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 (H(2)O(2)) 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 ([Ca(2+)](i)) and caspase-3 activity in parotid gland tissue were measured using fluorimetric methods. H(2)O(2) had little or no effect on amylase secretion compared to basal level. Combining H(2)O(2) 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 [Ca(2+)](i) comprising an initial peak followed by a plateau. In a Ca(2+)-free medium containing 1 mM EGTA, CCh evoked only the initial peak of [Ca(2+)](i). H(2)O(2) alone evoked a gradual and dose-dependent increase in [Ca(2+)](i). Combining H(2)O(2) with CCh resulted in a decrease in [Ca(2+)](i) compared to the effect of CCh alone. In a Ca(2+)-free medium, H(2)O(2) still evoked a small increase in [Ca(2+)](i), but this response was less compared to the results obtained with H(2)O(2) in normal [Ca(2+)](0). Combining H(2)O(2) with CCh resulted in only a small transient increase in [Ca(2+)](i). Following CCh stimulation, H(2)O(2) application resulted in a large increase in [Ca(2+)](i) in normal [Ca(2+)](0). This effect of H(2)O(2) was partially abolished in a nominally free Calcium medium containing EGTA. H(2)O(2) 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, H(2)O(2) can also mobilise Ca(2+) from intracellular stores and facilitate its influx into the cell from extracellular medium. This effect of H(2)O(2) may be due to its activity to induce apoptosis in the parotid gland, since H(2)O(2) can stimulate the activity of caspase-3, a marker of cellular apoptosis.
Mata
António
A
Cidade Universitária, Lisbon, Portugal. admata2@yahoo.com
Marques
Duarte
D
Martínez-Burgos
María A
MA
Silveira
João
J
Marques
Joana
J
Mesquita
Maria F
MF
Pariente
José A
JA
Salido
Gines M
GM
Singh
Jaipaul
J
D000681:Amylases / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015415:Biomarkers; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008297:Male; D008916:Miotics / Q000494:pharmacology; D009924:Organ Culture Techniques; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects*; D010306:Parotid Gland / Q000166:cytology / Q000378:metabolism*; D053978:Pentacyclic Triterpenes; D011448:Prostaglandin Antagonists / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D019284:Thapsigargin / Q000494:pharmacology; D014315:Triterpenes / Q000494:pharmacology; D000094062:Betulinic Acid
D015415:Biomarkers; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D008916:Miotics; D016877:Oxidants; D053978:Pentacyclic Triterpenes; D011448:Prostaglandin Antagonists; D014315:Triterpenes; D004533:Egtazic Acid; D019284:Thapsigargin; D002217:Carbachol; D006861:Hydrogen Peroxide; D000681:Amylases; C505410:Casp3 protein, rat; D053148:Caspase 3; D002118:Calcium; D000094062:Betulinic Acid