Morphine-induced nitric oxide production in PC12 cells

The role of nitric oxide [NO] in many well-known effects of morphine is well defined. NO is involved in the signaling pathway of the N-methyl-D-aspartate [NMDA] receptor, which is proposed to mediate some of morphine's effects. This research studies the effect of morphine and NMDA on lipopolysaccharide [LPS]-stimulated NO production by clonal rat pheochromocytoma [PC12] cells. We used the Griess reaction to measure NO concentrations in cell culture medium. PC12 cells that were incubated for 24 h with varying concentrations of morphine [0.1, 1, 10, 100, and 1000microM] plus LPS [1 microg/ml] did not significantly alter the concentration of NO in the medium. However, NO production increased when cells were treated for both 48 h with 100 and 1000 microM morphine and for 72 h with 10,100, and 1000 microM of morphine. After 72 h, 1 microM naloxone significantly decreased NO concentration. Naloxone, at doses of 0.1, 1, and 10microM prevented NO production by 1000 microM of morphine. NMDA [0.1, 1, and 10 microM] did not alter NO concentrations after 24, 48 or 72 h. Morphine [1 microM]-induced NO production was inhibited by 10 microM NMDA after 48 h. Inhibition of NO was also noted with1 and 10 microM concentrations of morphine after 72 h. Chronic treatment of PC12 cells with morphine significantly increases LPS-stimulated NO production via naloxone-sensitive receptors. The cells seem to release endogenous morphine in medium. NMDA does not affect NO production, which may be due to the lack of functional NMDA receptor expression in PC12 cells

Apoptotic and necrotic effects of pectic acid on rat pituitary GH3/B6 tumor cells

Pectin is composed of complex polysaccharides that can inhibit cancer metastasis and proliferation with no evidence of toxicity. In the present study, the apoptotic and necrotic effects of pectic acid [PA] on the rat pituitary GH3/B6 tumor cells has been investigated. GH3/B6 cells were cultured in the Ham's F12 medium enriched with 15% horse serum and 2.5% fetal bovine serum for 3 days. Then, they were treated by various amounts of PA in different periods [6, 24 and 48 hours]. Bromocriptine was used as positive control and the cell viability was detected by MTT test. The nuclear morphology of cells was explored by florescent stains including acridine orange [AO]/ethidium bromide [EB]. In addition, percentages of apoptotic and necrotic cells were studied with triphosphate nick-end labeling [TUNEL] assay, cell cycle analysis and propidium iodide [PI] staining. Long-term incubation with PA results in increased cell death and DNA damage as detected by MTT assay and AO/EB staining. TUNEL assay showed that PA [100 micro g/ml to 1 mg/ml] could induce apoptosis in a dose-dependent manner, while higher concentrations of PA [2.5 and 5 mg/ml] induced necrosis which was confirmed by PI staining. Furthermore, cell cycle analysis indicated that PA induced sub G1 events, and DNA fragmentation was also correlated with the number of the apoptotic cells. It can be concluded that PA is responsible for apoptosis in the rat pituitary tumor cells. Therefore, one may suggest that this group of polysaccharides can be used in treatment of pituitary tumors