Non-invasive single cell pH measurements in the isolated perfused pancreas

1. A new method was developed for non-invasive investigations of intracellular pH (pHi) regulation in different cell types of the isolated perfused pancreas using a confocal laser scanning technique. 2. After removal of the rat pancreas the coeliac artery was cannulated and the splenic segment of the pancreas was perfused with dextran (5%)-Ringer solution at a constant flow rate of 2 mL/min. In a temperature-controlled (37 degrees C) chamber, pH regulation was studied using the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(-6)-carboxyfluorescein (BCECF) with a confocal microscope (MRC-600; Bio-Rad, Hercules, CA, USA). 3. Image analysis permitted the identification and comparison of different cell types with a pHi of 7.26+/-0.1 in acinar cells and of 7.02+/-0.1 in endothelial cells. Increasing PCO2 from 5 to 20% resulted in a rapid decrease in pHi. Omission of sodium from the perfusate resulted in a smooth decline in pHi. Both decreases were found to be fully reversible. Increasing PCO2 under sodium-free conditions also resulted in a drop of pHi that was, however, not fully reversible, suggesting involvement of the Na+/H+ exchanger in the regulation of pHi in the intact organ. 4. The above method completely preserves tissue integrity and, therefore, allows the study of pH regulation in different cell types of the pancreas simultaneously and without interference with their functional arrangement. The technique should be of specific value to investigate experimental disease states of the pancreas.