Imaging of changes in sarcoplasmic reticulum [Ca(2+)] using Oregon Green BAPTA 5N and confocal laser scanning microscopy.

We describe experiments in which the low affinity indicator Oregon Green BAPTA 5N was used to record the spatially resolved changes in [Ca(2+)] from intracellular stores in rat gastric myocytes. Cells were loaded with the membrane permeant form of the indicator and imaged using a confocal scanning laser microscope. In doubly stained cells the Oregon Green signal colocalized with BIODIPY 558/568 Brefeldin A, a label for the endo/sarcoplasmic reticulum (SR) and Golgi apparatus. Oregon Green BAPTA 5N was calibrated in gastric myocytes, giving an in situ K(d) of 90 microM. The resting free [Ca(2+)] within the SR averaged 65 microM. A reversible decrease in Oregon Green fluorescence was observed on bath application of Inositol triphosphate (IP(3)) (10 microM) to permeabilized cells. Similar changes were also observed when cyclopiazonic acid (5 microM) was applied to intact myocytes, again with recovery of store [Ca(2+)] following drug washout. Identical patterns of Ca(2+) depletion were seen when caffeine (1 microM) and carbachol (10 microM) were applied sequentially to the same cells, suggesting that activation of ryanodine and IP(3)-sensitive channels can result in the release of Ca(2+) from the same regions of the SR.

Gastric motility following oesophagectomy.

The motility of the vagally denervated transposed stomach after oesophagectomy was examined by ambulatory gastric manometry and videofluoroscopy. Two groups of subjects were studied. Group 1 comprised ten patients who had undergone oesophagectomy 6-12 months previously and group 2 consisted of six normal control subjects. Studies were performed on fasting and fed subjects, and following injection of erythromycin 8 mg/kg. No distinguishable manometric wave activity was seen in either group while fasting. Feeding generated a measurable wave pattern in the patient group only. A significant increase in the mean (s.e.m.) distal wave amplitude was identified after infusion of erythromycin in both patients (34.0(15.1) versus 12.2(3.1) mmHg, P < 0.05) and controls (15.1(3.4) versus 5.0(0.0) mmHg, P = 0.05). The response to erythromycin was more rapid in patients than in controls (mean(s.e.m.) 113(16) versus 377(133) s, P < 0.05) and the effect persisted for longer (more than 1 h) in those who had undergone oesophagectomy. Videofluoroscopy confirmed purposeful motility in both the normal and vagally denervated stomach. It is concluded that the transposed stomach is a dynamic conduit. Enhancement of motility was greatest in the denervated stomach, indicative of denervation supersensitivity.