Effect of hydrogen peroxide on secretory response, calcium mobilisation and caspase-3 activity in the isolated rat parotid gland

The parotid glands are highly active secretory systems subjected to continuous stress, which in turn, can lead to several pathophysiological conditions. Damage of the parotid glands are caused by radical oxygen species (ROS) as by-products of oxygen metabolism. This study investigated the effect of hydrogen peroxide (H₂O₂) on Carbachol (CCh)-evoked secretory responses and caspase-3 activity in the isolated rat parotid gland to understand the role of oxidative stress on the function of the gland. Amylase secretion, cytosolic calcium concentration ([Ca²⁺]_i) and caspase-3 activity in parotid gland tissue were measured using fluorimetric methods. H₂O₂ had little or no effect on amylase secretion compared to basal level. Combining H₂O₂ with CCh resulted in an attenuation of the CCh-evoked amylase secretion compared to the effect of CCh alone. CCh can evoke a large increase in [Ca²⁺]_i comprising an initial peak followed by a plateau. In a Ca²⁺-free medium containing 1 mM EGTA, CCh evoked only the initial peak of [Ca²⁺]_i. H₂O₂ alone evoked a gradual and dose-dependent increase in [Ca²⁺]_i. Combining H₂ O₂ with CCh resulted in a decrease in [Ca²⁺] _i compared to the effect of CCh alone. In a Ca²⁺ -free medium, H₂O₂ still evoked a small increase in [Ca²⁺]_i, but this response was less compared to the results obtained with H₂O₂ in normal [Ca²⁺]₀. Combining H₂O₂ with CCh resulted in only a small transient increase in [Ca²⁺] _i. Following CCh stimulation, H₂O₂ application resulted in a large increase in [Ca²⁺]_i in normal [Ca²⁺]₀. This effect of H₂ O₂ was partially abolished in a nominally free Calcium medium containing EGTA. H₂O₂ can stimulate caspase-3 activity in parotid gland tissue. Similar response was obtained with betulinic acid and thapsigargin (TPS) on caspase-3 activity compared to basal. The results have demonstrated that like CCh, H₂ O₂ can also mobilise Ca²⁺ from intracellular stores and facilitate its influx into the cell from extracellular medium. This effect of H₂O₂ may be due to its activity to induce apoptosis in the parotid gland, since H₂ O₂ can stimulate the activity of caspase-3, a marker of cellular apoptosis.