Activation of apoptotic caspase‐3 and nitric oxide synthase‐2 in buccal mucosa with chronic alcohol ingestion

Abstract

SUMMARYApoptosis, the process of programmed cell death, involves activation of caspase proteases cascade that remains under the regulatory control of nitric oxide. In this study, we investigated the activity of a key apoptotic protease, caspase-3, and the expression of nitric oxide synthase-2 (NOS-2) associated with buccal epithelial cells apoptosis induced by chronic ethanol diet. The assays revealed that a 7.9-fold enhancement in buccal epithelial cells apoptosis, observed in the alcohol diet group, was accompanied by a 37.6-fold increase in caspase-3 activity and a 10.1-fold increase in NOS-2. Furthermore, the expression of NOS-2 showed a positive correlation (r = 0.92) with the extent of changes induced in caspase-3 activity. These results implicate caspase-3 in the process of alcohol-induced epithelial cells apoptosis, and point towards participation of NOS-2 in the amplification of the cell death signaling cascade.

A review of the causes of central pontine myelinosis: yet another apoptotic illness?

“…Excessive NO synthesis has been linked to cytotoxicity (apoptosis) in neurones, glia and myelin (Lancaster, 1995; Leist et al ., 1997). One way in which this effect is mediated is by the alcohol‐induced over‐expression of nitric oxide synthase‐2, which in turn activates the cell‐death signalling cascade via caspase‐3, a key apoptotic protease (Slomiany et al ., 1998). It may be noteworthy that cell death from excess NO and excitotoxicity are linked.…”

Suppression of Caspase‐3 and Nitric‐Oxide Synthase‐2 During Buccal Mucosal Ulcer Healing: Effect of Chronic Alcohol Ingestion

“…Caspase-3 activity assays were carried out with buccal epithelial cells and Quanti Zyme assay system (Biomol Research Lab., Inc.). The epithelial cells, prepared from buccal mucosal scrapings (5,7), were incubated at 4 ± C with the lysis buffer according to the manufacturer' s instruction, and the lysates were centrifuged at 10,000£ g for 10 min. The aliquots of the resulting cytosolic fraction, diluted with the reaction buffer, were incubated in the microtitrator wells with 50 l M of Asp-Glu-Val-Asp-p-nitroanilide (DEVD-pNA) substrate for 1 h at 37 ± C, and the caspase-3 activity was measured spectrophotometrically (7).…”

“…The aliquots of the resulting supernatants were incubated for 30 min at 25 ± C in the presence of L-[2,3,4.5-3 H]arginine (50 l Ci/l l), 10 mM NADPH, 5l M tetrahydrobiopterin, and 50 mM Tris-HCl buffer, pH 7.4, in a ® nal volume of 250 l l. The reaction was terminated by adding to each sample a 0.4-ml of stop buffer, followed by 0.1 ml of Dowex-50W Na + resin. The mixtures were transferred to centrifugation cups andcentrifuged, and the formed L-[ 3 H]citrulline contained in the¯owthrough was quantitated by scintillation counting (7).…”

“…Quantitative measurements of apoptosis werecarried out with epithelial cells prepared from buccal mucosal scraping (7). The cells were incubated in the lysis buffer in accordance with the manufacturer's (Boehringer Mannheim) instructions and centrifuged, and the diluted supernatant containing the cytoplasmic histone-associated DNA fragments were reacted in the microtitrator wells with immobilized anti-histone antibody.…”

“…Thecytotoxic effects of chronic alcohol abuse are manifested by a marked enhancement in buccal mucosal epithelial cells apoptosis triggered by the repression of the regulatory cytokine interleukin-4 (IL-4), 1 release of proin¯ammatory tumor necrosis factor TNF-a , and subsequent activation of proapoptotic caspase cascade ( 5± 7). Other, equally detrimental effects of alcohol abuse on the integrity of the soft oral tissue result from disturbances in nitric oxide signaling pathway, characterized by enhancement of a cytokine-inducible isoform of nitric-oxide synthase (NOS-2) expression and suppression of activity of theconstitutive (cNOS) isoenzyme (6,7).…”