Crucial role of intracellular effectors on glycogenolysis in the isolated rat heart: Potential consequences on the myocardial tolerance to ischemia

The role played by glycogenolysis in the ischemic heart has been recently put into question because it is suspected that a slowing down of this process could be beneficial for the tolerance of the myocardium to ischemia. The role of the intracellular effectors that control the rate of glycogenolysis has therefore regained interest. We aimed to understand the role played by those intracellular effectors which are directly related to the energy balance of the heart. To this end, we review some of the previously published data on this subject and we present new data obtained from P-31 and C-13 NMR spectroscopic measurement on isolated rat heart. Two conditions of ischemia were studied: 15 min global no-flow and 25 min low-flow ischemia. The hearts were isolated either from control animals or from rats pre-treated with isoproterenol (5 mg.kg^–1 b.w. i.p.) 1 h before the perfusion in order to C-13 label glycogen stores. Our main results are as follows: (1) the biochemically determined glycogenolysis rate during the early phase of ischemia (up to 10–15 min) was larger in no-flow ischemia than in low-flow conditions for both groups, (2) direct measurement of the glycogenolysis rate, as determined by C-13 NMR, after labelling of the glycogen pool in the hearts from isoproterenol-treated rats, confirms the estimations from the biochemical data, (3) glycogenolysis was slower in the hearts from pre-treated animals than in control hearts for both conditions of ischemia, (4) the total activity of glycogen phosphorylase (a + b) increased, by 50%, after 5 min no-flow ischemia, whereas it decreased by 42% after the same time of low-flow ischemia. However, the ratio phosphorylase a/a + b was not altered, whatever the conditions, (5) the concentration of inorganic phosphate (Pi) increased sharply during the first minutes of ischemia, to values above 8–10 mM, under all conditions studied. The rate of increase was larger during no-flow ischemia than during low-flow ischemia. The concentration of Pi was thereafter higher in controls than in the hearts from isoproterenol-treated animals.The calculated cytosolic concentration of free 5 AMP increased sharply at the onset of ischemia, reaching in a few minutes values above 30 M in controls and significantly lower values, around 15 M, in the hearts from isoproterenol-treated rats. (6) The hearts from isoproterenol-treated rats displayed a reduced intracellular acidosis, when compared to controls, under both conditions of ischemia.We conclude that the intracellular effectors, mainly free AMP, play an essential role in the control of glycogenolysis via allosteric control of phosphorylase b activity. The alteration in the concentration of free Pi, the substrate of both forms of phosphorylase, can also be considered as determinant in the control of the rate of glycogenolysis.The attenuation of ischemia-induced intracellular acidosis in the hearts from isoproterenol-treated rats could be a consequence of a reduced glycogenolytic rate and is likely to be related to a better resumption of the mechanical function on reperfusion.     

Metabolic alterations induced by hypoxia in the tortoise heart. Comparison between spongy and compact myocardium

Experiments were performed to check the tolerance to severe hypoxia of the tissue layers (compact and spongy) of the tortoise heart. The animals were subjected to hypoxia (7% O2) at 18 degrees C, 28 degrees C and 38 degrees C for 30, 6 and 2 hr respectively, or to anoxia for 30 hr at 18 degrees C and 2 hr at 38 degrees C. At 18 degrees C the metabolic alterations caused by a 30 hr hypoxia were mild whereas at 28 degrees C and 38 degrees C the cardiac glycogen was depleted, lactate had accumulated and the phosphate creatine and ATP content had decreased. The extent of these metabolic changes was similar in the compact and in the spongy layers of the heart.     

Energy metabolism response to calcium activation in isolated rat hearts during development and regression of T3-induced hypertrophy

The present paper shows that cultured bovine endothelial cells can be labeled with³H-carnitine by incubation. This process is slow and is uphill, requiring Na⁺/K⁺ATPase activity. After 3 days incubation isotopic equilibrium is reached, when the cells contain about 0.5 mM (total) carnitine at a medium concentration of about 3 M. The plasmamembrane barrier is rather resistant to acidosis and oxygen free radicals (OFR). The rate of carnitine release increases, significantly only at pH below 5.8 At pH 6.0 the release of stored carnitine can be initiated by the addition of D- or L-lactate. OFR, generated by the addition of xanthine and xanthine oxidase, did not affect carnitine release. Both mild acidosis and OFR left plasmamembranes of endothelial cells intact as judged by the absence of lactate dehydrogenase loss from the cells. Therefore, the known increase of capillary permeability during ischemia and reperfusion may not be due to plasmalemmal disruption of individual endothelial cells, but to increase of inter-endothelial spaces.     

Isotopic composition of nitrate-nitrogen as a marker of riparian and benthic denitrification at the scale of the whole Seine River system

Nitrogen budgets established for large river systems reveal that up to 60% of the nitrate exported from agricultural soils is eliminated, either when crossing riparian wetlands areas before even reaching surface waters, or within the rivers themselves through benthic denitrification. The study of nitrogen isotope ratios of riverine nitrates could offer an elegant means to assess the extent of denitrification and thus confirm these budgets, as it is known that denitrification results in a natural ¹⁵N enrichment of residual nitrates. The results reported here, for the Seine river system (France), demonstrate the feasibility of this isotopic approach at the scale of large watersheds. On the basis of in situ observations carried out in a large storage reservoir in the upstream Seine catchment (Der Lake), where intensive benthic denitrification occurs, as well as on the basis of laboratory experiments of denitrification under controlled conditions, it is shown that the isotopic discrimination associated with benthic denitrification is minimal ( of NO₃-N ranging from –1.5 to –3.6), probably because the rate-limiting step of the process consists of nitrate diffusion through the water-sediment interface. Riparian denitrification on the contrary, when it implies nitrate reduction during convective transfer through reducing environements, causes a much more significant isotopic enrichment of ¹⁵N of residual nitrate ( about –18). The authors report measurements of nitrogen isotopic composition of nitrate from rivers of various stream orders in the Seine river system under summer conditions. Anomalies in the data with respect to the values expected from the mixture of the various sources of nitrate are here attributed to riparian denitrification. However, the authors show that because of the patchy distribution of actively denitrifying riparian zones within the drainage network, the isotopic signature conferred to residual nitrate in river water intrinsically provides only a minimum estimate of the extent of denitrification.     

Enzyme activity of cardiac glycogen metabolism: study of an in situ hypoxia protocol in the rat

Myocardial hypoxia, induced by arrest of the artificial ventilation of anaesthetized open-chest rats, was utilized in order to study some aspects of the regulation of myocardial glycogen metabolism. Atenolol, a cardioselective beta-adrenergic receptor antagonist, and verapamil, an inhibitor of sarcolemmal calcium transfer, were used to determine the respective role of adenosine 3', 5'-cyclic monophosphate (cAMP) and calcium in the activation of the enzymes of glycogen phosphorolysis and synthesis. Glycogen degradation is reduced by atenolol treatment, as a consequence of a reduced activation of glycogen phosphorylase. Verapamil treatment has no significant effect, neither on the enzyme activation nor on the glycogen utilization. The activation of glycogen synthase, expressed by the conversion of the enzyme from the D to the I form, which results from the decrease in glycogen stores during hypoxia, is lowered under the effect of both drugs. However, in the beta-blocker treatment case, this effect results from a lower glycogen depletion while this effect is more specific in hearts from rats treated with verapamil. Under the effect of verapamil, the reduction of synthase activation, for a similar depletion of glycogen stores, was confirmed by experiments using isolated rat hearts submitted to ischaemia. These results show that: 1. the glycogenolysis in the hypoxic myocardium in situ is mainly controlled by a cAMP-dependent enzyme conversion or by metabolic allosteric effectors; 2. the activation of myocardial glycogen synthase, which is essentially correlated to the reduction of glycogen stores, is also calcium-dependent and most probably totally cAMP-independent.     

Assessing Nitrification and Denitrification in the Seine River and Estuary Using Chemical and Isotopic Techniques

Downstream from metropolitan Paris (France), a large amount of ammonium is discharged into the Seine River by the effluents of the wastewater treatment plant at Achères. To assess the extent of nitrification and denitrification in the water column, concentrations and isotopic compositions of ammonium (δ¹⁵N–NH₄⁺) and nitrate (δ¹⁵N–NO₃⁻, δ¹⁸O–NO₃⁻) were measured during summer low-flow conditions along the lower Seine and its estuary. The results indicated that most of the ammonium released from the wastewater treatment plant is nitrified in the lower Seine River and its upper estuary, but there was no evidence for water-column denitrification. In the lower part of the estuary, however, concentration and isotopic data for nitrate were not consistent with simple mixing between riverine and marine nitrate. A significant departure of the nitrate isotopic composition from what would be expected from simple mixing of freshwater and marine nitrates suggested coupled nitrification and denitrification in the water, in spite of the apparent conservative behavior of nitrate. Denitrification rates of approximately 0.02 mg N/L/h were estimated for this part of the estuary.     

Using antibodies against P2Y and P2X receptors in purinergic signaling research

The broad expression pattern of the G protein-coupled P2Y receptors has demonstrated that these receptors are fundamental determinants in many physiological responses, including neuromodulation, vasodilation, inflammation, and cell migration. P2Y receptors couple either G_q or G_i upon activation, thereby activating different signaling pathways. Ionotropic ATP (P2X) receptors bind extracellular nucleotides, a signal which is transduced within the P2X protein complex into a cation channel opening, which usually leads to intracellular calcium concentration elevation. As such, this family of proteins initiates or shapes several cellular processes including synaptic transmission, gene expression, proliferation, migration, and apoptosis. The ever-growing range of applications for antibodies in the last 30 years attests to their major role in medicine and biological research. Antibodies have been used as therapeutic tools in cancer and inflammatory diseases, as diagnostic reagents (flow cytometry, ELISA, and immunohistochemistry, to name a few applications), and in widespread use in biological research, including Western blot, immunoprecipitation, and ELISPOT. In this article, we will showcase several of the advances that scientists around the world have achieved using the line of antibodies developed at Alomone Labs for P2Y and P2X receptors.