The effects of various long-chain acyl-carnitines (AC) on mitochondrial functions and red cell membrane stability were studied. Lower concentrations slightly stimulate respiration-dependent functions such as phosphorylation rate and Ca++ uptake velocity, whereas higher concentrations inhibit these functions with concomitant depression of the ATP/O ratio. The order of effectiveness among the AC is very similar for different mitochondrial functions. The differences among AC in their actions on red cell stability in hypotonic media and their differences in influence on mitochondrial functions exhibit less resemblance. The relative order of erythrolytic concentrations of AC follows the order of their critical micellar concentrations. Model calculations indicate that the concentrations of AC found in ischemic hearts are below those which exhibit inhibitory effects in vitro. Ultrastructural changes in mitochondria incubated with AC are different from those found in ischemic tissue. From this, it seems questionable whether the elevated AC levels in ischemic hearts are indeed as important for the development of membrane damage as is often supposed.
Antibodies against the holo ecto-adenosinetriphosphatase (ATPase) of rat liver and antibodies against COOH-terminal peptides of the long isoform of this enzyme reacted in Western blots with a 105-kDa band from small intestinal brush-border membranes. Indirect immunofluorescence revealed reactive proteins predominantly at the apical surface of enterocytes with some staining of basolateral membranes and of vascular endothelium. Similar results were obtained with monoclonal antibodies against HA4, a protein from rat liver closely related to the ecto-ATPase. Since these results suggested the presence of an ecto-ATPase, ATP hydrolysis was studied in intact, right-side-out brush-border membrane vesicles. Nearly half of ATP hydrolysis was caused by alkaline phosphatase (AP). Besides purine and pyrimidine trinucleotides, AP also hydrolyzed ADP, AMP, pyrophosphate, and 4-nitrophenylphosphate. Inactivation of AP by cleavage of its membrane anchor and by removal of the Zn2+ necessary for its function left the ecto-ATPase that was activated by Ca2+ and Mg2+ and hydrolyzed purine and pyrimidine trinucleotides and dinucleotides, but not AMP, pyrophosphate, and 4-nitrophenylphosphate. These features are characteristic of an ATP diphosphohydrolase (EC 3.6.1.5, also called apyrase). The physiological role of the small intestinal ecto-ATPase may be the degradation of nutrient nucleotides.
The glycogenolytic potency of adenosine and ATP was studied in adult rat hepatocytes and compared with the action of glucagon and noradrenaline. In cells cultured for 48 h, adenosine and ATP as well as their analogues 2-chloroadenosine, phenylisopropyladenosine, N-ethylcarboxamidoadenosine and 6,y-methylene-substituted ATP (p[CH2] [14C]Glucose production from glycogen was stimulated only 3-fold by ATP and adenosine, compared with a 7-fold increase produced by the hormones. Stimulation of glucose production by glucagon or noradrenaline was almost completely abolished by ATP or adenosine, with half-maximal effects at around 10 /SM. The non-degradable adenosine analogues were equipotent with glucagon with respect to stimulation of glucose production, and their action was also inhibited by adenosine. ATP and p[CH2]ppA, which were both degraded to adenosine, showed comparable metabolic effects, whereas the a,,l-methylene analogue was without biological action and also was not degraded to adenosine. In the presence of the adenosine transport inhibitor nitrobenzyl thioinosine (NBTI), adenosine exerted an increased glycogenolytic potency, reaching 80 % of the maximal stimulation obtained by glucagon. The glucagon-antagonistic effect of adenosine could be completely abolished by NBTI, but was not affected by phenyltheophylline. It is concluded that, in the hepatocyte culture system, adenosine and ATP decrease the catalytic efficiency of phosphorylase a through signals arising from their uptake into the cell. INTRODUCTIONThe activation of hepatic glycogenolysis by adenosine and ATP has recently received much attention. It is well documented that both purines activate glycogen phosphorylase [1][2][3][4] and increase glucose production in perfused livers from fed rats, an action comparable with the effects of noradrenaline and glucagon and of hepatic nerve stimulation [5][6][7]. The effects of adenosine are thought to be mediated by an increase in cyclic AMP concentration via P1 receptors [3,8,9], whereas the signal chain initiated by ATP appears to involve the breakdown of phosphatidylinositol to InsP3 and mobilization of intracellular Ca2+ via P2y receptors in a cyclic AMP-independent manner [10][11][12][13][14][15]. A number of studies with hepatocyte suspensions report ATP-and adenosine-dependent activation of glycogen phosphorylase [3,4,13,15,16], but very rarely has glucose production also been determined in this isolated cell system. There are few and conflicting reports on the glycogenolytic potency of adenosine and ATP in hepatocyte suspensions. Basal glycogenolysis has been reported to be not affected [17], slightly decreased [18] or increased [19,20] by these purines.During an investigation into the interaction of ATP and insulin in their effects on glycogen metabolism, we found that in the system used, the primary cultured adult hepatocyte, ATP inhibited glucose production from glycogen and at the same time activated glycogen phosphorylase to the same degree as did glucagon. The present study investigates this p...
Activation of glycolysis by insulin in cultured rat hepatocytes is preceeded by an activation of phosphofructokinase 2 (PFK 2) and subsequent rise of the fructase 2,6-bisphosphate [Fru(2,6)P2] level. Extrdcelhlar addition of ATP or puromycin prevented the hormonal effect on glycolysis. The mechanism through which the purines abolished glycolytic stimulation was investigated.1. 50 pM ATP completely prevented the 3 -5-fold insulin-dependent increase of glycolysis, irrespective of whether the cells initially possessed a low or a high Fru(2,6)P2 content. 50 pM puromycin prevented the stimulation of glycolysis by insulin only in cells whose initial Fru(2,6)P2 levels were low and had to be increased by insulin prior to the increase in glycolysis. I t did not antagonize the action of insulin in cells with initial high Fru(2,6)P2 content.2. ATP exerted effects on its own; it decreased initially high Fru(2,6)P2 levels by 95% within 10 min and decreased the basal glycolytic rate by 60%. Half-maximal effects on the Fru(2,6)P2 level were obtained with about 25 pM ATP or 15 pM adenosine S'[fl,y-methyleneltriphosphate. ADP and adenosine-5-[y-thio]triphosphate were as effective as ATP, whereas 100 pM adenosine 5'[a,fl-methylene]triphosphate elicited no effect. Puromycin neither decreased high Fru(2,6)P2 levels nor inhibited basal glycolysis.3. Extracellular ATP (300 pM) led to inhibition of the active form of PFK 2. Intracellular levels of Glc6P, citrate, ATP, ADP and AMP were increased by extracellular ATP, the phosphoenolpyruvate content was decreased, Fru6P and glycerol 3-phosphate levels stayed constant. Puromycin did not inhibit PFK 2.4. Both puromycin and ATP prevented the insulin-dependent rise of the Fru(2,6)P2 level, they abolished the activation of PFK 2 by the hormone. Puromycin did not block the accumulation of Fru(2,6)P2 provoked by glucose addition; ATP also antagonized the glucose-dependent increase.5. 100 pM ATP elevated the CAMP-dependent protein kinase activity ratio from 0.1 to 0.38 and increased the level of inositol trisphosphate by 16-fold within 5 min, whereas puromycin was without effect on either level.It is concluded that the two purines block the insulin effect on glycolysis by preventing the hormone increasing the Fru(2,6)P2 level. The mode of action, however, seems to be different: ATP antagonizes insulin action in that it leads to increased inhibition of PFK 2 whereas puromycin prevents the activation of PFK 2 by insulin.Insulin as the principal hypoglycemic agent promotes disposal of external glucose in the liver in vivo [l]. For the study of its short-term effects on glucose metabolism, hepatocytes in primary culture have proved to be a suitable experimental tool. In cultured adult liver cells, insulin decreases glycogenolysis 12, 31 and increases glycolysis [4 -61. Acute stimulation of glycolysis by insulin proceeds only after a lag phase of 20 -
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