffeine-sensitive ryanodine binding, measured as the percent increase in ryanodine binding induced by caffeine (fetal LV: 148.8 +/- 16.9% vs. adult LV: 171.4 +/- 34.9%). The distinguishing property was the lower Ca(2+)-release channel density in the fetal heart (LV: 0.22 +/- 0.03 pmol/mg protein) compared with adult heart (LV: 0.59 +/- 0.04 pmol/mg protein; P < 0.05), as determined by [3H]ryanodine binding. The lower density of Ca(2+)-release channel is supported by the finding that there is very low ryanodine-sensitive oxalate-supported 45Ca2+ uptake in the fetal heart. The tested characteristics of the Ca(2+)-release channel were similar between LV and RV in both fetal and adult rat hearts. Ou results indicate that expression of Ca2+-release channels in sarcoplasmic reticulum increases during postnatal growth in the rat heart. This is consistent with previous physiological reports that Ca2+ available for excitation-contraction coupling in the fetal heart is derived mainly from transsarcolemmal Ca2+ influx.

STIMULATION OF SYNTHESIS OF GLYCOPROTEIN ENZYMES IN THE RAT UTERUS BY OESTRADIOL

1968 ◽  
Vol 42 (2) ◽  
pp. 261-NP ◽  
Author(s):  
F. A. DUGAN ◽  
B. RADHAKRISHNAMURTHY ◽  
R. A. RUDMAN ◽  
G. S. BERENSON
Keyword(s):  
Total Activity ◽  
Alkaline Phosphatases ◽  
Rat Uterus ◽  
Oestrogen Treatment ◽  
Immature Rat ◽  
Adult Rat ◽  
Electrophoretic Patterns ◽  
Electrophoretic Mobilities ◽  
Hydrolysis Of ◽  
Stimulation Of

SUMMARY Glycoproteins from immature and immature, oestrogen-stimulated and adult rat uteri were isolated and analysed by chemical and gel electrophoretic methods. Esterase, acid phosphatase, alkaline phosphatase and peroxidase activities were found. Changes in electrophoretic mobilities of certain enzyme bands in polyacrylamide gel were also observed after hydrolysis of the preparations with neuraminidase. These latter observations and chemical analyses provide additional evidence of the carbohydrate nature of the enzymes. The influence of 17β-oestradiol on immature rat uteri caused a significant increase in total protein and sialic acid per uterus compared with controls. Oestrogen treatment also resulted in an increase in the total activity of esterase and acid and alkaline phosphatases per uterus, but there was no increase in specific activities. Observations of electrophoretic patterns of glycoprotein preparations from untreated and oestrogen-stimulated, immature uteri did not show the evolution to a more adult pattern by oestrogen stimulation. These studies show that stimulation with oestrogen increases the synthesis of glycoprotein in the immature rat uterus. Factors which are involved in the more intricate control of glycoprotein biosynthesis need to be elucidated.

TRI-IODOTHYRONINE INCREASES INTRA-CELLULAR CALCIUM LEVELS IN SINGLE RAT MYOCYTES

1991 ◽  
Vol 7 (1) ◽  
pp. 77-79 ◽  
Author(s):  
R.B. Lomax ◽  
P.H. Cobbold ◽  
A.P. Allshire ◽  
K.S.R. Cuthbertson ◽  
W.R. Robertson
Keyword(s):  
Ventricular Myocytes ◽  
Single Cells ◽  
Mean Amplitude ◽  
Free Calcium ◽  
Acute Administration ◽  
Cytosolic Free Calcium ◽  
Rat Hearts ◽  
Rat Myocytes ◽  
Stimulation Of

ABSTRACT We have studied the effects of acute administration of tri-iodothyronine (T3) on cytosolic free calcium levels [Ca2+]i in single rat myocytes microinjected with aequorin. Ventricular myocytes were isolated by perfusing rat hearts with collagenase, and healthy, rod-shaped cells were injected to <1% of their volume with aequorin. The photons emitted from single cells were measured and a conversion to [Ca2+]i made on the basis of an in vitro calibration after the remaining aequorin had been discharged by cell lysis. Only cells that depolarized reversibly (showing elevated [Ca2+]i levels) when superfused with 80mM KC1, and which gave a substantial signal on lysis with distilled water were used. The [Ca2+]i rose from a resting value of 150±56nM (mean ± SD, n=14) by 127±47nM on depolarization with 80mM KC1. Application of T3 (1-100nM) led to an increase (P<0.05) in [Ca2+]i (mean amplitude of 152±35nM) before returning to baseline. The median duration of these events was 10 min (range = 1.4-34.4 min). The time to response was shorter when lOOnM T3 was applied (median and range; 6.8, 0-14 min) than when 1nM T3 was used (16, 7.0-56.1 min) (P<0.05). To conclude, physiological concentrations of thyroid hormones caused rapid but transient stimulation of [Ca2+]i in single rat myocytes.

Propofol Increases Contractility during α1a-Adrenoreceptor Activation in Adult Rat Cardiomyocytes

2005 ◽  
Vol 103 (2) ◽  
pp. 335-343 ◽  
Author(s):  
Brad D. Gable ◽  
Toshiya Shiga ◽  
Paul A. Murray ◽  
Derek S. Damron
Keyword(s):  
Phospholipase A2 ◽  
Ventricular Myocytes ◽  
Rho Kinase ◽  
Selective Inhibition ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Kinase C ◽  
Rat Hearts ◽  
Dependent Pathway ◽  
Exchange Activity

Background The objective of this study was to identify the extent to which propofol alters intracellular free Ca2+ concentration ([Ca2+]i), myofilament Ca sensitivity, and contraction of individual cardiomyocytes during activation of alpha1a adrenoreceptors and to determine the cellular mechanism of action. Methods Freshly isolated ventricular myocytes were obtained from adult rat hearts. Myocyte shortening and [Ca2+]i were simultaneously monitored in individual cardiomyocytes exposed to phenylephrine after treatment with chloroethylclonidine (alpha1b-adrenoreceptor antagonist) and BMY 7378 (alpha1d-adrenoreceptor antagonist). Data are reported as mean +/- SD. Results Phenylephrine increased myocyte shortening by 124 +/- 9% (P = 0.002), whereas peak [Ca2+]i only increased by 8 +/- 3% (P = 0.110). Inhibition of phospholipase A2 and phospholipase C attenuated the phenylephrine-induced increase in shortening by 84 +/- 11% (P = 0.004) and 15 +/- 6% (P = 0.010), respectively. Inhibition of protein kinase C (PKC) and Rho kinase attenuated the phenylephrine-induced increase in shortening by 17 +/- 8% (P = 0.010) and 74 +/- 13% (P = 0.006), respectively. In the presence of phenylephrine, propofol increased shortening by 40 +/- 6% (P = 0.002), with no concomitant increase in [Ca2+]i. PKC inhibition prevented the propofol-induced increase in shortening. Selective inhibition of PKCalpha, PKCdelta, PKCepsilon, and PKCzeta reduced the propofol-induced increase in shortening by 12 +/- 5% (P = 0.011), 36 +/- 8% (P = 0.001), 32 +/- 9% (P = 0.007), and 19 +/- 5% (P = 0.008), respectively. Na+ - H+ exchange inhibition reduced the propofol-induced increase in shortening by 56 +/- 7% (P = 0.001). Conclusion Activation of alpha1a adrenoreceptors increases cardiomyocyte shortening primarily via a phospholipase A2-dependent, Rho kinase-dependent increase in myofilament Ca2+ sensitivity. Propofol further increases myofilament Ca2+ sensitivity and shortening via a PKC-dependent pathway and an increase in Na+ - H+ exchange activity.

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