No abstract

Duck erythrocytes were incubated in hypotonic media at tonicities which do not produce hemolysis. The cells' response can be divided into two phases: an initial rapid phase of osmotic swelling and a second more prolonged phase (volume regulatory phase) in which the cells shrink until they approach their initial isotonic volume. Shrinkage associated with the volume regulatory phase is the consequence of a nearly isosmotic loss of KCI and water from the cell. The potassium loss results from a transient increase in K efflux. There is also a small reduction in Na permeability. Changes in cell size during the volume regulatory phase are not altered by 10 -4 M ouabain although this concentration of ouabain does change the cellular cation content. The over-all response of duck erythrocytes is considered as an example of "isosmotic intracellular regulation," a term used to describe a form of volume regulation common to euryhaline invertebrates which is achieved by adjusting the number of effective intracellular osmotic particles. The volume regulatory phase is discussed as the product of a membrane mechanism which is sensitive to some parameter associated with cell volume and is capable of regulating the loss of potassium from the cell. This mechanism is able to regulate cell size when the Na-K exchange, ouabain-inhibitable pump mechanism is blocked.

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Freshly prepared duck erythrocytes, incubated either in plasma or an isotonic synthetic medium containing norepinephrine ([K] of both media 2.5 m), maintain water and electrolyte composition in the steady state (upper steady state) for at least 90 min. If incubated in the synthetic medium without norepinephrine or in plasma to which a -adrenergic blocking agent (propranolol) is added, the cells lose both water and electrolyte (predominantly KCI) until a new steady state is reached (lower steady state). Reaccumulation of water and electrolyte from isotonic solutions toward the upper steady-state levels requires the addition of norepinephrine and KCI. Reaccumulation is maximal when the concentration of K and norepinephrine in the medium is 15 mM and 10-7 M, respectively. Dibutyryl cyclic-AMP (10-2 M) mimics norepinephrine in lower steady-state cells. Although an analogous effect in upper steady-state cells was not established with certainty, it is proposed that the catecholamine-induced net changes in water and electrolyte movement in duck erythrocytes are a consequence of stimulation of the activity of a membrane-bound adenyl cyclase system.

Amphiuma red cells were incubated for several hours in hypotonic or hypertonic media. They regulate their volume in both media by using ouabain-insensitive salt transport mechanisms . After initially enlarging osmotically, cells in hypotonic media return toward their original size by losing K, Cl, and H2O. During this volume-regulatory decrease (VRD) response, K loss results from a >10-fold increase in K efflux . Cells in hypertonic media initially shrink osmotically, but then return toward their original volume by gaining Na, Cl, and H2O. The volume-regulatory increase (VRI) response involves a large (>100-fold) increase in Na uptake that is entirely blocked by the diuretic amiloride (10-g M). Na transport in the VRI response shares many of the characteristics of amiloride-sensitive transport in epithelia: (a) amiloride inhibition is reversible ; (b) removal of amiloride from cells pretreated with amiloride enhances Na uptake relative to untreated controls ; (c) amiloride appears to act as a competitive inhibitor (K i = 1-3 AM) of Na uptake; (d) Na uptake is a saturable function of external Na (K m -29 mM); (e) Li can substitute for Na but K cannot . Anomalous Na/K pump behavior is observed in both the VRD and the VRI responses. In the VRD response, pump activity increases 3-fold despite a decrease in intracellular Na concentration, while in the VRI response, a 10-fold increase in pump activity is observed when only a doubling is predicted from increases in intracellular Na .

The addition of a hypertonic bathing medium to duck erythrocytes results in an initial instantaneous phase of osmotic shrinkage and, when the [K]o of the hypertonic solution is larger than "normal," in a second, more prolonged phase, the volume regulatory phase. During the latter, which also requires extracellular Na, the cells swell until they approach their initial isotonic volume. The increase in cell volume during the volume regulatory phase is accomplished by a gain in the cell content of K, C1, and H 2 0. There is also a smaller increase in the Na content of the cell. Potassium is accumulated against an electrochemical gradient and is therefore actively transported into the cell. This accumulation is associated with an increase, although dissimilar, in both K influx and efflux. Changes in cell size during the volume regulatory phase are not altered by 10 -4 M ouabain, although this concentration of ouabain does change the cellular cation content. The response is independent of any effect of norepinephrine. The changes in cell size during the volume regulatory phase are discussed as the product of a volume controlling mechanism identical in principle to the one reported in the previous paper which controls cell volume in hypotonic media. Similarly, this mechanism can regulate cell size, when the Na-K exchange, ouabain-inhibitable pump mechanism is blocked.

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This paper presents evidence that duck erythrocytes regulate their size in isotonic media by utilizing a previously reported "volume-controlling mechanism." Two different experimental situations are examined. In the first, cells enlarge in a solution containing norepinephrine and an elevated [K]o; and in the second, enlarged cells shrink to their original size if the norepinephrine and excess potassium are removed. As the erythrocytes enlarge, K, Cl, and H2O accumulate. Shrinkage, in contrast, is accompanied by the controlled loss of K, Cl, and H2O. These changes and the associated changes in membrane permeability resemble those reported previously when duck erythrocytes incubate in anisotonic media. There cells, after first shrinking or swelling, utilize a "volume-controlling mechanism" to reestablish their original size. The mechanism regulates cell size by adjusting the total number of osmotically active intracellular particles. The present studies indicate duck red cells use this mechanism to readjust their total monovalent cation content and thus their solute content in isotonic media as well. In addition, evidence is presented which indicates that the "volume-controlling mechanism" and ouabain-inhibitable cation pump differ functionally.