Hyperthyroid adult rat cardiomyocytes. I. Nucleotide content, beta- and alpha-adrenoreceptors, and cAMP production

1989 ◽  
Vol 257 (5) ◽  
pp. C948-C956 ◽  
Author(s):  
C. M. Hohl ◽  
S. Wetzel ◽  
R. H. Fertel ◽  
D. K. Wimsatt ◽  
G. P. Brierley ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Adenine Nucleotide ◽  
Phosphodiesterase Inhibitor ◽  
Cardiac Cells ◽  
Beta Agonist ◽  
Camp Production ◽  
Adult Rats ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Half Maximal Effective Concentration

Ventricular myocytes isolated from the hypertrophied hearts of thyrotoxic adult rats have an increase in mean protein content per myocyte (6.3 +/- 0.2 vs. 4.4 +/- 0.2 ng) compared with euthyroid cells. Viability and adenine nucleotide profiles are similar in both populations, but NAD content of the hyperthyroid myocytes is depressed (4.9 +/- 0.2 vs. 5.5 +/- 0.2 nmol/mg for controls) and UTP is higher (1.2 +/- 0.09 vs. 0.9 +/- 0.04 nmol/mg). Binding of (-)-[125I]iodocyanopindolol to intact hyperthyroid myocytes is increased by 42% compared with controls, with no change in the dissociation constant (Kd). This elevation in beta-receptor number is correlated to enhanced beta-agonist-induced adenosine 3',5'-cyclic monophosphate (cAMP) production. The half-maximal effective concentration (EC50) for the euthyroid isoproterenol dose-response curve is 2.14 x 10(-7) M but is decreased to 2.51 x 10(-8) M in hyperthyroid cardiac cells. Basal adenylate cyclase activity is apparently not affected by thyroid hormones, since basal cAMP levels for both groups are identical (5 pmol/mg) and both rise roughly twofold in the presence of a phosphodiesterase inhibitor. Forskolin-induced cAMP production and cAMP-specific phosphodiesterase activity are similar as well. In contrast to beta-adrenergic response, there are no significant differences in alpha 1-antagonist [3H]prazosin binding parameters between hyperthyroid and euthyroid cardiomyocytes.

scholarly journals A spliced variant of AE1 gene encodes a truncated form of Band 3 in heart: the predominant anion exchanger in ventricular myocytes

1999 ◽  
Vol 112 (10) ◽  
pp. 1519-1528
Author(s):  
S.M. Richards ◽  
M.E. Jaconi ◽  
G. Vassort ◽  
M. Puceat
Keyword(s):  
Ventricular Myocytes ◽  
Mrna Level ◽  
Band 3 ◽  
Cardiac Cells ◽  
Protein Isoforms ◽  
Truncated Form ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Protein Levels ◽  
Gene And Protein Expression

The anion exchangers (AE) are encoded by a multigenic family that comprises at least three genes, AE1, AE2 and AE3, and numerous splicoforms. Besides regulating intracellular pH (pHi) via the Cl-/HCO3- exchange, the AEs exert various cellular functions including generation of a senescent antigen, anchorage of the cytoskeleton to the membrane and regulation of metabolism. Most cells express several AE isoforms. Despite the key role of this family of proteins, little is known about the function of specific AE isoforms in any tissue, including the heart. We therefore chose isolated cardiac cells, in which a tight control of pHi is mandatory for the excitation-contraction coupling process, to thoroughly investigate the expression of the AE genes at both the mRNA and protein levels. RT-PCR revealed the presence of AE1, AE2 and AE3 mRNAs in both neonatal and adult rat cardiomyocytes. AE1 is expressed both as the erythroid form (Band 3 or eAE1) and a novel alternate transcript (nAE1), which was more specifically characterized using a PCR mapping strategy. Two variants of AE2 (AE2a and AE2c) were found at the mRNA level. Cardiac as well as brain AE3 mRNAs were expressed in both neonatal and adult rat cardiomyocytes. Several AE protein isoforms were found, including a truncated form of AE1 and two AE3s, but there was no evidence of AE2 protein in adult rat cardiomyocytes. In cardiomyocytes transfected with an AE3 oligodeoxynucleotide antisense, AE3 immunoreactivity was dramatically decreased but the activity of the Cl-/HCO3- exchange was unchanged. In contrast, intracellular microinjection of blocking anti-AE1 antibodies inhibited the AE activity. Altogether, our findings suggest that a specific and novel AE1 splicoform (nAE1) mediates the cardiac Cl-/HCO3- exchange. The multiple gene and protein expression within the same cell type suggest numerous functions for this protein family.

Do β2-adrenergic receptors modulate Ca2+ in adult rat ventricular myocytes?

1998 ◽  
Vol 274 (4) ◽  
pp. H1308-H1314 ◽  
Author(s):  
Michael A. Laflamme ◽  
Peter L. Becker
Keyword(s):  
Pertussis Toxin ◽  
Calcium Homeostasis ◽  
Adrenergic Receptors ◽  
Calcium Current ◽  
Ventricular Myocytes ◽  
Camp Accumulation ◽  
Camp Production ◽  
Rat Ventricular Myocytes ◽  
Adult Rat

We examined the role of β2-adrenergic receptors (ARs) in modulating calcium homeostasis in rat ventricular myocytes. Zinterol (10 μM), an agonist with a 25-fold greater affinity for β2-ARs over β1-ARs, modestly enhanced L-type calcium current ( I Ca) magnitude by ∼30% and modestly accelerated the rate of Ca2+ concentration ([Ca2+]) decline (∼35%) but had little effect on the magnitude of the [Ca2+] transient (a nonsignificant 6% increase). However, 1 μM of the highly selective β1-AR antagonist CGP-20712A completely blocked the I Ca increase induced by 10 μM zinterol. Pretreatment of cells with pertussis toxin (PTX) did not alter I Ca enhancement by 10 μM zinterol, although it did abolish the ability of acetylcholine to block the forskolin-induced enhancement of I Ca. Zinterol (10 μM) approximately doubled adenosine 3′,5′-cyclic monophosphate (cAMP) accumulation, although one-half of this increase was blocked by CGP-20712A. In contrast, 1 μM of the nonselective β-agonist isoproterenol increased cAMP production 15-fold. Thus we found no evidence that activation of β2-ARs modulates calcium homeostasis in rat ventricular myocytes, even after treatment with PTX.

scholarly journals Functional localization of cAMP signalling in cardiac myocytes

2006 ◽  
Vol 34 (4) ◽  
pp. 484-488 ◽  
Author(s):  
G. Vandecasteele ◽  
F. Rochais ◽  
A. Abi-Gerges ◽  
R. Fischmeister
Keyword(s):  
Spatial Organization ◽  
Molecular Mechanisms ◽  
Ventricular Myocytes ◽  
Cardiac Cells ◽  
Functional Responses ◽  
Adult Rat ◽  
Cyclic Nucleotide Gated Channels ◽  
Camp Pathway ◽  
Functional Localization ◽  
Heart Physiology

The cAMP pathway is of cardinal importance for heart physiology and pathology. The spatial organization of the various components of the cAMP pathway is thought to allow the segregation of functional responses triggered by the different neuromediators and hormones that use this pathway. PDEs (phosphodiesterases) hydrolyse cAMP (and cGMP) and play a major role in this process by preventing cAMP diffusion to the whole cytosol and inadequate target activation. The development of olfactory cyclic nucleotide-gated channels to directly monitor cAMP beneath the plasma membrane in real time allows us to gain new insights into the molecular mechanisms responsible for cAMP homoeostasis and hormonal specificity in cardiac cells. The present review summarizes the recent results we obtained using this approach in adult rat ventricular myocytes. In particular, the role of PDEs in the maintenance of specific cAMP signals generated by β-adrenergic receptors and other Gs-coupled receptors will be discussed.

scholarly journals Elevation of the adenylate pool in rat cardiomyocytes by S-adenosyl-L-methionine.

2000 ◽  
Vol 47 (4) ◽  
pp. 1171-1178 ◽  
Author(s):  
R T Smolenski
Keyword(s):  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Normal Function ◽  
Cell Protein ◽  
Nucleotide Synthesis ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Adenylate Pool ◽  
Collagenase Perfusion ◽  
Subsequent Incorporation

Rapid resynthesis of the adenylate pool in cardiac myocytes is important for recovery of contractility and normal function of regulatory mechanisms in the heart. Adenosine and adenine are thought to be the most effective substrates for nucleotide synthesis, but the possibility of using other compounds has been studied very little in cardiomyocytes. In the present study, the effect of S-adenosyl-L-methionine (SAM) on the adenylate pool of isolated cardiomyocytes was investigated and compared to the effect of adenine and adenosine. Adult rat cardiomyocytes were isolated using the collagenase perfusion technique. The cells were incubated in the presence of adenine derivatives for 90 min followed by nucleotide determination by HPLC. The concentrations of adenine nucleotides expressed in nmol/mg of cell protein were initially 22.1 +/- 1.4, 4.0 +/- 0.3 and 0.70 +/- 0.08 for ATP, ADP and AMP, respectively (n = 10, +/- S.E.M.), and the total adenylate pool was 26.8 +/- 1.6. In the presence of 1.25 mM SAM in the medium, the adenylate pool increased by 5.2 +/- 0.4 nmol/mg of cell protein, but only if 1 mM ribose was additionally present in the medium. No changes were observed with SAM alone. A similar increase (by 4.9 +/- 0.6 nmol/mg protein) was observed after incubation with 1.25 mM adenine plus 1 mM ribose, but no increase was observed if ribose was omitted. Adenosine at 0.1 or 1.25 mM concentrations also caused an increase in the adenylate pool (by 5.2 +/- 1.0 and 5.2 +/- 0.9 nmol/mg protein, respectively), which in contrast to the SAM or adenine was independent of the additional presence of ribose. Thus, S-adenosyl-L-methionine could be used as a precursor of the adenylate pool in cardiomyocytes, which is as efficient in increasing the adenylate pool after 90 min of incubation as adenosine or adenine. Nucleotide synthesis from SAM involves the formation of adenine as an intermediate with its subsequent incorporation by adenine phosphoribosyltransferase.

Hyperthyroid adult rat cardiomyocytes. II. Single cell electrophysiology and free calcium transients

1989 ◽  
Vol 257 (5) ◽  
pp. C957-C963 ◽  
Author(s):  
Q. Li ◽  
Z. Guan ◽  
B. A. Biagi ◽  
B. T. Stokes ◽  
R. A. Altschuld
Keyword(s):  
Single Cell ◽  
Action Potentials ◽  
Mechanical Performance ◽  
Ventricular Myocytes ◽  
Calcium Transients ◽  
Calcium Handling ◽  
Free Calcium ◽  
Half Time ◽  
Rat Cardiomyocytes ◽  
Adult Rat

The effects of hyperthyroidism on electrophysiological properties and intracellular free calcium transients in single adult rat cardiomyocytes were studied using conventional microelectrodes and time-resolved single cell fura-2 fluorescence microscopy. Under control conditions, resting membrane potentials and triggered action potentials were not different in euthyroid and hyperthyroid myocytes. Calcium transients produced by electrical stimulation, however, were markedly abbreviated in hyperthyroid myocytes. During a train of stimuli, the duration of the calcium transients at half peak amplitude (half time) was 124 +/- 14 ms at the fifth beat in hyperthyroid cells vs. 287 +/- 35 ms in euthyroid cells. Isoproterenol (1 microM) prolonged time to 50% repolarization (APD50) of the action potentials and increased the peak calcium transients in both euthyroid and hyperthyroid myocytes. It also shortened the half time of the calcium transients in euthyroid myocytes but had little effect on the half time in hyperthyroid cells. These data are consistent with the electrophysiology and mechanical performance in intact euthyroid and hyperthyroid cardiac tissues, and the intrinsic changes in hyperthyroid tissues can therefore be illustrated in single ventricular myocytes. Furthermore, the results suggest that alterations in intracellular calcium handling by sarcoplasmic reticulum may account for contractile changes of the heart induced by hyperthyroidism.

Adenosine stimulation of AMP deaminase activity in adult rat cardiac myocytes

1993 ◽  
Vol 264 (1) ◽  
pp. C48-C53 ◽  
Author(s):  
B. Hu ◽  
R. A. Altschuld ◽  
C. M. Hohl
Keyword(s):  
Ventricular Myocytes ◽  
Cardiac Cells ◽  
Adenosine A1 Receptor ◽  
Nucleoside Transport ◽  
Amp Deaminase ◽  
Adult Rat ◽  
Adenosine A1 ◽  
Adenosine Transport ◽  
A1 Receptor

Using an in situ assay for analyzing AMP deaminase activity in isolated adult rat ventricular myocytes, we have shown that IMP production is stimulated approximately twofold in cardiac cells incubated with 10 microM adenosine. This effect of adenosine was not blocked by the adenosine A1-receptor antagonist 8-cyclophenyl-1,3-dipropylaxanthine (0.01-1 microM) except at a concentration (100 microM) that may inhibit adenosine transport. Similarly, in situ AMP deaminase activity was not enhanced by treatment with the specific adenosine A1-receptor agonists N6-phenylisopropyl adenosine or cyclopentyladenosine, nor was it sensitive to prior treatment of cells with pertussis toxin. The nucleoside transport blockers S-4-nitrobenzyl-6-thioinosine, dipyridamole, and papaverine inhibited adenosine-induced increases in IMP production by 75-85%, suggesting an intracellular site of action. Modulation of enzyme activity via the transmethylation pathway could not be implicated since incubation of cardiac cells under conditions known to elevate intracellular S-adenosyl-L-homocysteine had no demonstrable effect on AMP deaminase. Furthermore, a direct allosteric effect of adenosine on the partially purified rat cardiac enzyme was not observed. The results indicate that intracellular adenosine modulates rat cardiac AMP deaminase by an unknown mechanism.

Subcellular localization of the delayed rectifier K+ channels KCNQ1 and ERG1 in the rat heart

2004 ◽  
Vol 286 (4) ◽  
pp. H1300-H1309 ◽  
Author(s):  
Hanne Borger Rasmussen ◽  
Morten Møller ◽  
Hans-Günther Knaus ◽  
Bo Skaaning Jensen ◽  
Søren-Peter Olesen ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Rat Heart ◽  
Ventricular Myocytes ◽  
Cardiac Cells ◽  
Delayed Rectifier ◽  
Transverse Tubular System ◽  
Adult Rat ◽  
Confocal Immunofluorescence Microscopy ◽  
Ventricular Cells ◽  
Tubular System

In the heart, several K+ channels are responsible for the repolarization of the cardiac action potential, including transient outward and delayed rectifier K+ currents. In the present study, the cellular and subcellular localization of the two delayed rectifier K+ channels, KCNQ1 and ether- a- go- go-related gene-1 (ERG1), was investigated in the adult rat heart. Confocal immunofluorescence microscopy of atrial and ventricular cells revealed that whereas KCNQ1 labeling was detected in both the peripheral sarcolemma and a structure transversing the myocytes, ERG1 immunoreactivity was confined to the latter. Immunoelectron microscopy of atrial and ventricular myocytes showed that the ERG1 channel was primarily expressed in the transverse tubular system and its entrance, whereas KCNQ1 was detected in both the peripheral sarcolemma and in the T tubules. Thus, whereas ERG1 displays a very restricted subcellular localization pattern, KCNQ1 is more widely distributed within the cardiac cells. The localization of these K+ channels to the transverse tubular system close to the Ca2+ channels renders them with maximal repolarizing effect.

Adult rat cardiomyocytes exhibit capacitative calcium entry

2004 ◽  
Vol 286 (3) ◽  
pp. H1124-H1132 ◽  
Author(s):  
Dacia L. Hunton ◽  
LuYun Zou ◽  
Yi Pang ◽  
Richard B. Marchase
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Ventricular Myocytes ◽  
Physiological Responses ◽  
Atpase Inhibitor ◽  
Capacitative Calcium Entry ◽  
Rat Cardiomyocytes ◽  
Inward Current ◽  
Adult Rat ◽  
Adult Cardiomyocytes ◽  
Inositol 1,4,5 Trisphosphate

Capacitative Ca2+ entry (CCE) refers to the influx of Ca2+ through plasma membrane channels activated on depletion of endoplasmic-sarcoplasmic reticulum Ca2+ stores. We utilized two Ca2+-sensitive dyes (one monitoring cytoplasmic free Ca2+ and the other free Ca2+ within the sarcoplasmic reticulum) to determine whether adult rat ventricular myocytes exhibit CCE. Treatments with inhibitors of the sarcoplasmic endoplasmic reticulum Ca2+-ATPases were not efficient in releasing Ca2+ from stores. However, when these inhibitors were coupled with either Ca2+ ionophores or angiotensin II (an agonist generating inositol 1,4,5 trisphosphate), depletion of stores was observed. This depletion was accompanied by a significant influx of extracellular Ca2+ characteristic of CCE. CCE was also observed when stores were depleted with caffeine. This influx of Ca2+ was sensitive to four inhibitors of CCE (glucosamine, lanthanum, gadolinium, and SKF-96365) but not to inhibitors of L-type channels or the Na+/Ca2+ exchanger. In the whole cell configuration, an inward current of ∼0.7 pA/pF at –90 mV was activated when a Ca2+ chelator or inositol (1,4,5)-trisphosphate was included in the pipette or when Ca2+ stores were depleted with a Ca2+-ATPase inhibitor and ionophore. The current was maximal at hyperpolarizing voltages and inwardly rectified. The channel was relatively permeant to Ca2+ and Ba2+ but only poorly to Mg2+ or Mn2+. Taken together, these data support the existence of CCE in adult cardiomyocytes, a finding with likely implications to physiological responses to phospholipase C-generating agonists.

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.

HCO 3 − -dependent alkalinizing transporter in adult rat ventricular myocytes: characterization and modulation

1997 ◽  
Vol 273 (6) ◽  
pp. H2596-H2603 ◽  
Author(s):  
Karine Le Prigent ◽  
Dominique Lagadic-Gossmann ◽  
Emmanuel Mongodin ◽  
Danielle Feuvray
Keyword(s):  
Ventricular Myocytes ◽  
Intracellular Acidification ◽  
Total Acid ◽  
Adult Rats ◽  
Adult Rat ◽  
Ventricular Cells ◽  
Intracellular Ca ◽  
Acid Load ◽  
Decreasing Ph ◽  
Intracellular Acid

The present work was designed to identify the [Formula: see text]-dependent alkalinizing carrier in ventricular myocytes of normal and diabetic adult rats and to determine to what extent this system contributes to acid-equivalent extrusion after an intracellular acidification. We also examined the possible influence of intracellular Ca2+([Formula: see text]) and glycolytic inhibition on the carrier activation. Intracellular pH (pHi) was recorded using seminaphthorhodafluor-1. The [Formula: see text] method was used to induce an intracellular acid load. Evidence is provided for the existence of a Cl−-independent Na+-[Formula: see text]cotransport contributing to pHirecovery from an intracellular acid load in ventricular cells of adult rats. Na+-[Formula: see text]cotransport accounts for 33% of the total acid-equivalent efflux ([Formula: see text]) from normal adult myocytes after intracellular acidification at pHi 6.75 in CO2/[Formula: see text]-buffered solution. In addition, the activity of this carrier, which is not affected either by decreasing [Formula: see text] or by inhibiting Ca2+/calmodulin protein kinase II, is downregulated by inhibition of glycolysis. Under pathophysiological conditions such as diabetes, although total[Formula: see text] was significantly decreased compared with normal myocytes,[Formula: see text] carried by Na+-[Formula: see text]cotransport remained unchanged. However, because of a decrease in Na+/H+exchange, the contribution of this carrier to total[Formula: see text] increased with decreasing pHi (i.e., under conditions that may be associated with an ischemic episode), reaching ∼58% of total[Formula: see text] at pHi 6.75 (vs. ∼33% in normal myocytes).

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