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.     

Opposite effects of uracil and adenine nucleotides on the survival of murine cardiomyocytes

We previously showed that the human heart expresses all known P2X and P2Y receptors activated by extra-cellular adenine or uracil nucleotides. Despite evidence that, both in humans and rodents, plasma levels of ATP and UTP markedly increase during myocardial infarction, the differential effects mediated by the various adenine- and uracil-preferring myocardial P2 receptors are still largely unknown. Here, we studied the effects of adenine and uracil nucleotides on murine HL-1 cardiomyocytes. RT-PCR analysis showed that HL-1 cardiomyocytes express all known P2X receptors (except for P2X(2)), as well as the P2Y(2,4,6,14) subtypes. Exposure of cardiomyocytes to adenine nucleotides (ATP, ADP or BzATP) induced apoptosis and necrosis, as determined by flow-cytometry. Cell death was exacerbated by tumour necrosis factor (TNF)-alpha, a cytokine implicated in chronic heart failure progression. Conversely, uracil nucleotides (UTP, UDP and UDPglucose) had no effect 'per se', but fully counteracted the deleterious effects induced by adenine nucleotides and TNF-alpha, even if added to cardiomyocytes after beginning exposure to these cell death-inducing agents. Thus, exposure of cardiomyocytes to elevated concentrations of ATP or ADP in the presence of TNF-alpha contributes to cell death, an effect which is counteracted by uracil-preferring P2 receptors. Cardiomyocytes do not need to be 'primed' by uracil nucleotides to become insensitive to adenine nucleotides-induced death, suggesting the existence of a possible 'therapeutic' window for uracil nucleotides-mediated protection. Thus, release of UTP during cardiac ischaemia and in chronic heart failure may protect against myocardial damage, setting the basis for developing novel cardioprotective agents that specifically target uracil-preferring P2Y receptors.     

Effect of progesterone on rat plasma and liver lipid levels (author's transl)

The effect of progesterone upon several lipidic parameters on rat liver and plasma was studied. Progesterone administration led to a significant increase in the hepatic triacylglyceride and cholesterol esterified levels and to a decrease in the phospholipid content. After progesterone treatment decreases in plasma total lipids, phospholipids, cholesterol and cholesterol esterified were observed, whereas plasma triacylglyceride and free fatty acid levels increased. The hormonal treatment altered the lipoprotein pattern, which significantly reduced the beta-lipoprotein fraction.     

The effect of acetate oxidation on the endogenous adenine nucleotides of rat heart mitochondria

When citrate cycle substrates or glutamate are oxidized by rat heart mitochondria, AMP comprises only a few percent of the endogenous pool of adenine nucleotides. However, when acetate is oxidized, greater than half or about 30% of the total mitochondrial pool of adenine nucleotides is converted to AMP in the ADP-stimulated or resting state, respectively. Supporting substrates which form GTP as a result of their oxidation partially, but not completely, reverse the accumulation of AMP which results from acetate metabolism.     

Purine catabolism in rat brain (author's transl)

In soluble rat brain fraction, the specific activities of purine nucleoside phosphorylase, guanine deaminase, 5'Nucleotidase and adenosine deaminase, decrease in their mentioned order. A kinetic parameter comparison between these enzymes shows that 5'Nucleotidase with AMP has the lowest KM and the greatest Vmax values, while purine nucleoside phosphorylase has its lowest KM and its greatest Vmax values with guanosine and with inosine, respectively. The enzymes activity is not modified by the metabolic intermediates differently from their own reaction products which behave as competitive inhibitors.     

Effect of p-chlorophenylalanine on the reproduction of the rat (author's transl)]

The effect of p-chlorophenylalanie (p-CPA) --300 mg/kg-- on reproduction has been studied in the female rat. Groups of animals were injected with a dose of 300 mg/kg of p-CPA 48 hours before proper copulation conditions at different moments along the ovarian cycle. Presence of spermatozoa in the vaginal frotis was negative in treated rats; in the control groups however, positivity was found in variable proportions according to the phase of the ovarian cycle: 30 ad 90% in diestrus and proestrus respectively. Treated animals showed continuous diestrus phases and diffuse luteinitation of the ovary. The results may indicate that a decrease of cerebral 5-HT, caused by p-CPA, lessens the reproductive behaviour of the female rat through mechanisms depending probably on the liberation of gonadrotrophins.     

Effect of hypothalamic extracts and synthetic LH-RH on proteic synthesis by rat hypothalamus in vitro (author's transl)]

The incorporation of 14C-Leucine in pituitary proteins in rats, in vitro, has been studied. In absence of stimulation, the pituitaries of adult female rats have shown approximately twice the capacity of protein biosynthesis in vitro than the pituitaries of prepuberal female rats (21 days old). For the stimulation in vitro of the pituitaries, synthetic LH-RH or hypothalamic extracts from adult or prepuberal female rats were used. The pituitaries of adult female rats did not respond to any of the stimulation tests employed. The pituitaries of prepuberal female rats increased their biosynthetic activity significantly, when synthetic LH-RH or adult female rat hypothalamic extract was added to the culture medium. The addition of prepuberal female rat hypothalamic extract did not alter the basic response. The female prepuberal rats injected during 5 consecutive days with FSH and LH, have shown a greater sensibility to LH-RH in vitro than the ones injected with estradiol and progesterone, or with synthetic LH-RH.