N A Tobey1, G Koves, R C Orlando. 1. Department of Medicine, Tulane University School of Medicine, Veterans Administration Medical Center, New Orleans, Louisiana, USA.
Abstract
BACKGROUND & AIMS: In a prior study, esophageal epithelial cells within intact epithelium were observed to swell after exposure to low extracellular pH (pHo) caused by activation of a bumetanide-sensitive NaK2Cl cotransporter. Because these results were obtained by correlation of tissue wet-weight gain with morphological evidence of cell swelling, another method was selected to confirm and extend these findings. METHODS: Primary cultured rabbit esophageal cells were loaded with the fluorescent dye 2'-7'-bis(carboxyethyl)-5,6-carboxyfluorescein so that changes in intracellular pH (pHi) and cell volume could be simultaneously monitored by microfluorimetry. RESULTS: Cells exposed to low pHo in both bicarbonate-free and bicarbonate-containing buffers produced a decline in pHi to as low as 6.5, but this occurred without change in volume. However, when pHo was lowered to < or = 2, pHi declined to <6.5 and volume increased by 25% in 3 minutes. Removal of Na+, K+, or Cl- or adding bumetanide inhibited acid-induced swelling. Also, swelling was inhibited when cells were acidified in Ca2+-free media and eliminated by adding 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid to Ca2+-free media. CONCLUSIONS: Esophageal cells swell when low pHo reduces pHi to <6.5. At pHi <6.5, swelling occurs through excess osmolyte (and consequently water) uptake via a bumetanide-sensitive NaK2Cl cotransporter. Activation of this enzyme at low pHi seems to be mediated by increases in cell Ca2+.
BACKGROUND & AIMS: In a prior study, esophageal epithelial cells within intact epithelium were observed to swell after exposure to low extracellular pH (pHo) caused by activation of a bumetanide-sensitive NaK2Cl cotransporter. Because these results were obtained by correlation of tissue wet-weight gain with morphological evidence of cell swelling, another method was selected to confirm and extend these findings. METHODS: Primary cultured rabbit esophageal cells were loaded with the fluorescent dye 2'-7'-bis(carboxyethyl)-5,6-carboxyfluorescein so that changes in intracellular pH (pHi) and cell volume could be simultaneously monitored by microfluorimetry. RESULTS: Cells exposed to low pHo in both bicarbonate-free and bicarbonate-containing buffers produced a decline in pHi to as low as 6.5, but this occurred without change in volume. However, when pHo was lowered to < or = 2, pHi declined to <6.5 and volume increased by 25% in 3 minutes. Removal of Na+, K+, or Cl- or adding bumetanide inhibited acid-induced swelling. Also, swelling was inhibited when cells were acidified in Ca2+-free media and eliminated by adding 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid to Ca2+-free media. CONCLUSIONS: Esophageal cells swell when low pHo reduces pHi to <6.5. At pHi <6.5, swelling occurs through excess osmolyte (and consequently water) uptake via a bumetanide-sensitive NaK2Cl cotransporter. Activation of this enzyme at low pHi seems to be mediated by increases in cell Ca2+.