Impaired protein kinase C activation is associated with decreased hepatic alpha 1-adrenoreceptor responsiveness in adrenalectomized rats.

The present work aimed to study the influence of corticosteroids on the alpha 1-adrenoreceptor-induced activation of hepatic metabolic functions. The experiments were performed in a nonrecirculating liver perfusion system featuring continuous monitoring of pO2, pCa2+, Ca2+, pH, and portal pressure. The alpha 1-adrenergic-induced stimulation of respiration, H+ and Ca2+ release, glycogen breakdown, and gluconeogenesis, were diminished in livers from adrenalectomized animals. The normal liver responsiveness was restored on administration of exogenous corticosteroids but not mineralocorticoids. The following observations support the conclusion that corticosteroids control a hepatocyte-specific early postreceptor step in the alpha 1-adrenergic signaling pathway: 1) the alpha 1-adrenergic stimulation of vascular smooth muscle contraction was not impaired by corticosteroid deficiency; 2) the alpha 1-adrenoreceptor ligand-binding affinity does not seem to be altered by adrenalectomy; 3) the alpha 1-adrenergic-induced intracellular alkalosis, protein kinase C activation, and Ca2+ mobilization were diminished in hepatocytes from adrenalectomized rats, indicating that both Ca(2+)-dependent and -independent processes were altered; and 4) non-receptor-mediated homeostatic mechanisms of metabolic or intracellular pH control were not impaired by adrenalectomy.

Loss of fatty acid control of gluconeogenesis and PDH complex flux in adrenalectomized rats.

This work aimed to determine the role played by the adrenal gland in the fatty acid control of gluconeogenesis in isolated perfused rat livers. The gluconeogenic substrate concentration responses were not altered in adrenalectomized (ADX) rats. This observation indicates that glucocorticoids are not essential to maintain normal basal gluconeogenic rates. In contrast, fatty acid failed to stimulate gluconeogenesis from lactate and elicited attenuated stimulation with pyruvate as substrate in livers from ADX rats. Fatty acid-induced stimulation of respiration and ketone body production were similar in control and ADX rats. Thus the diminished responsiveness of the gluconeogenic pathway to fatty acid cannot be the result of different rates of energy production and/or generation of reducing power. Fatty acids did not inhibit pyruvate decarboxylation in livers from ADX rats. Even though mitochondria isolated from livers of ADX rats showed normal basal rates of pyruvate metabolism, fatty acids failed to inhibit pyruvate decarboxylation and the activity of the pyruvate dehydrogenase complex. This novel observation of the glucocorticoid effect in controlling the pyruvate dehydrogenase complex responsiveness indicates that the mitochondrial partitioning of pyruvate between carboxylation and decarboxylation reactions may be altered in livers from ADX rats. We propose that the diminished effect of fatty acid in stimulating gluconeogenesis in livers from ADX rats is the result of a limited pyruvate availability for the carboxylase reaction due to a lack of inhibition of flux through the pyruvate dehydrogenase complex.

Functional coupling of Na+/H+ and Na+/Ca2+ exchangers in the alpha 1-adrenoreceptor-mediated activation of hepatic metabolism.

The purpose of this study was to characterize the role of ions other than Ca2+ in hepatic responses to alpha 1-adrenergic stimulation. We report that the alpha 1-adrenoreceptor activation of hepatic functions is accompanied by extracellular acidification and an increase in intracellular pH. These effects are dependent on extracellular Na+ concentration and are inhibited by the Na+/H+ antiporter blocker 5-(N-ethyl-N-isopropyl) amiloride under conditions that preclude antagonistic effects on agonist binding. Thus, the activation of plasma membrane Na+/H+ exchange is an essential feature of the hepatic alpha-adrenoreceptor-coupled signaling pathway. The following observations indicate that the sustained hepatic alpha 1-adrenergic actions rely on a functional coupling between the plasma membrane Na+/H+ and Na+/Ca2+ exchangers, resulting in the stimulation of Ca2+ influx. 1) Inhibition of the Na+/K(+)-ATPase does not prevent the alpha 1-adrenergic effects. However, alpha 1-adrenoreceptor stimulation fails to induce intracellular alkalinization and to acidify the extracellular medium in the absence of extracellular Ca2+. 2) A non-receptor-induced increase in intracellular Na+ concentration, caused by the ionophore monensin, stimulates Ca2+ influx and increases vascular resistance. 3) Inhibition of Na+/Ca2+ exchange prevents, in a concentration-dependent manner, most of the alpha 1-agonist-induced responses. 4) The actions of Ca(2+)-mobilizing vasoactive peptide receptors or alpha 2-adrenoreceptors, which produce neither sustained extracellular acidification nor release of Ca2+, are insensitive to Na+/H+ exchange blockers.

Reciprocal changes in gluconeogenesis and ureagenesis induced by fatty acid oxidation.

Fatty acids produced a stimulation of gluconeogenesis and either inhibition or no effect on ureagenesis in livers perfused with gluconeogenic substrates and having NH4Cl plus ornithine as the nitrogen source. This finding indicates that stimulation of flux through pyruvate carboxylase is not sufficient to enhance urea production from ammonia. The metabolic action of fatty acids showed the following characteristics: (1) it was concentration-dependent, showing saturation-type kinetics similar to those described for fatty acid oxidation; (2) the stimulatory action on gluconeogenesis was constant and independent of NH4Cl concentration, whereas the inhibition of ureagenesis was variable and dependent on NH4Cl concentration and the degree of reduction of the gluconeogenic substrate; and (3) fatty acids produced apparent reciprocal changes in the state of reduction of the cytosolic and mitochondrial NAD systems. Fatty acid oxidation exerted its effect mainly, if not exclusively, by preventing the gluconeogenic substrate-induced stimulation of ureagenesis. Fatty acids also inhibited ureagenesis without stimulating gluconeogenesis (lactate < 1 mmol/L), ruling out a limiting energy availability as the cause of the inhibition. One or both of the following two mechanisms seem to account for the fatty acid-induced inhibition of ureagenesis from NH4Cl. First, a decreased uptake of ornithine, and second, decreased flux through pyruvate dehydrogenase and probably other NAD(P)-linked mitochondrial dehydrogenases. The correlation found between the ability of fatty acids to inhibit ureagenesis and the state of activation of pyruvate dehydrogenase supports the latter point.

Role of protein kinase-C in the alpha 1-adrenoceptor-mediated responses of perfused rat liver.

The present work aimed to determine the role played by protein kinase-C (PKC) in the alpha 1-adrenoceptor-induced activation of hepatic metabolism. The following observations indicate that activation of PKC is a condition necessary for alpha 1-adrenoceptor activation of hepatic functions, but not sufficient to mimic the receptor-mediated effects in the absence of external physiological stimuli. 1) alpha 1-Adrenoceptor activation promoted the translocation of PKC from the cytosol to its active form in the plasma membrane. 2) Activation of PKC by the phorbol ester 12-myristate 13-acetate or exogenous diacylglycerols or by elevation of endogenous levels of diacylglycerols by inhibiting diacylglycerol kinase mimicked the alpha 1-adrenoceptor-mediated actions. However, the time course and magnitude of the nonreceptor responses differ from those mediated by alpha 1-adrenoceptor activation. In addition, nonreceptor-mediated activation of PKC decreased the alpha 1-adrenoceptor responsiveness. 3) Inhibition of PKC by either H-7 [1-(5-isoquinolinilsulfonyl)2-methylpiperazine] or staurosporine inhibited all of the alpha 1-adrenoceptor-induced responses, except gluconeogenesis. The vasopressin effects were not inhibited by H-7, indicating that PKC activation is a distinct feature of the hepatic alpha 1-adrenoceptor activation that is not shared by all the Ca(2+)-mobilizing agonists. The diacylglycerol-PKC branch of the alpha 1-adrenoceptor signaling pathway seems to control the sustained phase of stimulation of hepatic functions. In these studies we have also observed that phorbol 12-myristate 13-acetate produces a concentration-dependent inhibition of hepatic respiration. However, decreased energy availability does not seem to be the cause of its action to decrease alpha 1-adrenoceptor responsiveness.

Interrelationships between ureogenesis and gluconeogenesis in perfused rat liver.

Stimulation of ureogenesis by ornithine and/or NH4Cl inhibited gluconeogenesis from lactate but not from equimolar concentrations of pyruvate in perfused rat liver. Neither a shortage of energy nor a decrease in alpha-ketoglutarate availability seems to be responsible for this inhibition. With lactate as substrate the extracellular concentration of pyruvate attained was approximately equal to 0.15 mM that assuming reflects its cytosolic concentration it would be limiting for its mitochondrial transport. Stimulation of ureogenesis from NH4Cl enhances flux through pyruvate dehydrogenase. Furthermore, activation of pyruvate dehydrogenase by dichloroacetate led to stimulation of ureogenesis and inhibition of glucose production. Conversely, inhibition of pyruvate dehydrogenase flux by fatty acid enhanced glucose production and inhibited ureogenesis. Thus, ornithine and/or NH4Cl seem to inhibit lactate to glucose flux by shifting the mitochondrial partitioning of pyruvate from carboxylation towards decarboxylation with the result of a decreased oxaloacetate formation. Gluconeogenic substrates enhanced the hepatic uptake of ornithine. However, no correlation seems to exist between the uptake of ornithine, ornithine-induced stimulation of ureogenesis and total rates of urea production. Ornithine produced a concentration-dependent acidification of the hepatic outflow perfusate, suggesting that it may be transported in exchange for H+.

On the mechanism of stimulation of ureagenesis by gluconeogenic substrates: role of pyruvate carboxylase.

Gluconeogenic substrates, lactate or pyruvate, or ornithine produced 100% increase of urea synthesis from NH4Cl. The combined administration of ornithine and lactate (or pyruvate) produced more than additive effects, indicating that they acted at different steps in a potentiating manner. The uptake of ornithine was enhanced by gluconeogenic substrates. This finding may explain, at least in part, the stimulating effect of these substrates on ureagenesis from NH4Cl and ornithine. The gluconeogenic substrate-induced stimulation of ureagenesis from NH4Cl was still observed under conditions of reduced flux through pyruvate carboxylase, ruling out that their action was exclusively mediated by the anaplerotic effect of this enzyme. Pyruvate was a more potent stimulator of ureagenesis than lactate and its effect less sensitive to pyruvate carboxylase inhibition. These observations indicate that a correlation exists between stimulation of ureagenesis by gluconeogenic substrates and flux through pyruvate dehydrogenase. It is concluded that gluconeogenic substrates may stimulate ureagenesis from NH4Cl by 1) increasing intracellular ornithine availability and/or 2) enhancing flux through pyruvate dehydrogenase and consequently the tricarboxylic acid cycle activity.

A quantitative study of chiasma terminalization in the grasshopper Chorthippus jucundus.

The possible existence of chiasma terminalization in the grasshopper Chorthippus jucundus was tested in four males by means of comparisons between chiasma locations at diplotene and metaphase I within L3, M4 and M5 bivalents. Diplotene cells were stained by a C-banding technique to recognize heterochromatic regions, especially the centromeric ones, whereas metaphase I cells were stained by a silver staining technique that visualizes a core-like structure that extends through each homologous chromosome. The core is very pronounced at kinetochores and forms cross-shaped configurations at chiasmata. No evidence of chiasma terminalization has been found.