Fluorescent measurements of intracellular free calcium in isolated toad urinary bladder epithelial cells

Abstract

Sodium-calcium exchange has been suggested to play a pivotal role in the regulation of cytosolic free calcium (Caf) by epithelial cells. Using isolated epithelial cells from the toad urinary bladder, Caf has been measured using the intracellular Ca-sensitive fluorescent dyes Fura 2 and Quin 2. Dye loading did not alter cell viability as assessed by measurements of ATP and ADP content or cell oxygen consumption. When basal Caf was examined over a wide range of cell dye content (from 0.04 to 180 nmol dye/mg protein) an inverse relationship was observed. At low dye content, Caf was 300-380 nM and, as dye content was increased, Caf progressively fell to 60 nM. Using low dye content cells, in which minimal alteration in Ca steady state would be expected, the role for plasma membrane Na-Ca exchange was examined using either medium sodium substitution or ouabain. While medium sodium substitution increased Caf, prolonged treatment with ouabain had no effect on Caf despite a clear increase in cell sodium content. The lack of effect of ouabain suggests that Na-Ca exchange-mediated Ca efflux plays a minimal role in the regulation of basal Caf. However, exchange-mediated Ca efflux may play a role in Caf regulation when cytosolic calcium is elevated.

Free cytosolic calcium in renal proximal tubules from the spontaneously hypertensive rat.

“…This issue is supported by the lack of a significant effect of ouabain on [Ca 2+ ], when added in vitro to our preparation of renal proximal tubules (see Figure 3). This finding is in agreement with other studies in cultured renal epithelial cells, 22 -47 toad urinary bladder, 28 and platelets, 48 although contrary evidence has been presented in renal cells. 49 The lack of effect of ouabain on [Ca 2+ ], in our preparation of renal proximal tubules is in contrast with the marked increase caused by removal of external Na + (J. Llibre, C. Gutterman, and D.C. Batlle, unpublished observation, 1988 50 and strophanthidin, another cardiotonic steroid, has been shown to increase contractile tension in small rings of rat aorta, suggesting increased Ca 2+ delivery to the contractile apparatus.…”

“…Measurements of fluorescence were corrected for cell autofluorescence measured in unloaded cells. [Ca 1+ ], was determined using the previously described formula 28 -&:…”

Na+/K+ATPase activity inhibition and isoform-specific translocation of protein kinase C following angiotensin II administration in isolated eel enterocytes

“…The method of measuring intracellular calcium ([Ca 2+ ] i ) was derived from one that was previously described (Jacobs & Mandel 1987), using fura-2, acetoxymethyl ester, with modifications (Poggioli et al 1992). Isolated enterocytes were incubated for 30 min at 25 C in 2 ml PSS buffer supplemented with 5 mM pyruvate, 0·1% fatty acid-free BSA, and 4 µM fura-2/acetoxymethyl ester (pH 7·4).…”

Alpha1-adrenergic and muscarinic receptors in adult and neonatal rat type II pneumocytes

“…The same masking effect occurs if localized [Ca2+] i changes within individual cells occur out of synchrony or within only a subpopulation of cells [7]. Furthermore, Fura-2 may buffer Ca 2+, and blunt responses to [Ca2+]i changes [13].…”