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.

Purine metabolism in isolated rat hepatocytes

1977 ◽  
Vol 55 (11) ◽  
pp. 1134-1139 ◽  
Author(s):  
Camilla M. Smith ◽  
Liisa M. Rovamo ◽  
Martti P. Kekomäki ◽  
Kari O. Raivio
Keyword(s):  
Cell Function ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Purine Metabolism ◽  
Nucleotide Synthesis ◽  
Isolated Rat Hepatocytes ◽  
Collagenase Perfusion ◽  
Adenine Nucleotide Pool

The metabolism of adenine, hypoxanthine, guanine, and adenosine was studied in rat liver cell suspensions, prepared by collagenase perfusion. Oxygen supply was a critical variable in the preparation and subsequent incubation of the cells, as judged on the basis of the ratio of radioactivity in ATP to that in ADP after incubation with [14C]adenine. This ratio is suggested as an additional criterion of cell function. Adenine nucleotides synthesized from [14C]adenine were slowly catabolized to allantoin, with little incorporation of radioactivity into other purine compounds. [14C]Adenine is thus suitable for prelabelling the adenine nucleotide pool. [14C]Guanine and [14C]hypoxanthine were rapidly catabolized to allantoin, whereas nucleotide synthesis was low. [14C]Adenosine was initially phosphorylated and deaminated at about equal rates, but with continued incubation catabolic products predominated. Isolated hepatocytes were found suitable for studies of purine metabolism, in which the liver has important functions for the whole organism.

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.

Metabolism of adenine nucleotides in the cultured fetal mouse heart

1977 ◽  
Vol 233 (2) ◽  
pp. H282-H288
Author(s):  
I. A. Kaufman ◽  
N. F. Hall ◽  
M. A. DeLuca ◽  
J. S. Ingwall ◽  
S. E. Mayer
Keyword(s):  
Organ Culture ◽  
Adenosine Deaminase ◽  
De Novo ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Energy Charge ◽  
Mouse Heart ◽  
Nucleotide Synthesis ◽  
Fetal Mouse ◽  
Deprivation Period

Intact beating fetal mouse hearts in organ culture were deprived of oxygen and glucose for up to 4 h, resulting in loss of beating, an 80% fall in ATP, reduction of energy charge from 0.85 to 0.48, and doubling of total nucleoside concentration. Radiolabeled adenine nucleotides were degraded to hypoxanthine and inosine, which were lost from the hearts into the medium during the deprivation period. Adenosine and adenine also appeared in the medium when adenosine deaminase was inhibited. After 24 h of O2 and glucose resupply, ATP returned to 60% of control, and energy charge rose to 0.76. Labeled nucleosides and bases remaining in the heart or exogenous labeled adenine were utilized to resynthesize ATP. [14C]glycine was rapidly taken up by recovering hearts but was not used for de novo adenine nucleotide synthesis. Ability to recover ATP and spontaneous contraction appear related to residual nucleotide and nucleoside content rather than to energy charge.

Adenine nucleotide synthesis from inosine during normoxia and after ischaemia in the isolated perfused rat heart

1985 ◽  
Vol 63 (9) ◽  
pp. 1159-1164 ◽  
Author(s):  
J. Aussedat ◽  
M. Verdys ◽  
A. Rossi
Keyword(s):  
Rat Heart ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Low Flow ◽  
Nucleotide Synthesis ◽  
Perfused Rat Heart ◽  
Isolated Perfused Rat Heart ◽  
Nucleotide Content ◽  
Glycogen Stores

[14C]inosine in a range of concentrations of 20 μM to 1 mM was administered-to the isolated perfused rat heart for 30 min. The incorporation of the nucleoside into myocardial adenine nucleotides increased for extracellular concentrations of the precursor up to 50 μM, reaching a plateau at 60 nmol∙g−1∙30 min−1 with concentrations ranging between 50 and 200 μM. The supply of 500 μM and 1 mM of inosine induced a further increase in cardiac adenine nucleotide synthesis to about 200 nmol∙g−1∙30 min−1. When supplied during low flow ischaemia (0.5 mL∙min−1, 30 min.), 1 mM of inosine protected the heart against ATP degradation, while 100 μM of inosine was inefficacious. In the presence of 1 mM of inosine on reperfusion the adenine nucleotide content of the heart was similar to that observed in the absence of the nucleoside. The incorporation of [14C]inosine into adenine nucleotides was, in this last condition, below the value measured before ischaemia. Inosine administration was effective in protecting the heart against ischaemic breakdown of glycogen and favoured postischaemic restoration of glycogen stores.

Natriuretic peptide receptor-B in adult rat ventricle is predominantly confined to the nonmyocyte population

2002 ◽  
Vol 282 (6) ◽  
pp. H2117-H2123 ◽  
Author(s):  
Donald D. Doyle ◽  
Judy Upshaw-Earley ◽  
Eric L. Bell ◽  
H. Clive Palfrey
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Natriuretic Peptide ◽  
Rat Heart ◽  
Cardiac Fibroblasts ◽  
Muscle Cells ◽  
Neonatal Rat ◽  
Cell Protein ◽  
Rat Cardiomyocytes ◽  
Adult Rat

We assessed the cellular localization and relative concentration of the C-type natriuretic peptide (CNP) guanylate cyclase-B (GC-B) receptor in the adult rat heart ventricle by several techniques. In frozen sections of the ventricle, anti-receptor antibody stained the vasculature and cells interstitial to myocytes, but not the myocytes themselves. The same antibody detected GC-B in immunoblots of protein extracts of nonmyocytes, but not myocytes and recognized an equivalent protein in extracts of cultured cardiac fibroblasts, but not A7r5 rat smooth muscle cells. In functional assays, CNP-induced cGMP accumulation per milligram cell protein was an order of magnitude greater in cultured cardiac fibroblasts than in A7r5 smooth muscle cells and two orders of magnitude greater than in freshly isolated cardiac myocytes. Modulation of cGMP accumulation by phosphodiesterases (PDEs) was cell specific as determined by antagonist pharmacological profiles, PDE1 in fibroblasts, PDE2 in A7r5 cells, and PDE3 in myocytes, suggesting that significant but low-level cGMP response to CNP measured in heart myocytes is not due to nonmyocyte contamination. Fibroblasts of cardiac origin do not show an interactive relationship between receptor responsiveness to CNP, cGMP levels, and proliferation-related mitogen-activated signal transduction pathways. Whereas previous reports suggest CNP exerts significant effects in neonatal rat cardiomyocytes, our results suggest that fibroblasts are likely the most responsive cell type (cGMP production) in the adult rat heart.

BDM drives protein dephosphorylation and inhibits adenine nucleotide exchange in cardiomyocytes

1998 ◽  
Vol 275 (4) ◽  
pp. H1260-H1266 ◽  
Author(s):  
Mary T. Stapleton ◽  
Claudia M. Fuchsbauer ◽  
Ashley P. Allshire
Keyword(s):  
Atp Hydrolysis ◽  
Adenine Nucleotide ◽  
Atp Depletion ◽  
Metabolic Inhibitors ◽  
Nucleotide Exchange ◽  
Futile Cycle ◽  
Intact Cells ◽  
Rat Cardiomyocytes ◽  
Permeabilized Cells ◽  
Adult Rat

Contractile dysfunction plays a key role in injury sustained by ischemic myocardium at reperfusion, whereas interventions that impede hypercontracture enhance recovery. In permeabilized adult rat cardiomyocytes, the negative inotrope 2,3-butanedione monoxime (BDM; 10–50 mM) inhibited rigor at low MgATP concentration but stimulated net ATP hydrolysis. Hydrolysis was attenuated by H-7, kaempferol, chelerythrine, and genistein. Evidently BDM opposed phosphorylation of both serine/threonine and tyrosine kinase target proteins, either directly or by enhancing protein phosphatase activity, in a futile cycle of ATP hydrolysis independent of cross-bridge cycling. Although 20 mM BDM did not affect the onset of rigor contracture in permeabilized cells at low MgATP, in intact cells exposed to the metabolic inhibitors cyanide and 2-deoxyglucose rigor onset was accelerated, indicating that BDM increases ATP depletion in quiescent cardiomyocytes. Conversely, in cells exposed to the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, BDM delayed the onset of contracture and hence ATP depletion, consistent with an inhibition of adenine nucleotide movement across the mitochondrial inner membrane. Such effects will limit the value of BDM as a cardioprotective agent at physiological temperature.

scholarly journals Hypoxia and the energy charge of the cerebral adenylate pool

1972 ◽  
Vol 127 (2) ◽  
pp. 351-355 ◽  
Author(s):  
J. W. Ridge
Keyword(s):  
Rat Brain ◽  
Feedback Inhibition ◽  
Adenine Nucleotide ◽  
Energy Charge ◽  
Nucleotide Synthesis ◽  
Limited Ability ◽  
Adenylate Pool ◽  
The Brain

A brief period of anoxia in vivo causes a transitory decrease in the size of the adenylate pool in the rat brain. This is probably caused by feedback inhibition by AMP of adenine nucleotide synthesis. Exposing rats to various degrees of hypoxia suggests that the sensitivity of the brain to lack of O2 results from the brain's limited ability to maintain an adequate energy charge in unfavourable circumstances.

The Platelet of the Newborn Infant – Adenine Nucleotide Metabolism and Release

1980 ◽  
Vol 43 (02) ◽  
pp. 099-103 ◽  
Author(s):  
J M Whaun ◽  
P Lievaart ◽  
Keyword(s):  
Newborn Infant ◽  
Adenine Nucleotide ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Newborn Infants ◽  
Specific Radioactivity ◽  
Nucleotide Metabolism ◽  
Breakdown Product ◽  
Term Infants ◽  
Adenine Nucleotide Metabolism

SummaryBlood from normal full term infants, mothers and normal adults was collected in citrate. Citrated platelet-rich plasma was prelabelled with 3H-adenine and reacted with release inducers, collagen and adrenaline. Adenine nucleotide metabolism, total adenine nucleotide levels and changes in sizes of these pools were determined in platelets from these three groups of subjects.At rest, the platelet of the newborn infant, compared to that of the mother and normal adult, possessed similar amounts of adenosine triphosphate (ATP), 4.6 ± 0.2 (SD), 5.0 ± 1.1, 4.9 ± 0.6 µmoles ATP/1011 platelets respectively, and adenosine diphosphate (ADP), 2.4 ± 0.7, 2.8 ± 0.6, 3.0 ± 0.3 umoles ADP/1011 platelets respectively. However the marked elevation of specific radioactivity of ADP and ATP in these resting platelets indicated the platelet of the neonate has decreased adenine nucleotide stores.In addition to these decreased stores of adenine nucleotides, infant platelets showed significantly impaired release of ADP and ATP on exposure to collagen. The release of ADP in infants, mothers, and other adults was 0.9 ± 0.5 (SD), 1.5 ± 0.5, 1.5 ± 0.1 umoles/1011 platelets respectively; that of ATP was 0.6 ± 0.3, 1.0 ± 0.1,1.3 ± 0.2 µmoles/1011 platelets respectively. With collagen-induced release, platelets of newborn infants compared to those of other subjects showed only slight increased specific radioactivities of adenine nucleotides over basal levels. The content of metabolic hypoxanthine, a breakdown product of adenine nucleotides, increased in both platelets and plasma in all subjects studied.In contrast, with adrenaline as release inducer, the platelets of the newborn infant showed no adenine nucleotide release, no change in total ATP and level of radioactive hypoxanthine, and minimal change in total ADP. The reason for this decreased adrenaline reactivity of infant platelets compared to reactivity of adult platelets is unknown.Infant platelets may have different membranes, with resulting differences in regulation of cellular processes, or alternatively, may be refractory to catecholamines because of elevated levels of circulating catecholamines in the newborn period.

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