Protective effect of saffron extract and crocin on reactive oxygen species-mediated high glucose-induced toxicity in PC12 cells.

Diabetic neuropathy is one of the most frequent complications of diabetes. Despite some studies, the exact mechanism of glucose neurotoxicity has not been fully elucidated. Increased reactive oxygen species (ROS) has proposed as a possible mechanism. Crocus sativus L. (saffron) has been known as a source of antioxidants. Therefore, neuroprotective effect of saffron extract, its active component crocin and gamma-glutamylcysteinylglycine (GSH) was studied in glucose-induced neurotoxicity, using PC12 cells as a suitable in vitro model of diabetic neuropathy. Cell viability was quantitated by MTT assay. ROS was measured using DCF-DA by flow cytometry analysis. The result showed that glucose (13.5 and 27 mg/ml) reduced the cell viability of PC12 cells after 4 days. Saffron extract (5 and 25 mg/ml), crocin (10 and 50 muM) and GSH (10 muM) could decrease this toxicity. Glucose toxicity was consistent with increased ROS production which reduced by saffron, crocin and GSH pretreatment. These results suggest saffron and its carotenoid crocin could be potentially useful in diabetic neuropathy treatment.

Effects of aqueous and macerated extracts from Nigella sativa on guinea pig isolated heart activity.

Several therapeutic effects including those on digestive and gynaecological disorders, against asthma and dyspnea have been described for the seeds of Nigella sativa. In the present study, the effects of aqueous and macerated extracts from Nigella sativa on heart rate and contractility of the isolated heart were examined. Isolated guinea-pig hearts were perfused through aorta in the Langendorff mode. Heart rate (HR) and contractility were determined on the presence of four concentrations of aqueous and macerated extract from Nigella sativa (0.5, 1.0, 2.0 and 5.0 mg%) and diltiazem, a calcium channel blocker (0.1, 1, 10 and 100 microM) in comparison with baseline values in two different groups of experiments as follows: 1) Perfused heart with ordinary Krebs solution (group 1 experiments, n = 9). 2) Perfused heart with calcium free Krebs solution (group 2 experiments, n = 8). In group 1 three higher concentrations of diltiazem (1, 10 and 100 microM) and both extracts (1.0, 2.0 and 5.0 mg%) showed a significant reduction in heart rate (P < 0.001). However, only two larger concentrations of diltiazem (10 and 100 [microM) and macerated extract (2.0 and 5.0 mg%) and three concentrations of the aqueous extract (1.0, 2.0 and 5.0 mg%) caused a significant reduction in heart contractility in this group (P < 0.001). In group 2 only 100 microM diltiazem, caused significant reduction in heart contractility. However, two concentrations of macerated extract (2.0 and 5.0 mg%) and three higher concentrations of aqueous extract (1.0, 2.0 and 5.0 mg%) caused significant reductions in heart rate and contractility in this group (p < 0.05 to p < 0.001). There were significant negative correlations between concentrations of both extracts and diltiazem and their effect on heart rate and contractility in both groups (p < 0.01 to p < 0.001). These results showed a potent inhibitory effect of both extracts from Nigella sativa on both heart rate and contractility of guinea pig heart that was comparable and even higher than that of diltazem. The results of the present study may be due to calcium channel inhibitory or an opening effect for the plant on potassium channels of the isolated heart.

Bradykinin-induced release of PGI2 from aortic endothelial cell lines: responses mediated selectively by Ca2+ ions or a staurosporine-sensitive kinase.

1. Bradykinin (100 nM) triggers release of nitric oxide and prostacyclin from both AG07680A and AG04762 bovine cultured aortic endothelial cells. The exposure of these cells to bradykinin is in each case associated with a striking rise in intracellular calcium ion concentration. 2. Exposure of AG07680A cells to 250 nM ionomycin was followed also by a significant release of prostacyclin, whereas 250 nM ionomycin had no capacity to stimulate release of prostacyclin from AG04762 cells. 3. There was a similar concentration-dependent increase in intracellular calcium ion concentration on exposure of AG07680A and AG04762 cells to ionomycin. 4. Exposure of AG04762 cells for 10 min to staurosporine produced a concentration-dependent inhibition (IC50 = 107 +/- 14 nM) in bradykinin-stimulated prostacyclin release. There was no similar inhibitory effect of staurosporine in AG07680A cells. 5. Bradykinin (10 nM) triggered release of nitric oxide from both AG07680A and AG04762 cells, and the effect was not inhibited by 500 nM staurosporine. There was a similar ionomycin-dependent release of nitric oxide from both cell types. 6. These results identify a common pathway for bradykinin-dependent nitric oxide release from both AG07680A and AG04762 cells, involving increases in intracellular calcium ion concentration. In contrast, the bradykinin-dependent release of prostacyclin may involve one of two pathways (involving an increase in intracellular calcium or activation of a staurosporine-sensitive kinase), and the two pathways are selectively exploited in AG07680A and AG04762 cells, respectively.

Differential sensitivities of the prostacyclin and nitric oxide biosynthetic pathways to cytosolic calcium in bovine aortic endothelial cells.

1. Bovine aortic endothelial cells were cultured in vitro, and shown to release both prostacyclin (PGI2; Kact = 24.1 nM) and endothelium-derived relaxing factor (EDRF, NO; Kact = 0.7 nM) in a concentration-dependent manner when exposed to bradykinin. 2. The bradykinin-dependent release of PGI2 (but not EDRF) was inhibited by 1 microM isoprenaline or 5 microM forskolin, and the inhibitory effect of isoprenaline could be reversed by the beta 2-adrenoceptor antagonist, ICI 118551. In contrast, isoprenaline had no capacity to inhibit PGI2 release stimulated by exogenous arachidonic acid. 3. Exposure of cells to bradykinin increased the cytosolic concentration of Ca2+ ions ([Ca2+]i; Kact = 4.8 nM), and the effect was inhibited by both 1 microM isoprenaline and 5 microM forskolin. 4. In similar experiments, exposure of cells to ionomycin also increased [Ca2+]i and the values of [Ca2+]i were calibrated in terms of the ionomycin concentration. In subsequent experiments involving exposure of endothelial cells to selected concentrations of ionomycin, it was possible to show that the biosynthesis of NO was triggered at ionomycin concentrations about one tenth of the required for PGI2 biosynthesis and that these corresponded to a [Ca2+]i threshold of 350 nM for PGI2 release while that for EDRF release was less than 200 nM. 5. These differences in Ca2+ ion sensitivity explain the selective inhibition of bradykinin-stimulated PGI2 biosynthesis (to the exclusion of NO biosynthesis) by isoprenaline or forskolin, both of which attenuate bradykinin-dependent increases in [Ca2+]i.

Activation of bovine endothelial thromboxane receptors triggers release of prostacyclin but not EDRF.

OBJECTIVE: The aim was to examine the capacity of U46619 (a stable thromboxane A2 mimetic) to mediate release of endothelium derived relaxing factor (EDRF) from bovine aortic endothelial cells, and compare the response to the U46619 dependent release of prostacyclin (PGI2). METHODS: Bovine aortic endothelial cells (AG4762) were cultured in vitro on microcarrier beads, which were then loaded onto a column and perfused. The cells were challenged with U46619, bradykinin, or the Ca2+ ionophore ionomycin in the perfusate, and measurements made of the release of 6-oxo-PGF1 alpha (the stable hydrolysis product of PGI2, measured by radioimmunoassay) and EDRF (bioassay). Cells were also cultured on glass cover slips, loaded with Fura 2-AM, and measurements made of the rise in intracellular Ca2+ after challenge with U46619, bradykinin or ionomycin. RESULTS: U46619 triggered release of 6-oxo-PGF1 alpha but not EDRF from AG4762 cells, contrasting with bradykinin which released both 6-oxo-PGF1 alpha and EDRF. Ionomycin had little or no capacity to mimic and trigger release of 6-oxo-PGF1 alpha, although ionomycin mediated large increases in intracellular Ca2+. In contrast, staurosporine (a putative inhibitor of protein kinase C) substantially inhibited the U46619 and bradykinin dependent release of 6-oxo-PGF1 alpha. CONCLUSIONS: In contrast to bradykinin linked receptors on AG4762 endothelial cells, which are coupled to the release of both prostacyclin and EDRF, activation of thromboxane A2 receptors on these cells selectively triggers release of PGI2 but not EDRF. Further, based on the distinct effects of ionomycin and staurosporine, it appears that agonist stimulated PGI2 release from these cells is mediated predominantly by protein kinase C, rather than by rises in intracellular Ca2+. This observation contrasts with previously described mechanisms of PGI2 release from endothelium obtained from other sources.