Accumulation and salvage of adenosine and inosine by isolated mature cardiac myocytes

Reoxygenation of ischaemic, energy-depleted heart does not result in sufficiently rapid regeneration of normal adenine nucleotide concentrations for preservation of cardiac function and structure. Salvage of nucleoside as a mechanism for restoration of ATP in the post-ischaemic myocardium is limited by efflux of adenosine during ischaemia. Isolated cardiac myocytes have been used to establish the kinetics of uptake and salvage of adenosine and inosine, measuring the distribution of radioactive nucleoside incorporated into ATP, ADP and AMP. Maximum rates of catalysis of reactions on the salvage pathway, and of enzymes competing for substrates on the pathway, have been established in myocyte extracts. Myocytes have little capacity to salvage or catabolise inosine. Enzyme measurements indicate that salvage of adenosine should proceed at 7-8-times the rate exhibited by intact myocytes dependent upon extracellular adenosine as substrate. The data indicate that the rate of transport of adenosine is not determined by its metabolic utilization, but is the rate-limiting step in the salvage of adenosine.     

Metabolism of hypoxanthine in isolated rat hepatocytes.

The hepatic metabolism of hypoxanthine was investigated by studying both the fate of labelled hypoxanthine, added at micromolar concentrations to isolated rat hepatocyte suspensions, and the kinetic properties of purified hypoxanthine/guanine phosphoribosyltransferase from rat liver. More than 80% of hypoxanthine was oxidized towards allantoin; less than 5% of the label was incorporated into the purine mononucleotides, and a similar proportion appeared transiently in inosine. The maximal velocity of oxidation (approx. 750nmol/min per g of cells) was in close agreement with the known activity of xanthine oxidase in liver extracts. In contrast, the maximal velocity of the incorporation of labelled hypoxanthine into mononucleotides reached only 30nmol/min per g of cells, compared with an activity of hypoxanthine/guanine phosphoribosyltransferase, measured at substrate concentrations analogous to those prevailing intracellularly, of 500nmol/min per g of cells. Hypoxanthine incorporation into the mononucleotides was decreased by allopurinol, anoxia and ethanol, despite inhibition of its oxidation under these conditions; it was increased by incubation of the cells in supraphysiological concentrations of Pi. Allopurinol and anoxia decreased the concentration of phosphoribosyl pyrophosphate inside the cells by respectively 40 and 60%, ethanol had no effect on the concentration of this metabolite and Pi increased its concentration up to 10-fold. The kinetic study of purified hypoxanthine/guanine phosphoribosyltransferase showed that a mixture of ATP, IMP, GMP and GTP, at the concentrations prevailing in the liver cell, decreased the V max. of the enzyme 6-fold, increased its Km for hypoxanthine from 1 to 4 microM and its Km for phosphoribosyl pyrophosphate from 2.5 to 25 microM. In the presence of 5 microM-hypoxanthine and 2.5 microM-phosphoribosyl pyrophosphate, the mixture of nucleotides inhibited the activity of purified hypoxanthine/guanine phosphoribosyltransferase by 95%. It is concluded that this inhibition results in a limited participation of hypoxanthine/guanine phosphoribosyltransferase in the control of the production of allantoin by the liver.     

5'-Nucleotidase activity of isolated mature rat cardiac myocytes

Specific location of 5'-nucleotidase in the heart has been uncertain, some authors citing evidence for an exclusively non-myocyte location, while other data point to the existence of cytoplasmic and membrane-bound fractions. Single myocytes isolated from mature rat heart, and free of endothelial or interstitial cells, have been used to establish that muscle cells of the myocardium are rich in 5'-nucleotidase, exhibiting activity sufficient to account for the total myocardial content of this enzyme. All 5'-nucleotidase is accessible to extracellular AMP. Inhibitors of 5'-nucleotidase and adenosine transport have been used to establish that only the adenosine component of adenine nucleotides is taken up by myocytes, but hydrolysis of AMP by 5'-nucleotidase does not commit the adenosine formed to transport across the sarcolemmal membrane. Myocytes also have ecto-phosphatases which hydrolyse ADP and ATP.     

Degradation and resynthesis of adenine nucleotides in adult rat heart myocytes

The degradation and short-term resynthesis of adenine nucleotides have been examined in a preparation of isolated rat heart myocytes. These myocyte preparations are essentially free of vascular and endothelial cells, contain levels of adenine nucleotides quite comparable to those of intact heart tissue, and retain these components remarkably well for up to 2 h of aerobic incubation in the presence of 1 mM Ca2+. When the cells are rapidly and synchronously de-energized by addition of uncoupler, an inhibitor of respiration and iodoacetate, cellular ATP is degraded almost quantitatively to AMP. The AMP is then converted to either intracellular adenosine, which accumulates to high concentrations before release to the cell exterior, or to IMP. The relative contribution of these two pathways depends on the metabolic state of the cells just prior to de-energization, with IMP production favored when respiring cells are de-energized and adenosine formation predominant when glycolyzing myocytes are subjected to this treatment. Cells de-energized by anaerobiosis in the absence of glucose lose ATP and adenine nucleotides with the production of IMP and adenosine. Upon reoxygenation, these cells restore a high adenylate energy charge and about 60% of control levels of GTP. There is a net resynthesis of 5-7 nmol of adenine nucleotides.mg-1 protein with a corresponding decline in IMP. Added [14C]adenosine labels the adenine nucleotide pool, but little net resynthesis of adenine nucleotides via adenosine kinase can be detected. It therefore appears that a rapid regeneration of adenine nucleotides can occur via the enzymes of the purine nucleotide cycle in heart myocytes and is limited by the size of the IMP pool retained.     

5′-Nucleotidase activity of isolated mature rat cardiac myocytes

Specific location of 5′-nucleotidase in the heart has been uncertain, some authors citing evidence for an exclusively non-myocyte location, while other data point to the existence of cytoplasmic and membrane-bound fractions. Single myocytes isolated from mature rat heart, and free of endothelial or interstitial cells, have been used to establish that muscle cells of the myocardium are rich in 5′-nucleotidase, exhibiting activity sufficient to account for the total myocardial content of this enzyme. All 5′-nucleotidase is accessible to extracellular AMP. Inhibitors of 5′-nucleotidase and adenosine transport have been used to establish that only the adenosine component of adenine nucleotides is taken up by myocytes, but hydrolysis of AMP by 5′-nucleotidase does not commit the adenosine formed to transport across the sarcolemmal membrane. Myocytes also have ecto-phosphatases which hydrolyse ADP and ATP.     

Mitochondrial enzyme retention by irreversibly damaged rectangular isolated adult rat heart myocytes

A one hour hypoxic incubation causes the release of a small but significant amount of cytosolic lactic dehydrogenase from glucose-deprived isolated adult rat heart myocytes. However, enzymes associated with the mitochondria are not liberated, and there is no increase in the number of hypercontracted cells. These observations led Piper et al. (Life Sciences 35, 127-134 [1984]) to conclude that reversibly injured myocytes can release cytosolic proteins. This conclusion was based on the supposition that irreversibly hypoxic injury must cause mitochondrial enzyme efflux and hypercontracture. The present study establishes that this supposition is invalid.     

Alterations of purine salvage pathways during differentiation of rat heart myoblasts towards myocytes

Enzyme activities of purine catabolism and salvage, the concentrations of high-energy phosphates and the reutilisation of purine bases and purine nucleosides were studied in rat heart myoblasts and myocytes. Rat heart myoblasts H9c2(2-1) were grown in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% fetal calf serum. Reduction of fetal calf serum to 2% for 1 week resulted in a differentiation into myocytes with respect to their morphological features and their enzyme pattern. In differentiated myocytes, activity of 5'-nucleotidase was increased more than 2-fold, and AMP deaminase and creatine kinase activities were more than 10-fold elevated. The concentration of creatine phosphate in differentiated myocytes was doubled compared to that in myoblasts. The uptake into myoblasts and myocytes and the incorporation into adenine nucleotides was highest using adenosine, inosine and adenine uptake rates were intermediate, and hypoxanthine was utilised least. Differentiation of myoblasts into myocytes resulted in a slightly lower overall uptake of adenosine and adenine, whereas about 40% more inosine and hypoxanthine were utilised by myocytes. Increasing the phosphate concentration in the incubation medium up to 50 mmol/l resulted in a stimulation of uptake of all purine compounds tested. This stimulation was more pronounced in myoblasts.     

Immunological quantitation and localization of tubulin in adult rat heart isolated myocytes

Isolated myocytes were purified from adult rat heart. Identification and localization of microtubules and quantitation of tubulin in these cells were performed by immunochemical procedures. Antibodies were raised against brain tubulin and purified by affinity chromatography. An enzyme-linked immunosorbent assay, ELISA, was developed for quantitation of tubulin. It allowed the measurement of 10 to 500 ng of tubulin. Tubulin content in adult rat cardiac myocytes was found to be approximately 10 micrograms per 100 mg of the total protein content. By means of a double immunofluorescence technique, the microtubule network, identified with antitubulin, was studied in reference to the sarcomeric A band labeled with antibodies specific to myosin heavy chains. The basis for identifying the microtubule network have included the use of specific antitubulin immunoglobulins and the sensitivity of the specific labeling of the network to antimitotic drugs and low temperature. It was found that microtubules were organized mainly around the nuclei, with important concentrations at the poles, showing extensions in the cone and in the cytoplasm as loosely organized loops. The shape of adult cardiac myocyte was not dependent upon the integrity of the microtubule network.     

Tolerance to physiological calcium by isolated myocytes from the adult rat heart; An improved cellular preparation

The preparation of isolated adult rat heart myocytes able to tolerate physiological calcium concentrations is described. The use of tissue culture medium as the buffer for the enzymic perfusion and digestion of the heart and, subsequently, not exposing isolated myocytes to temperatures below room temperature, proved necessary for the preparation of isolated myocytes able to maintain integrity in the presence of 2 mM calcium. After 60 minutes, 85 percent of the myocytes incubated at 37° with 2 mM calcium exclude the dye trypan blue. Levels of ATP and related compounds, although depressed, were maintained for an extended period. Oxidation of glucose and pyruvate was greater and succinate oxidation lower in calcium-resistant mycoytes compared to myocytes not resistant to calcium. The two types of myocytes were equally rapid in converting succinate to malate. Oxygen utilization increased following the addition of calcium. Myocytes demonstrated both Pasteur and Randle effects and responded to plasma levels of insulin by increasing glucose oxidation. This is the first report of isolated adult rat heart myocytes able to tolerate millimolar calcium concentrations in physiologic medium.     

大鼠肥厚心肌细胞钙电流及 Na~ /Ca~(2 )交换电流的研究(英文)

本文观察了心肌肥厚对大鼠心肌细胞Na ／Ca2 交换电流的影响。我们采用Goldblatt两肾一夹方法诱发大鼠心肌肥厚,应用全细胞膜片钳技术记录电流。结果表明：肥厚心肌细胞的Ni2 -敏感Na ／Ca2 交换电流密度大于正常细胞。在钳制电压为＋50mV时,正常细胞的外向交换电流密度为1．53±0．31pA／pF,而肥厚细胞则为2．62±0．53pA／pF（P＜0．01）;钳制电压为-100mV时,正常细胞的内向交换电流密度为0．42±0．14pA／pF,肥厚细胞达1．12±0．33pA／pF（P＜0．001）。这些结果提示,肥厚心肌细胞的Na ／Ca2 交换电流发生了改变,其意义有待进一步探讨。     

A potential prebiotic route to adenine from hypoxanthine

A possible reaction mechanism for the dehydration of glycinamide (3) and N,N'-diformylurea (4) yielding hypoxanthine (2) has been investigated. Furthermore, a potential prebiotic route converting hypoxanthine (2) into adenine (1) via phosphate activation followed by substitution reaction with NH3 was studied. This reaction mimics the proposed biochemical mechanism for the conversion of IMP to AMP.     

Effects of amiloride on calcium uptake by myocytes isolated from adult rat hearts

Amiloride at high concentrations inhibits the uptake of Ca by rat heart myocytes containing elevated levels of intracellular Na and retards the development of Ca-dependent hypercontracture in these cells. In contrast, amiloride enhances the net uptake of Ca in Ca-tolerant myocytes containing normal levels of Na. The results suggest that amiloride may inhibit Na-Ca exchange across the sarcolemma of cardiac myocytes.     

Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes.

Nicotinamide adenine dinucleotide (NADH) plays a critical role in oxidative phosphorylation as the primary source of reducing equivalents to the respiratory chain. Using a modified fluorescence microscope, we have obtained spectra and images of the blue autofluorescence from single rat cardiac myocytes. The optical setup permitted rapid acquisition of fluorescence emission spectra (390-595 nm) or intensified digital video images of individual myocytes. The spectra showed a broad fluorescence centered at 447 +/- 0.2 nm, consistent with mitochondrial NADH. Addition of cyanide resulted in a 100 +/- 10% increase in fluorescence, while the uncoupler FCCP resulted in a 82 +/- 4% decrease. These two transitions were consistent with mitochondrial NADH and implied that the myocytes were 44 +/- 6% reduced under the resting control conditions. Intracellular fluorescent structures were observed that correlated with the distribution of a mitochondrial selective fluorescent probe (DASPMI), the mitochondrial distribution seen in published electron micrographs, and a metabolic digital subtraction image of the cyanide fluorescence transition. These data are consistent with the notion that the blue autofluorescence of rat cardiac myocytes originates from mitochondrial NADH.     

Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes

A technique is presented that allows neonatal rat cardiac myocytes to form spontaneously and coherently beating 3-dimensional engineered heart tissue (EHT) in vitro, either as a plane biconcaval matrix anchored at both sides on Velcro-coated silicone tubes or as a ring. Contractile activity was monitored in standard organ baths or continuously in a CO(2) incubator for up to 18 days (=26 days after casting). Long-term measurements showed an increase in force between days 8 and 18 after casting and stable forces thereafter. At day 10, the twitch amplitude (TA) of electrically paced EHTs (average length x width x thickness, 11 x 6 x 0.4 mm) was 0.51 mN at length of maximal force development (L(max)) and a maximally effective calcium concentration. EHTs showed typical features of neonatal rat heart: a positive force-length and a negative force-frequency relation, high sensitivity to calcium (EC(50) 0.24 mM), modest positive inotropic (increase in TA by 46%) and pronounced positive lusitropic effect of isoprenaline (decrease in twitch duration by 21%). Both effects of isoprenaline were sensitive to the muscarinic receptor agonist carbachol in a pertussis toxin-sensitive manner. Adenovirus-mediated gene transfer of beta-galactosidase into EHTs reached 100% efficiency. In summary, EHTs retain many of the physiological characteristics of rat cardiac tissue and allow efficient gene transfer with subsequent force measurement.