Oxygen-sensitive regulatory volume increase and Na transport in red blood cells from the cane toad,Bufo marinus

Abstract

SUMMARY 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 β-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 P50of ∼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. marinusRBCs 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 PCO2of 1 and 3 kPa or washed RBCs, and 10–4 mol l-1amiloride reduced the RVI under all conditions, whereas 10–5mol 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 β-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.

Regulation of erythrocyte function: Multiple evolutionary solutions for respiratory gas transport and its regulation in fish

“…12,42,106 Adrenergically stimulated Na + /H + exchange has also been found in erythrocytes of amphibians [107][108][109] ; however, activation in these cells is not rapid 109 precluding a role in the short-term regulation of O 2 transport. 12,42,106 Adrenergically stimulated Na + /H + exchange has also been found in erythrocytes of amphibians [107][108][109] ; however, activation in these cells is not rapid 109 precluding a role in the short-term regulation of O 2 transport.…”

“…113,114 The increase in erythrocyte pH i is dependent upon the absence of CA in the plasma which would otherwise short-circuit this response by rapidly catalyzing the conversion of plasma HCO − 3 and extruded H + s to CO 2 . 115 The extremely rapid H + extrusion and its O 2 dependency appear unique for teleost erythrocytes, although O 2 dependency in the adrenergically stimulated Na + /H + exchange of the amphibian Bufo marinus 109 and in other ion transport pathways of vertebrate erythrocytes [116][117][118][119] has been described. Indeed, the buffering capacity of teleost Hbs is much lower than that of other vertebrate groups.…”

“…Erythrocyte pH can be rapidly increased in teleost fish through an adrenergically stimulated Na + /H + exchange. 12,42,106 Adrenergically stimulated Na + /H + exchange has also been found in erythrocytes of amphibians [107][108][109] ; however, activation in these cells is not rapid 109 precluding a role in the short-term regulation of O 2 transport. The adrenergic Na + /H + exchange activity of teleost erythrocytes is strongly oxygenation dependent, [110][111][112] and results in rapid net H + extrusion following stimulation.…”

“…Indeed, the buffering capacity of teleost Hbs is much lower than that of other vertebrate groups. 115 The extremely rapid H + extrusion and its O 2 dependency appear unique for teleost erythrocytes, although O 2 dependency in the adrenergically stimulated Na + /H + exchange of the amphibian Bufo marinus 109 and in other ion transport pathways of vertebrate erythrocytes [116][117][118][119] has been described. Common to those ion transport pathways is that they seem to be involved in volume regulation, which does not appear to require as rapid a response as that in the O 2 transport cascade.…”

Erythrocytes as regulators of blood vessel tone

“…In fishes, along with Na + ,K + ,2Clco transport, haemoglobin deoxy genation increases the rate of Na + /H + exchange and inhibits K + ,Clco transport [90,94]. Oxygen sensi tive ion transporters were also found in several amphibian species [90,95]. It has been proposed that in nucleated erythrocytes, pO 2 sensing is accom plished not only by haemoglobin but also by hypoxia inducible factor (HIF 1a) [90,91].…”