Further studies on volume regulation and effects of copper in relation to pH and EDTA in the naked marine flagellate Dunaliella marina

Abstract

The internal K+ concentration in the marine flagellate Dunaliella marina has been found to be 4.8 to 5.5 times higher than in the extracellular fluid. D. marina cells transferred to isotonic media with Na+ replaced by K+ initially swell, but tend to establish a new constant volume at a higher level depending on the K+ concentration. D. marina cells transferred to hypotonic media with elevated K+ show: (1) an initial phase of swelling, (2) a cell volume regulatory phase in which the rate of volume readjustment is progressively slower as the concentration of K+ in the media becomes higher, followed by (3) a swelling phase in which the cells establish a new constant volume progressively higher as the K+ concentration becomes higher. Transfer of D. marina from 30‰ S to hypotonic medium of 15‰ S results in loss of 20 to 30% of total intracellular K+ content during the following 90 min. In hypertonic media elevated K+ does not influence volume regulation. It is suggested that K+ is the volume-regulating factor in hypotonic media and Na+ in hypertonic media. The measured changes in steady-state volume in D. marina transferred to media with elevated K+ is consistent with the “pump and leak” hypothesis, and the mechanism of volume regulation in anisotonic media is found to be comparable to that in animal cells. The inhibitory effect of copper on volume regulation in hypotonic media is reduced when Na+ is replaced by Cholin-Cl and it is suggested that the effect of copper is to increase the permeability to Na+ which enters the cell and makes it stay swollen. The instantaneous shrinkage in D. marina cells transferred to hypertonic media with copper becomes smaller and the initial rate of volume readjustment faster and the new steady-state volume progressively bigger as the copper concentrations becomes higher; probably due to increased permeability to Na+. The effect of 5 ppm copper concentrations on volume regulation mechanism in hypotonic media is influenced by pH and EDTA: (1) at pH 8.4 volume regulation is completely inhibited while volume readjustment takes place at pH 7.5 and 6.5. Change in pH from 8.4 to 7.5 in the volume readjustment phase suspends complete inhibition 5 min, but not 30 min after transfer of flagellate cells to hypotonic media (2) when the synthetic chelator EDTA is added in the volume readjustment phase the inhibiting effect of copper is to a certain degree suspended, less pronounced the later it is added. This work shows that the inhibiting effects of copper on volume regulation mechanism is influenced by the copper speciation.