Biochemical medicine and metabolic biology最新文献

Eicosapentaenoic acid (EPA) caused elevations of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) measured by fura-2 fluorescence in cultured human endothelial cells. The EPA induced increase in [Ca²⁺]_i could still be observed when either cyclooxygenase or lipoxygenase inhibitors were added. These results suggest that EPA itself rather than its metabolites has direct effects on intracellular Ca²⁺ mobilization, causing the elevation of [Ca²⁺]_i.

Palmitate and oleate (0.5 to 1.0 mM) caused a time-and concentration-related augmentation of insulin release evoked by D-glucose (6.0 to 16.7 mM) in rat isolated pancreatic islets. This contrasted with an inhibitory action of the fatty acids upon L-[4-³H] phenylalanine incorporation into TCA-precipitable material, but coincided with an increased biosynthesis of proinsulin relative to that of other islet peptides. The failure of palmitate to cause an immediate increase in insulin output at a low glucose concentration (6.0 mM) coincided with an unchanged rate of O₂ uptake over a 10- to 15-min exposure to this fatty acid. Over prolonged incubation (90 min), however, both palmitate and oleate (1.0 mM) stimulated ⁴⁵Ca net uptake by islets exposed to 6.0 mM D-glucose. Like their insulinotropic effect, the time course for the oxidation of [U-¹⁴C]palmitate and [U-¹⁴C]oleate was characterized by a progressive buildup in ¹⁴CO₂, production rate. Moreover, palmitate and oleate decreased D-[5-³H]glucose conversion to ³HOH and D-[U-¹⁴C]glucose conversion to radioactive acidic metabolites over short (30 min) but not prolonged (120 min) incubation periods. The two fatty acids also interfered with the generation of ¹⁴CO₂, from islets prelabeled with [U-¹⁴C]palmitate, but not L-[U-¹⁴C]glutamine. It is concluded that, at least during prolonged exposure to either palmitate or oleate, the secretory, cationic, and metabolic response to these fatty acids displays features comparable to those usually found in islets stimulated by nutrient secretagogues.

The activity of glycogen synthase and glycogen phosphorylase was measured in rat pancreatic islet homogenates. For this purpose, the sensitivity of current radioisotopic procedures for the assay of these enzymes in liver extracts was increased by about two orders of magnitude. Even so, the measurement of glycogen synthase and phosphorylase in islet homogenates was hampered by a potent amylase-like activity, resulting in the hydrolysis of preformed or newly formed ¹⁴C-labeled glycogen. Acarbose suppressed the latter phenomenon which was found attributable to both minute contamination of isolated islets by acinar cells and genuine α-amylase activity in purified islet β-cells. As measured by the more sensitive method in the presence of acarbose, the a/(a + b) ratio for glycogen synthase activity in islet homogenates was increased in islets preincubated in the presence as distinct from absence of D-glucose and decreased after preincubation with forskolin. These changes represented a mirror image of those evoked by D-glucose and forskolin in the a/(a + b) ratio for glycogen phosphorylase activity. It is concluded that glycogen synthesis and breakdown are regulated in the endocrine pancreas in a manner qualitatively comparable to that prevailing in hepatocytes, the possible participation of an amylase-like activity to glycogen metabolism in intact islet β-cells requiring further investigation.

Mitochondrial creatine kinase in brain mitochondria appears to be located at two different intramitochondrial sites. By using immunogold-labeling techniques, a peripheral immunoreactivity was localized between the two boundary membranes, while an additional, central immunoreactivity was found at the crista surface. The peripheral enzyme was accessible to the antibodies after treatment of the brain mitochondria with 100-300 μg digitonin/mg mitochondrial protein, which left 75% of the activity bound to the membranes. Electron microscopic analyses revealed that 43% of the labeled, peripheral creatine kinase was bound at those places where outer membrane vesicles remained attached to the inner envelope membrane, suggesting that the enzyme is in involved in contact formation between outer and inner mitochondrial membranes. Postembedding staining of mitochondria on thin sections of brain tissue or in the isolated state led to the observation of a second location of creatine kinase inside the mitochondria, along the cristae, which was not accessible to the antibodies in isolated, digitonin-treated mitochondria.

Endotoxin and other bacterial products induce the release of mediators which alter the circulation and cellular metabolism. Recent evidence suggests nitric oxide (NO) is one such mediator. The proposed mechanism by which NO produces hypotension is the activation of guanylate cyclase with subsequent biosynthesis of 3′:5′ cyclic guanosine monophosphate (cGMP). We studied the production of cGMP during Escherichia coli-induced septic shock in two experiments; the first with sepsis alone and the second using N^G-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of nitric oxide synthase. Animals in both experiments experienced significant bacteremia (P < 0.05), endotoxemia (P < 0.05), and lactic acidosis (P < 0.03). Mean arterial blood pressure decreased (P < 0.03) and heart rate increased (P < 0.05) within both groups but did not differ between groups. A significant increase in the production of circulating whole blood cGMP occurred at 3-5 h (P < 0.03). There was significantly less cGMP produced by the L-NMMA-treated animals (P < 0.01). These results demonstrate an elevation in cGMP during septic shock which is attenuated by the addition of L-NMMA. This suggests that NO may be present during gram-negative septic shock and its effects mediated through cGMP.

We previously showed that recurrent calcium renal stone formers have enhanced urinary excretions of calcium and oxalate resulting from malabsorption of citrate. In the present investigation, the mechanism of the citrate-induced increased calcium uptake was studied using guinea pig ileal brush border membrane vesicles. In this model, calcium is absorbed in a concentration dependent, single mechanism uptake with a K_m of 275 ± 30 umol/liter (SD) and a V_max of 4.0 ± 0.5 nmol/min · mg protein. Under conditions of maximal calcium uptake, both citrate and phosphate inhibited calcium absorption into brush border membrane vesicles (BBMVs). In contrast, when phosphate and citrate were added together, calcium absorption normalized. Citrate inhibition of calcium absorption appeared to be due to free citrate ions, and phosphate ions overcame this inhibition. Phosphate inhibition was mostly due to decreased concentrations of ionized calcium and partly to precipitation of insoluble calcium phosphate. These studies confirm that the effects of citrate in humans in enhancing calcium absorption occur in the lumen of the gut and are not related to further biochemical conversions of citrate by the gut cells, to effects of citrate on calcium-related hormones, or to the renal handling of calcium. Also, the effects of citrate on increasing calcium absorption should be increased or attenuated in patients who malabsorb citrate, and this explains the increased urinary calcium and oxalate excretions reported for recurrent calcium stone formers.

We investigated the priming effects of protein kinase C (PKC) activators such as phorbol 12-myristate 13-acetate (PMA), 1,2-DiC₈ and OAG, and 1,3-DiC₈ (a poor activator of PKC) on thromboxane A₂ (TxA₂)-independent phospholipase A₂ (PLA₂) activation in human platelets using collagen and A23187 as agonists. We measured PLA₂ activation in collagen-stimulated platelets in the presence of BW755C, which abolished TxA₂ synthesis, rise in cytosolic Ca²⁺, and aggregation. In the presence of PMA (50 nM), the amount of arachidonic acid (AA) released in platelets stimulated with collagen and A23187 represented 300% (13.85 nmol versus 4.5 nmol) and 400% (28 nmol versus 7 nmol) of controls (without PMA), respectively, while 1,2-DiC₈, OAG, and 1,3-DiC₈ increased TxA₂-independent AA release by 50% in A23187-stimulated platelets and had no effect on the release of AA in collagen-stimulated platelets. Interestingly, 1,3-DiC₈, which is a poor activator of PKC, was as effective as the other two DAGs (OAG and 1,2-DiC₈) in priming TxA₂-independent PLA₂ activation, but was less effective than PMA in platelets stimulated with A23187. These results suggest that the TXA₂-dependent IP₃-mediated rise in cytosolic Ca²⁺ may not be obligatory for priming PLA₂ activation in the presence of PMA in collagen-stimulated platelets. In contrast, 1,2-DiC₈, OAG, and 1,3-DiC₈ likely enhance PLA₂ activation via intracellular Ca²⁺ as they selectively affect this enzyme only in A23187-stimulated platelets. We also observed a significant increase in both saturated (palmitic and stearic acids) and unsaturated fatty acids (oleic and linoleic acids) in platelets stimulated by collagen or A23187 in the presence of PMA (50 nM), but not in the presence of DAGs. These findings imply that PMA may also affect the activation of DAG/MAG lipases, PLA₁, or nonspecific PLA₂. Since both 1,2-DiC₈ and OAG exert no significant effect on the release of these fatty acids, the effects observed with PMA on DAG lipase/PLA₁ may not involve a PKC dependent mechanism. We, therefore, conclude that the mechanisms by which PMA and DAGs prime PLA₂ activation are different and that the priming mechanism by DAGs may not involve PKC, but may require a rise in intracellular Ca²⁺.