Heidi K. Baumgartner

Calcium Elevation in Mitochondria Is the Main Ca2+ Requirement for Mitochondrial Permeability Transition Pore (mPTP) Opening

We have investigated in detail the role of intra-organelle Ca2+ content during induction of apoptosis by the oxidant menadione while changing and monitoring the Ca2+ load of endoplasmic reticulum (ER), mitochondria, and acidic organelles. Menadione causes production of reactive oxygen species, induction of oxidative stress, and subsequently apoptosis. In both pancreatic acinar and pancreatic tumor AR42J cells, menadione was found to induce repetitive cytosolic Ca2+ responses because of the release of Ca2+ from both ER and acidic stores. Ca2+ responses to menadione were accompanied by elevation of Ca2+ in mitochondria, mitochondrial depolarization, and mitochondrial permeability transition pore (mPTP) opening. Emptying of both the ER and acidic Ca2+ stores did not necessarily prevent menadione-induced apoptosis. High mitochondrial Ca2+ at the time of menadione application was the major factor determining cell fate. However, if mitochondria were prevented from loading with Ca2+ with 10 μm RU360, then caspase-9 activation did not occur irrespective of the content of other Ca2+ stores. These results were confirmed by ratiometric measurements of intramitochondrial Ca2+ with pericam. We conclude that elevated Ca2+ in mitochondria is the crucial factor in determining whether cells undergo oxidative stress-induced apoptosis.

Endogenous cyclo-oxygenase activity regulates mouse gastric surface pH

In the stomach, production of prostaglandins by cyclo‐oxygenase (COX) is believed to be important in mucosal defence. We tested the hypothesis that endogenous COX activity is required for protective gastric surface pH control. Intact stomachs of anaesthetized mice were perfused with a weakly buffered solution (150 mmNaCl+ 4 mm Homopipes) at pH values from 2.5 to 7.0. Gastric effluents were collected to measure pH and estimate amounts of acid or alkali secretion in nanomoles secreted per minute. A switch from net acid to net alkali secretion was seen in response to acidifying luminal pH with an apparent ‘set point’ between pH 4 and 5. At luminal pH 3, the net alkali secretion (12.7 ± 2.8 nmol OH− equivalents min−1) was abolished (2.2 ± 1.7 nmol OH− min−1) by the non‐specific COX inhibitor indomethacin (5 mg kg−1 I.P.). Similar inhibition was observed using a COX‐1 inhibitor (SC‐560; 10 mg kg−1 I.P.), but not a COX‐2 inhibitor (NS‐398; 10 mg kg−1 I.P.). Subsequent treatment with 16,16‐dimethyl prostaglandin E2 (dm‐PGE2; 1 mg kg−1 I.P.) rescued the alkali secretion (21.8 ± 2.7 nmol OH− min−1). In either the absence or presence of the H+,K+‐ATPase inhibitor omeprazole (60 mg kg−1 I.P.), indomethacin blocked similar amounts of net alkali secretion (10.5 ± 2.7 and 16.4 ± 3.4 nmol OH− min−1, respectively). We also used in vivo confocal microscopy to examine pH near the mucosal surface. The gastric mucosal surface of anaesthetized mice was exposed and mucosal surface pH was imaged using the fluorescence intensity ratio of Cl‐NERF as a pH indicator. Results showed a switch from a continuous net acid to net alkali secretion by the stomach in response to changing superfusate pH from 5 to 3. At luminal pH 3, the relatively alkaline surface pH (4.3 ± 0.1) was acidified (3.6 ± 0.2) by indomethacin, and subsequent dm‐PGE2 restored surface pH (4.2 ± 0.2). We conclude that the pre‐epithelial alkaline layer is regulated by endogenous COX activity.

Cyclooxygenase 1 is required for pH control at the mouse gastric surface

Background: Endogenous cyclooxygenase (COX) activity is required to maintain a relatively alkaline surface pH at the gastric luminal surface. Aims: The purpose of this study was to determine which COX isoform, COX-1 or COX-2, is responsible for regulating the protective surface pH gradient and to test if COX inhibitors also had non-COX mediated effects in vivo. Methods: Immunofluorescence and western blot analysis showed constitutive expression of both COX isoforms in the normal mouse stomach. We used in vivo confocal microscopy to measure pH near the mucosal surface of anaesthetised COX-1 (−/−), COX-2 (−/−), or wild-type mice of the same genetic background. Results: When the gastric mucosal surface was exposed and superfused (0.2 ml/min) with a weakly buffered saline solution (pH 3) containing the pH indicator Cl-NERF, the pH directly at the gastric surface and thickness of the pH gradient were similar in wild-type and COX-2 (−/−) mice, but COX-1 (−/−) mice had a significantly thinner pH gradient. Addition of indomethacin had minimal effects on the residual surface pH gradient in COX-1 (−/−) mice, suggesting no role for COX-2 in surface pH regulation. Whole stomach perfusion studies demonstrated diminished net alkali secretion in COX-1 (−/−) mice, and application of SC-560 or rofecoxib to wild-type mice and mutant mice confirmed that only COX-1 inhibition reduced alkali secretion. Conclusion: COX-1 is the dominant isoform regulating the normal thickness of the protective surface pH gradient in mouse stomach.