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First published online June 15, 2007
Journal of Experimental Biology 210, 2290-2299 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.002824
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Oxygen-sensitive regulatory volume increase and Na transport in red blood cells from the cane toad, Bufo marinus

Karina Kristensen1, Pia Koldkjær2,*, Michael Berenbrink2 and Tobias Wang1

1 Department of Zoophysiology, Aarhus University, Denmark
2 School of Biological Sciences, The Biosciences Building, Crown Street, Liverpool L69 7ZB, UK

* Author for correspondence (e-mail: piak{at}liv.ac.uk)

Accepted 24 April 2007

The red blood cells (RBCs) of cane toad, Bufo marinus, are only partially saturated with oxygen in most of the circulation due to cardiac shunts that cause desaturation of arterial blood. The present study examines the oxygen dependency of RBC ouabain-insensitive unidirectional Na transport, using 22Na, in control cells and in cells exposed to hyperosmotic shrinkage or the ß-adrenergic agonist isoproterenol. Deoxygenation per se induced a slow, but significant Na influx, which was paralleled by a slow increase in RBC volume. Hyperosmotic shrinkage by a calculated 25% activated a robust Na influx that in the first 30 min had a strong PO2 dependency with maximal activation at low PO2 values and a P50 of ~5.5 kPa. This activation was completely abolished by the Na/H exchanger (NHE) inhibitor EIPA (10–4 mol l-1). Hyperosmotic shrinkage is particularly interesting in B. marinus as it withstands considerable elevation in extracellular osmolarity following dehydration. Parallel studies showed that deoxygenated B. marinus RBCs had a much faster regulatory volume increase (RVI) response than air-equilibrated RBCs, reflecting the difference in magnitude of Na influxes at the two PO2 values. The extent of RVI (~60%) after 90 min, however, was similar under the two conditions, reflecting a more prolonged elevation of the shrinkage-induced Na influx in air-equilibrated RBCs. There were no significant differences in the ability to perform RVI between whole blood cells at a PCO2 of 1 and 3 kPa or washed RBCs, and 10–4 mol l-1 amiloride reduced the RVI under all conditions, whereas 10–5 mol l-1 bumetanide had no effect. Isoproterenol (10–5 mol l-1) induced a significant and prolonged increase in an EIPA-sensitive and bumetanide-insensitive Na influx at low PO2 under iso-osmotic conditions, whilst there was no stimulation by isoproterenol for up to 45 min in air-equilibrated RBCs. The prolonged ß-adrenergic activation of the Na influx at low PO2 is distinctly different from the rapid and transient stimulation in teleost RBCs, suggesting significant differences in the signal transduction pathways leading to transporter activation between vertebrate groups.

Key words: Bufo marinus, oxygen-dependent ion transport, erythrocyte, NHE






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