Effects of endothelin-1 and nitric oxide on glucokinase activity in isolated rat hepatocytes.

To test the hypothesis that endothelin-1 (ET-1) and nitric oxide (NO) influence glucokinase (GK) activity in an opposite manner, we evaluated the effects of ET-1, L-NAME, an inhibitor of NO synthase, and L-arginine, a substrate for NO synthase, on GK activity and glycogen content in isolated rat hepatocytes. Moreover, to understand the receptor involved in the process, the effects of BQ 788, a specific antagonist of ETB receptor, and PD 142893, an antagonist of ETA-ETB receptors, were also evaluated. GK activity, cyclic guanosine monophosphate (cGMP), and glycogen intracellular content were measured on isolated hepatocytes, while glucose levels and NO as NO2-/NO3- were determined in the medium. High ET-1 levels induced a 20% decrease of NO2-/NO3- levels and cGMP intracellular content, followed by a 49% reduction of GK activity and a 15% decrease of glycogen. In parallel, a 10% increase of glucose in the medium was observed. In the presence of L-NAME, GK activity and glycogen levels showed analogous decrements as observed with ET-1. Also in this case, a significant decrease of the intracellular content of cGMP was observed. No synergistic effects of ET-1 and L-NAME were observed. L-Arginine was able to counteract the inhibitory effect of ET-1 on cGMP and GK activity. Glycogen content was slightly but not significantly reduced, and under those conditions, a significant decrease of glucose in the medium was observed. When hepatocytes were incubated with ET-1 plus BQ 788 or ET-1 plus PD 142893, GK activity was unchanged. Interestingly, no changes were observed in NO2-/NO3- levels and the intracellular content of cGMP was not modified when the antagonists of ET-1 receptors were added to the medium. In conclusion, the present study shows that the NO pathway seems to be an important regulator of GK activity and glycogen content through cGMP activity. In addition, ET-1 seems to be not active per se, but its activity seems mediated by a simultaneous decrease of NO levels.

Effects of low-dose heparin infusion on arterial endothelin-1 release in humans.

BACKGROUND: The aim of this study was to evaluate the effect of low-dose heparin infusion on arterialized endothelin-1 (ET-1) release in the presence of fasting or high insulin levels in healthy humans. METHODS AND RESULTS: Eleven normal subjects underwent two tests in random order lasting 240 minutes. A primed (250 IU), continuous heparin (600 IU/h) infusion was performed in test 1; saline was infused in test 2 as control. At 120 minutes, a euglycemic hyperinsulinemic clamp (25 mU.kg-1.h-1) was started that lasted 2 hours in both tests. Two hours after heparin infusion (test 1), ET-1 levels decreased by 32% (3.52 +/- 0.60 to 3.02 +/- 0.73 pg/mL), while nitric oxide (NO) and forearm blood flow increased by 29% and 14%, respectively. During saline infusion, ET-1, nitric oxide, and forearm blood flow remained unchanged. There was a significant interaction between the effect of decreasing ET-1 levels and the heparin treatment (F, 4.06; df, 3.30; P < .01). The decrease in ET-1 levels was significantly correlated with the increase in forearm blood flow in test 1 (r = .74; P < .01) but not in test 2. During the heparin/insulin period, ET-1 increased by 25%, returning to fasting values; nitric oxide levels increased by 12%; and forearm blood flow remained unchanged. CONCLUSIONS: The present study showed that it is possible to decrease ET-1 levels by use of low-dose heparin infusion in humans. This effect seems mediated by a simultaneous increase in nitric oxide levels and is completely reversed by a mild increase in insulin concentrations.

Hypertriglyceridemia and hyperinsulinemia are potent inducers of endothelin-1 release in humans.

The purpose of the study was to evaluate fasting endothelin-1 levels in subjects with syndrome X, in subjects with insulinoma, and in normal subjects. The single and synergistic contributions of insulin and triglyceride levels to endothelin-1 release were studied in normal subjects. This was achieved by the evaluation of endothelin-1 levels in response to an insulin bolus combined with a euglycemic clamp (protocol A) and during intralipid (test 1) or saline (test 2) infusions lasting 360 min (protocol B). In protocol B, a euglycemic two-step hyperinsulinemic (25 and 125 mU x kg-1 x h-1) clamp was started at 120 min. Subjects with syndrome X showed significantly higher endothelin-1 levels than normal subjects and subjects with insulinoma (7.22 +/- 0.89 vs. 2.61 +/- 0.38 and 2.49 +/- 0.24 pg/ml, P < 0.01). After an insulin bolus, endothelin-1 levels peaked at 10 min (3.71 +/- 0.96 pg/ml). The incremental area of endothelin-1 was significantly higher after insulin than after a saline bolus. In test 1, an acute increase in triglyceride levels significantly enhanced endothelin-1 levels, with were further increased by the synergistic contribution of high insulin and triglyceride levels. In test 2, endothelin-1 release was achieved at high insulin levels but remained significantly lower than in test 1. In conclusion, subjects with syndrome X showed higher endothelin-1 levels than normal subjects and subjects with insulinoma. These levels were reproduced in normal subjects by a simultaneous increase in insulin and triglyceride levels.

Formation and spectral characterization of Cu(II)-poly(L-ornithine) complexes.

Cu(II)-Poly-(1-ornithine) complexes in aqueous solution have been studied using potentiometric titration and absorption and circular dichroism spectra. As in the case of Cu(II)-poly(L-arginine) complexes studied previously, two types of compounds have been detected, labeled complexes I and II. Complex I contains two amine nitrogens and two water molecules coordinated to the copper. Complex II, two amine and two amide nitrogens. Amide nitrogen coordination confers optical activity to the copper d-d transitions. Furthermore, amine and amide nitrogen coordination to the copper are characterized by charge transfer transitions at 250 and 320 nm respectively which were already identified in Cu(II)-poly(L-arginine) systems.