Camp. Biochem. Physiol.Vol. 102A. No. 4. pp. 625-630, 1992 Printed in Great Britain
0
ION TRANSPORT
0300-9629/92 $5.00 + 0.00 1992 Pergamon Press Ltd
IN SHEEP RED BLOOD CELLS
PHILIP R. DUNHAM Department of Biology, Syracuse University, Syracuse, NY 13244, U.S.A. (Received
I6 December 199 1)
Abstract-There
is a polymorphism (HK/LK or high potassium/low potassium) of the cation concentrations in sheep red cells which also affects the cation transport pathways in these cells. The current status of understanding of two of these pathways in LK red cells, the Na/K pump and the K-Cl cotransporter, is summarized here. Recent results are presented on stimulation of the Na/K pump by insulin-like growth factor, and on the transduction mechanism by which changes in cell volume modulate the K-Cl cotransporter.
INTRODUCTION
The special interest in cation transport in sheep red cells stems from the unusual HK/LK* polymorphism of their cellular cation concentrations. Red cells from sheep of the HK phenotype have the typical high K and low Na concentrations; cells from sheep of the LK phenotype have atypically low K and high Na concentrations (Kerr, 1937), as shown in Table 1. These differences were explained in terms of differences in fluxes of Na and K, both through the Na/K pump and through “passive” transport pathways (Tosteson and Hoffman, 1960), and differences in numbers of Na/K pumps per cell (Dunham and Hoffman, 1971). The relatively high pump fluxes in HK cells are explained in part by a relatively high number of pumps per cell, and relatively low ouabain-insensitive (“passive”) cation fluxes, which for K is mostly K-Cl cotransport in both HK and LK cells (Table 1). The association of another polymorphism of red cells, the M and L blood group antigens, with the HK/LK polymorphism is explained below. The HK/LK polymorphism is determined by a single genetic locus with two alleles, the LK allele being dominant. It has been found in six of seven bovid species tested; it has been sought and not found in three related families of artiodactyls (see Georgiades et al., 1988, Georgiades and Dunham, 1990, for brief reviews). The polymorphism has been argued to be of ancient, monophyIetic origin, and to have been maintained by selection, though it is of unknown adaptive significance (Georgiades et al., 1988).
*Abbreviations: HK and LK-phenotypes of sheep with high and low potassium concentrations, respectively, in their red blood cells; (IQ, [Mg],, and [&-con~entrations of intracellular K, intraceilu~ar Me. and extracellular K, respectively; K, and K,, intr&eiiular and extracefluiar K without respect to concentration; J,,,maximum flux velocity; &--substrate concentration at half-maximal flux velocity; k,, and k,,-unimnlecular rate constants for the forward and reverse reactions, respectively, for conversion from the A-state of the transporter to the B-state; k,, and k,,+orresponding rate constants for the B-state to C-state conversion; IGF-I-insulin-like growth factor I. 625
The M/L antigen polymo~hism is associated with the HK!LK polymorphism as follows: HK cells have only M antigen, and L antigen is only on LK cells. LK cells from heterozygous sheep have both M and L antigens (enabling determination of genotype of animals of the dominant phenotype). The result which compelled particular interest in the Na/K pump in LK cells was the demonstration that anti-L antiserum (made by immunizing HK sheep with LK cells) caused a several-fold stimulation of the Na/K pump flux in LK cells treated briefly with the antiserum (Ellory and Tucker, 1969). In addition, the antiserum inhibited ouabain-insensitive K transport (Ellory et al., 1972), later shown to be K-Cl cotransport (Ellory and Dunham, 1980; Dunham and Ellory, 1981). It was shown subsequently that anti-L antisera contained two distinct antibodies, anti-L, and anti-L,, which stimulated the pump and inhibited cotransport at two different antigens, L, and L,, associated with pumps and cotransporters, respectively (Dunham, 1976). It remains unclear how a single genetic locus can control such a complex phenotype (two transporters, two antigens). Nevertheless, these features of the polymorphism have made LK sheep cells a useful system for the study of the Na/K pump and the K-Cl cotransporter. These studies are aided by the absence in LK cells of other K transporters such as Ca-activated K channels (Brown et al., 1978) and the Na-KC2 cotransporter (Dunham and Ellory, 1981).
Na/K PUMP Stimulation by anti-L antibody After the first demonstration of stimulation of the pump by anti-L (Ellory and Tucker, 1969), several attempts were made to explain the stimulation. Lauf et af. (1970) suggested that it was due in part to the activation of latent pump sites. Subsequently, Joiner and Lauf (1978) found no increase in the number of pump sites after anti-L stimulation. If true, there must be an alteration in affinities for one or more ligands of the pump (substrates, inhibitors, activators), and/or an increase in turnover number of the pumps. Lauf ei al. (1970) showed that stimulation was not a consequence of an increase in affinity for
626
PHILIPB. DUNHAM
Table I. HK/LK ~l~orphism of cation content and transport of sheen red blood cells Phenotype
HK LK
Ircp
[Nap
Pumpt
Leakt
88 12
9 85
0.5 0.1
0.05 0.4
Pumps/cellf
Antigen$
120 40
M L
*mmol/l cells; tK influx, mm&/l x hr, ouabain-inhibitable (pump) and ouabain-insensitive (leak): ~~3H]ouabain binding: sblood group antigen type.
K,, as a substrate. Glynn and Ellory (19’72) suggested that pumps of LK cells have a higher affinity for K as a dead-end inhibitor than for Na, as a substrate. They suggested further that stimulation was a consequence of altering the relative affinities for K, and Na,, perhaps by raising the affinity for Na, We re-examined the basis for this stimulation of the pump (Dunham and Anderson, 1987). We found that anti-L caused a two-fold increase of the number of functioning pump sites per cell from 41 to 85 pumps/cell (functioning pumps were determined as the number of ouabain molecules bound per cell necessary to cause 100% inhibition of the pump, measured at subsaturating ouabain concentrations, the method used by Lauf et al., 1970). The difference between our result and that of Joiner and Lauf (1978) was partially resolved (Dunham and Anderson, 1987). Joiner and Lauf (1978) used saturating ouabain concentrations, which may have counted some inactive pumps with low ouabain affinity. We also studied the kinetics of the pump flux by varying [Na], at three fixed [K],s. We made the surprising observation that K, is a noncompetitive inhibitor of the pump: raising [K], from 0.2 to 9 mM (near physiolo~cal [Xc],) reduced J,,, of the pump three-fold (Dunham and Anderson, 1987). Treatment with anti-L abolished this noncompetitive inhibition, but had no effect on the affinity for K, as a competitive inhibitor or on the affinity for Na, as a substrate. Thus, anti-L stimulates, in part, by a specific alteration in the kinetic properties of the pump. This is not the whole story because anti-L also stimulates with no K, present without causing further increase in the number of functioning pumps. Therefore, anti-l probably also raises the turnover number of the pumps. Since anti-L, acting extracellularly, inhibits binding of intracellular IS, we predicted that KCwould modify anti-L binding. We demonstrated modification (Farquharson and Dunham, 1986), but not in the direction expected: K, promoted binding of anti-L. The interactions between anti-L and K, can be understood in terms of a reversible membrane complex of pump and antigen, L-P, to which both KCand anti-L (A) bind in preference to the free pump and antigen, respectively, However, anti-L promotes dissociation Tabie 2. Stimulation of IGF-I of NajK pump in LK sheep red mlfs: effects of varying INa], and [ici,
lK1, n 30
Pump flux (qnol /I x hr) Control IGF-I Stimulation 44 29
62 j,
+1X +7
[IGF-II: 3 x IO-* M. Pump flux: ouabain-inhibitabie S6Rb influx. Media: 145mM NaCl, 5 mM KC1 ?r 0.1 mM ouabain. INaL: 30 mmolil cells; [Na], and w], varied -as wwdescribed before (Dunham and Anderson, 1987).
of the complex, thereby reducing K, binding, whereas KCstabilizes the complex, and this promotes antibody binding. This scheme is summarized as follows: A+L-P-K-A-L+P+K. In this way anti-L can inhibit the binding of K, while K, promotes the binding of anti-L. Stimulation of the pump by hormones The question arises, is there any general significance of stimulation of the pump by anti-L? It is of no obvious consequence to the sheep. There is no physiolo~cal signi~cance of stimulation of the pump by antibodies, but the phenomenon may relate to modulation of the pump by other ligands. Lytton (1985) reported stimulation by insulin of the Na/K pump in rat adipocytes. Insulin raised the affinity for Na, of one of the two isoforms of the pump. Though this is not how anti-L stimulates, this observation prompted us to test for stimulation of the pump by insulin and also insulin-iike growth factor I (IGF-I) in LK sheep cells. We observed stimulation by both hormones. Table 2 shows stimulation by IGF-I at a physiological concentration. At [K], of 30 mmol/l. there was less stimulation of the pump than in K-free cells, suggesting that relief of inhibition by KC is not the basis for stimuiation, unlike stimulation by antiL. On the other hand, stimulation was never seen in HK cells, suggesting that the presence of the L antigen is necessary for stimulation by the hormones. IGF-I may react with a different functionai consequence than anti-L or with a different region of the L antigen. Alternatively, the L antigen may promote interaction of IGF-I receptors (which are on red cells) with pumps. Insulin stimulated as well as IGF-I did, but concentrations -. IO-fold higher were required. Stimulation by the hormones was observed frequently in cells from a number of sheep, but unfortunately not in every experiment, even on cells from the same sheep. The hormones never inhibited transport, so the phenomenon is real enough, but it is difficult to work out in detail until the basis for the variability is determined (and corrected). K-Cl COTRANSPORT
The K-Cl cotransporter, found in a number of cell types (Dunham, 1990), mediates a net KC1 efllux from cells in an obligatorily coupled fashion when the transporter is activated. It can participate in transcellular salt transport in epithelia. It can also transport solute at a sufficient rate to promote osmotically obliged water efflux, and thereby participate in regulation of cell volume. It is responsible for the reduction in cell volume during the differentiation of reticulocytes to erythrocytes in red cells of sheep &auf and Bauer, 1983) and people (O’Neiil, 1989). LK sheep red cells provide a convenient system for study of K-Cl cotransport because it comprises the majority of K transport in these cells (Dunham and Ellory, 1981). To play a role in regulation of cell volume, K-Cl cotransport (or any solute transporter) must be associated with a sensor of cell volume. A small (N 10%) osmotically-induced swelling results in a
627
Ion transport in sheep red blood cells several-fold increase in K transport and shrinkage inhibits it (Dunham and Ellory, 1981). In our view, such a system requires the following: (1) a signal of the volume change, (2) a receptor for the signal, (3) a transducer of the signal to the transporter, and (4) changes in the transporter. It is possible that a single molecule or molecular complex serves more than one of these roles. None of these functions is well understood for the K-Cl cotransporter or for any other volume-sensitive transporter. However, we have made a little progress recently toward understanding all four of these features of the control of K-Cl cotransport. Kinetics of activation by swehng From measurements of steady state of K-Cl cotransport, we showed that both J,, and K,,, for I(, are increased by swelling; see Table 3 (Bergh et al., 1990). Furthermore, these two changes are separable in that changes in J,,,,, and K,,* can be induced independently. Reducing [Mg], caused an increase in J,,, without affecting K,,2. Swelling of low-Mg cells reduced K,,z without further effect on J,,,,, (Table 3). In cells swollen 50% (rather than 25% as in Table 3), the fluxes into control and low-Mg cells were the same, showing that the effects of swelling and reduced [Mgl, on J,,, are not additive. Since the changes in J,,, and Kli2 were separable, we undertook to determine if they occurred in sequence. Though swelling after a hypotonic challenge is complete in seconds, there is a delay of several minutes in the increase in K-Cl cotransport. This was first observed by Kregenow (197 1) in duck red cells, and was subsequently reported for red cells of pigs (Kim et ai., 1989) and sheep (Dunham, 1990). It was first studied in detail by Jennings and Al-Rohil(l990) in rabbit red cells. A two-state model based on chemical relaxation kinetics was applied. In this model, most of the transporters are in a slow state, A, in cells of normal volume. The fraction in a fast state, B, is increased in swollen cells: kl2 AB. kz1 The distribution between the two states is given by two first-order rate constants. After a volume change, the sum of the rate constants evaluated in the new steady state gives the rate of relaxation to that state. The reciprocal of the sum of the rate constants, (k,, + k,,)-‘, gives the time delay of the relaxation process. By fitting to the model results on changes in time course of K fluxes provoked by volume changes, tentative conclusions can be drawn about which rate constants are volume-~nsitive. The swelling-indu~d shift in the distribution of states of the transporter, increased B/A, was Table 3. Steady state kinetics of K-Cl cotransport in LK sheep red cells: effects of osmotic swelling and reduced [MgL
Control
Normal J mm K,,2 3.7 * 0.g 87 * 10 10.5* 2.8 85 f 44
Swollen J mm hi2 5.7 + 1.0 48+6 9.0 f 0.2 28_+2
Low lM& Cl-dependent K influxes in mmol/o~~naI 1 eelis; &, mM [K],. Swollen cells: 1.25 x volume of control ceils. Low (MA ~41s: 20 min preincubation, 10 PM A23187 (from Bergh er af., 1990).
swollen 50%
swollen 20%
/
.L Y
0
Iv
/
ficontro L
IO
1
20 minutes
I
30
Fig. I. K influxes in cells of constant volume or swollen at time zero. Curves were computer fits from mean constants calculated from results of eight experiments using the twostate model (Jennings and Al-Rohil, 1990). The mean constants were: (1) initial steady state influx in cells of constant volume, 4.3 * 0.5 pmol/l cells x min (control); (2) final steady state fluxes, 20% swelling, 16 f 3 pmol/original 1 cells, and 50% swelling, 27 f 4 pmol/original 1 cells; (3)
delays, 20% swelling, 27 & 8 min, and 50% swelling, 20 f 4 min. Curves are shown to 30 min; values were calculated from data collected to 90min.
envisioned to result either from increasing kll or decreasing k,, (or both). These possibilities can be distinguished by determining the relationship between extent of swelling and the length of the delay. If swelling increases k,z, the delay would decrease with increasing extents of swelling, because (k,, + kz,)-‘, the time delay, would progressively decrease as k,, increased. Conversely, if transport was activated by decreasing k,, , the delay would increase with increased swelling. We determined in a series of experiments the effect of the extent of swelling on the delay. The results are summarized in Fig. 1, showing mean curves calculated from eight experiments. The lower curve (control) shows the constant time course of unidirectional K influx into cells of constant volume in isotonic medium. The upper two curves show time courses of K influx into cells swollen 20 or 50% at time zero. By fitting the data to the two-state model, the delays in reaching the new steady state influxes were calculated. In some experiments, greater swelling increased the delay, and in others greater swelling decreased the delay, but the mean delays from all the experiments were similar (see legend, Fig. 1). Therefore both the forward and reverse reactions in the two-state model must be sensitive to changes in cell volume. Specifically, kt2 increases with swelling and k2, decreases, though to varying extents. Jennings and Al-Rohil(I990) arrived at a different conclusion about rabbit red cells. With increased extents of swelling, the delay of relaxation to the new steady state also increased, suggesting that k,, is decreased by swelling and that k,, need not change. When swollen rabbit cells were shrunken, there was no measurable delay, suggesting that shrinkage increases k,, , with no effect on k12. We compared delays of relaxation to a new steady state in sheep cells swollen 50% and in swollen cells shrunken to
628
PHILIPB. DUNHAM
normal volume at time zero. We found a delay of - 10 min when cells were swollen. In contrast to the results with rabbit cells, we observed a delay of the same duration when swollen cells were shrunken, supporting our conclusion that both rate constants are volume-sensitive. To determine whether the difference between results with sheep and rabbit cells is real, or a consequence of differences in techniques, we carried out the experiment just described on rabbit cells. Gratifyingly, we obtained the same results reported by Jennings and Al-Rohil (1990): a delay after swelling, but no delay when swollen cells are shrunken. Therefore rabbit and sheep cells are different: both k,, and k,, appear to be volume sensitive in sheep cells, but only k,, need be in rabbit cells.
Another level of complexity in the time course is expected from the results on steady state kinetics in sheep cells with low-Mg: lowering [Mg], increased -I,,, but not K,/2, while swelling low-Mg cells decreased KIj2 (Table 3). We looked at the time course of swelling-activation in low-Mg cells (Fig. 2). circles show influx in of normal of constant (solid circles) swollen at zero (open circles), with which there was activation with the usual delay. The solid triangles show K influx in low-Mg cells of unaltered volume. The flux is higher than in control cells, presumably due to an increase in J,,,. The open triangles show K influx into low-Mg cells swollen at time zero. The influx is increased further, presumably due to a decrease in and with no discernible delay. The results in K Fig.: 2 suggest that the delay after swelling is in the process whereby Jmaxis increased, and therefore this must precede the process of increase in K,,, which proceeds with little delay. It was important to look at the time course of sh~nkage-inactivation of the flux in swollen low-Mg cells. Interestingly, there was a in delay of - 10 min which was indistinguishable extent from the delay in swollen cells with normal [Mg] (results not shown). These various results with low-Mg cells indicate the necessity of a three-state model: ki2 k23 A-B-C. k;i ksz Most of the transporters in cells of normal volume and with normal [Mg] are in the A-state. In low-Mg cells of normal volume, the fraction of the transporters in the B-state is increased. In swollen cells, normal or low-Mg, the fraction of the transporters in the C-state is increased. We recently estimated that in cells swollen -5O%, about half of the transporters are in the C-state (Dunham and Logue, unpublished results). The rapid swelling-activation in low-Mg cells (Fig. 2) and the slow shrinkage-inactivation in these cells lead to the conclusion that both k,, and k,z increase with swelling, though to different extents, and both decrease with shrinkage. This conclusion is based on these two observations on time courses (B-C conversions in the three state model), and on the recent conclusion that k,, and k,, are approximately equal in low-Mg cells swollen SO%, a conclusion drawn from the estimate that approximately
minutes
Fig. 2. Effect of swelling on K influx in cells of normal Mg, (0, 0) and with M& reduced (A, A) (see Table 3 for method of reducing Mg,). Cells were either of constant volume (*, A) or swollen 20% at time zero (0, A). Lines were fitted by eye (redrawn from Dunham, 1990).
half of the transporters are in the C-state in this condition. The lower limit for k,, + k,,, the rate of activation by swelling of low-Mg cells, is about 1-l min [the delay, (k,, + k32)--L,would be 1 min, and this could have been resolved, so the rate is at least 1 min-‘1. Assuming this rate, kX3+ k,, = I min-’ and k 23= kjZ = 0.5 min-‘. The delay for the inactivation (C-B) was - 10 min, so k,, + k,, after shrinkage = -0.1 min-‘. Since 0.5 min-’ > 0.1 min-‘, both rate constants must decrease with shrinkage, even if k,, > k,,, and both must increase with swelling. Rate constants changing in the same direction is consistent with, but does not prove, a single catalytic process promoting Bw C conversions. The time courses observed after shrinking and swelling of nodal-Mg cells (A-C’) indicate that a rate constant for a forward reaction increase with swelling (Fig. 1); this must be k,,. In addition a rate constant for a reverse reaction must decrease with swelling, and this is k,, if kj2 increases with swelling, as just concluded. k,z need not be volume-sensitive. The same conclusions were reached about k,? and k,, by Jennings and Al-Rohil (1990) for rabbit cells and a two-state model. These conclusions are consistent with two catalytic processes responsible for the A+-+B conversions. Role of phosphate metabolism
The next step in the analysis of the transduction process is to attempt to assign the volume-sensitive rate constants to biochemical processes. Jennings and Schulz (1991) found that okadaic acid, a specific phosphatase inhibitor, inhibited swelling-activation of K influx in rabbit red cells, so protein dephosphorylation must be necessary for activation by sweiling. Similar results were reported for human red cells (Kaji and Tsukitani, 1991). It follows that the phosphatase is volume-insensitive (k,*) and that swellingactivation occurs by inhibition of a volume-sensitive protein kinase (k,, ). Jennings and Schulz (1991) were unable to assign this kinase to any of the known classes of protein kinases. Sheep and rabbit cells differ in that sheep cells appear to have a swelling-activated process (in the three-state model, the increases in k,, and k,, with
529
Ion transport in sheep red blood cells
swelling). Our working hypothesis about A-B conversions in sheep cells is that k12 is the rate constant for a volume-sensitive phosphatase and k,, is the rate constant for a protein kinase inhibited by swelling. We have shown in preliminary experiments that okadaic acid inhibits K-Cl cotransport and its activation in sheep red cells. We speculate below on a possible basis for the 3-C conversions,
In an attempt to gain further insight into the control of K-Cl cotransport, we employed a simplified experimental system, inside-out vesicles (IOVs) made from LK sheep red eel1 membranes. We demonstrated Cl-dependent K effluxes from these IOVs, and showed that the efflux was increased by swelling the vesicles and decreased by shrinking them (Kracke and Dunham, 1990). This is a convenient system for study of the cotransporter because there is essentiaiiy no cytoplasm, and yet at least some of the regulatory machinery remains associated with the membrane. Therefore, one can make measurements of the flux with simultaneous and continuous access to the cytoplasmic membrane surface, the presumed locus of the regulatory machinery. These results provide clues about the nature of the signal of volume changes. One can envision two classes of signals of cell swelling: (I) a mechanical change in the membrane, and (2) a decrease in the concentration of a critical cytoplasmic solute. The results with the IOVs rule out a change in concentration of a solute, and therefore indicate a mechanical change as the signal. The results also place limits on the nature of the m~hanical change which can serve as a signal. Swelling of both intact cells and IOVs increases the flux. Since the membranes of these two systems have opposite orientations, change in degree of curvature of the membrane is not likely to be the signal. Other possibilities are stretch, which is unlikely, and a change in relative orientation of integral and peripheral regions of the membrane, which we favor. In preliminary experiments, we have looked at the time course of swelling-activation of K influx in IOVs. There was no indication of a delay in the approximately six-fold increase of the flux after -40% swelling. Therefore, the vesicles behaved as though [Mg] was low even though it was 1 mM. We next looked at the dependence on [Mg] of K flux in the IOVs of unchanged or swollen volume, and found no effect when varying [Mgl from nominally zero to 1 mM. Apparently, in preparing the vesicles, there was a conversion of most of the transporters to the B-state, which have no delay in swelling-activation (expected from results on intact cells), and an insensitivity to Mg (not expected from intact cebs). This latter characteristic suggests an irreversible conversion of A to B, perhaps owing to a loss of constituent(s) of the membrane and/or cytoplasm. Multiple volume sensors
Jennings and Schulz (1990) presented evidence that the signal of swelling in rabbit red ceils is not a m~hanical change in the membrane, but rather a change in concentration of a critical cytoplasmic solute. Echinocytes and discocytes have the same rate
of K-Cl cotransport as the erythrocytes from which they were made. This and our results on IOVs raise the possibility of two types of signals of swelling. In terms of the three-state model, this could mean two signals operating in sequence, one promoting A -+ B and a second promoting B --f C. The first, a decrease in concentration of a ~ytoplasmic solute, would inhibit a volume-sensitive protein kinase (rate constant k,,) resulting in an increase in JmaX. Since Mg-ATP is a substrate for the kinase, a reduction in Mg activity caused by swelling could be the signal (if [Mg] is rate limiting). The resultant inhibition of the kinase would activate cotransport (since Mg may also be required by the phosphatase, the Mg activity need be limiting only for the kinase and not for the phosphatase). The argument for two signals is that reducing cell [Mg] does not fully activate cotransport; swelling is required. The results on IOVs indicate that the second signal is mechanical It was argued above that the Bc+C conversions are controlled by a single catalyst. A protein kinase is unlikely to be involved since neither Mg (nor ATP) is required. Indeed, the catalyst does not even need to be an enzyme. Thus, the reduction in K,,* for K associated with the B -+ C conversion could be due to a swelling-induced structural rearrangement in the membrane. Despite this speculation about the transduction mechanism, a dimly perceived picture of the regulation of K-Cl cotransport is beginning to emerge. Acknowiedgement-ale original work reported here was supported by NIH grant DK-33640. REFERENCES
Bergh C., Kefley S. J. and Dunham P. B. (1990) K-Cl cotransport in LK sheep erythrocytes: kinetics of stimulation by swell swelling. J. ~e~~r~~~ Biol. 117, 177-188. Brown A. M., Ellory J. C., Young J. D. and Lew V. L. (1978) A calcium activated potassium channel present in foetal red cells of the sheep and goats. &c&m. hio$z~~.r. Acta 511, 163-179. Dunham P. B. (1976) Anti-L serum: two populations of antibodies affecting cation transport in LK erythrocytes of sheep and goats. Biochim. biophys. Acta 443,219-226. Dunham P. B. (1990) K-Cl cotransport in mammalian erythrocytes. In Regulation of Potassium Transport across Bio~og~caimembranes (Edited by Reuss L., Russell J. M. and Sxabo G.), pp. 333-362. University of Texas Press, Austin, TX. Dunham P. B. and Anderson C. (1987) On the mechanism of stimulation of the Na/K pump of LK sheep erythrocytes by anti-L antibody. J. gen. Physiol. 90, 3-25. Dunham P. B. and Ellory J. C. (1981) Passive potassium transport in low potassium sheep red cells: dependence upon cell volume and chloride, J. Physiol., Land. 318, 511-530. Dunham P. B. and Hoffman J. F. (1971) Active cation transport and ouabain binding in high potassium and low potassium red blood cells of sheep. J. gen. Physiol. 58, 94416. Ellory J. C. and Dunham P. B. (1980) Volume dependent passive potassium transport in LK sheep red cells. In Membrane Transport in Erythrocytes (Edited by Lassen U. V., Ussing H. H. and Wieth J. O.), pp. __ 409-427. Alfred Benzon Symposium 14. Munksgaard, Co~nhagen. Ellory 3. C., Sachs J. R., Dunham P. B. and Hoffman J. F. (1972) The L antibody and potassium fluxes in LK red ceils of sheep and goats. In B~omembranes, Vol. 3, Passice
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PHILIP B. IWiiH~t4
~er~e~~lity of Red Ceil membranes (Edited by Kreuzer F. and Slegers J. F. G.), pp. 237-245. Plenum, New York. Ellory J. C. and Tucker E. M. (1969) Stimulation of the potassium transport system in low potassium type sheep red cells by a specific antigen antibody reaction. Nafure 222, 477-478. Farquharson B. E. and Dunham P. B. (1986) Intracellular potassium promotes antibody binding to an antigen associated with the Na/K pump of sheep erythrocytes. Biochem. biophys. Res. Common. 134, 982-988. Georgiades N. and Dunham P. B. (1990) The HIQLK polymorphism of erythrocyte cation content in two wild species of East African bovids: demonstrated in wiidebeest but not in African buffalo. Animal Genefics 21, 1999205. Georgiades N., Dunham P. B., Read B. and Templeton A. (1988) HK/LK polymorphism and its genetic determination in Speke’s gazelle. J. Hered. 79, 325-33 1. Glynn I. M. and Ellory J. C. (I 972) Stimulation of a sodium pump by an antibody that increases the apparent affinity of sodium ions of the sodium loading sites. In Role of Membranes in Secretory Processes (Edited by Bolis L., Kevnes R. D. and Wilbrandt W.), vv. . . 2244237. Elsevier. Noith Holland. Jennings M. L. and Al-Rohil N. (1990) Kinetics of and inactivation of swelling-stimulated activation K+/Cl- transport. Volume-sensitive parameter is the rate constant for inactivation. J. gen. Physiol. 95, 1021-1040. Jennings M. L. and Schulz R. K. (1990) Swelling-activated KC1 cotransport in rabbit red cells: flux is determined mainly by cell volume rather than cell shape. Am. J. Physiol. 259, C960-C967.
Jennings M. L. and Schulz R. K. (1991) Okadaic acid inhibition of KC1 cotransport. J. gen. Ph_rsiol. 97, 799-818.
Joiner C. H. and Lauf P. K. (1978) The correlation between ouabain binding and potassium pump inhibition in human and sheep erythrocytes. J. Phykol.,-Lond. 283, 155-177. Kaii D. M. and Tsukitani Y. 11991) Role of nrotein phosphatase in activation of Kdl cotransport in human erythrocytes. Am. J. Physiol. 260, Cl76C180. Kerr S. E. (1937) Studies on the inorganic composition of blood. IV. The relationship of potassium to the acidsoluble phosphorus fractions. J. biol. Chem. 117,227-235. Kim H. D.. Sergeant S.. Forte L. R.. Sohn D. H. and Im J. H. (1989) ~tivation of Cl-dependent K flux by CAMP in pig red cells. Am. J. Physiol. 256, C772X778. Kracke G. R. and Dunham P. B. (1990) Volume-sensitive K-Cl cotransport in inside-out vesicles made from erythrocyte membranes from sheep of low-K phenotype. Proc. natn. Acad. Sci. U.S.A. 87. 8575-8579.
Kregenow F. M. (1971) The response of duck erythrocytes to nonhemolytic hypotonic media: Evidence of a volume controlling mechanism. 1. gen. Physiol. 58, 372-395. Lauf P. K. and Bauer J. (1983) Direct evidence for chloridedependent volume reduction in macrocytic sheep reticulocytes. Biochem. biophys, lies. Comman. 144, 849-855. Lauf P. K., Rasmusen B. A., Hoffman P. G., Dunham P. B., Cook P., Parmalee M. L. and Tosteson D. C. (1970) Stimulation of active potassium transport in LK sheep red cells by blood group-L-antiserum. J. Membrane Biol. 3, i-15.
Lytton J. (1985) Insulin affects the sodium affinity of the rat adipocyte (Na+, K+)-ATPase. J. biol. Chem. 260, 1~75-1~80.
O’Neill W. C. (1989) Cl-dependent K transport in a pure population of volume regulating human erythrocytes. Am. J. Physiol. 256, C858-C864.
Tosteson D. C. and Hoffman J. F. (1960) Regulation of cell volume by active cation transport in high and low potassium sheep red cells. J. gen. Phsviol. 44, 1599194.
0
ION TRANSPORT
0300-9629/92 $5.00 + 0.00 1992 Pergamon Press Ltd
IN SHEEP RED BLOOD CELLS
PHILIP R. DUNHAM Department of Biology, Syracuse University, Syracuse, NY 13244, U.S.A. (Received
I6 December 199 1)
Abstract-There
is a polymorphism (HK/LK or high potassium/low potassium) of the cation concentrations in sheep red cells which also affects the cation transport pathways in these cells. The current status of understanding of two of these pathways in LK red cells, the Na/K pump and the K-Cl cotransporter, is summarized here. Recent results are presented on stimulation of the Na/K pump by insulin-like growth factor, and on the transduction mechanism by which changes in cell volume modulate the K-Cl cotransporter.
INTRODUCTION
The special interest in cation transport in sheep red cells stems from the unusual HK/LK* polymorphism of their cellular cation concentrations. Red cells from sheep of the HK phenotype have the typical high K and low Na concentrations; cells from sheep of the LK phenotype have atypically low K and high Na concentrations (Kerr, 1937), as shown in Table 1. These differences were explained in terms of differences in fluxes of Na and K, both through the Na/K pump and through “passive” transport pathways (Tosteson and Hoffman, 1960), and differences in numbers of Na/K pumps per cell (Dunham and Hoffman, 1971). The relatively high pump fluxes in HK cells are explained in part by a relatively high number of pumps per cell, and relatively low ouabain-insensitive (“passive”) cation fluxes, which for K is mostly K-Cl cotransport in both HK and LK cells (Table 1). The association of another polymorphism of red cells, the M and L blood group antigens, with the HK/LK polymorphism is explained below. The HK/LK polymorphism is determined by a single genetic locus with two alleles, the LK allele being dominant. It has been found in six of seven bovid species tested; it has been sought and not found in three related families of artiodactyls (see Georgiades et al., 1988, Georgiades and Dunham, 1990, for brief reviews). The polymorphism has been argued to be of ancient, monophyIetic origin, and to have been maintained by selection, though it is of unknown adaptive significance (Georgiades et al., 1988).
*Abbreviations: HK and LK-phenotypes of sheep with high and low potassium concentrations, respectively, in their red blood cells; (IQ, [Mg],, and [&-con~entrations of intracellular K, intraceilu~ar Me. and extracellular K, respectively; K, and K,, intr&eiiular and extracefluiar K without respect to concentration; J,,,maximum flux velocity; &--substrate concentration at half-maximal flux velocity; k,, and k,,-unimnlecular rate constants for the forward and reverse reactions, respectively, for conversion from the A-state of the transporter to the B-state; k,, and k,,+orresponding rate constants for the B-state to C-state conversion; IGF-I-insulin-like growth factor I. 625
The M/L antigen polymo~hism is associated with the HK!LK polymorphism as follows: HK cells have only M antigen, and L antigen is only on LK cells. LK cells from heterozygous sheep have both M and L antigens (enabling determination of genotype of animals of the dominant phenotype). The result which compelled particular interest in the Na/K pump in LK cells was the demonstration that anti-L antiserum (made by immunizing HK sheep with LK cells) caused a several-fold stimulation of the Na/K pump flux in LK cells treated briefly with the antiserum (Ellory and Tucker, 1969). In addition, the antiserum inhibited ouabain-insensitive K transport (Ellory et al., 1972), later shown to be K-Cl cotransport (Ellory and Dunham, 1980; Dunham and Ellory, 1981). It was shown subsequently that anti-L antisera contained two distinct antibodies, anti-L, and anti-L,, which stimulated the pump and inhibited cotransport at two different antigens, L, and L,, associated with pumps and cotransporters, respectively (Dunham, 1976). It remains unclear how a single genetic locus can control such a complex phenotype (two transporters, two antigens). Nevertheless, these features of the polymorphism have made LK sheep cells a useful system for the study of the Na/K pump and the K-Cl cotransporter. These studies are aided by the absence in LK cells of other K transporters such as Ca-activated K channels (Brown et al., 1978) and the Na-KC2 cotransporter (Dunham and Ellory, 1981).
Na/K PUMP Stimulation by anti-L antibody After the first demonstration of stimulation of the pump by anti-L (Ellory and Tucker, 1969), several attempts were made to explain the stimulation. Lauf et af. (1970) suggested that it was due in part to the activation of latent pump sites. Subsequently, Joiner and Lauf (1978) found no increase in the number of pump sites after anti-L stimulation. If true, there must be an alteration in affinities for one or more ligands of the pump (substrates, inhibitors, activators), and/or an increase in turnover number of the pumps. Lauf ei al. (1970) showed that stimulation was not a consequence of an increase in affinity for
626
PHILIPB. DUNHAM
Table I. HK/LK ~l~orphism of cation content and transport of sheen red blood cells Phenotype
HK LK
Ircp
[Nap
Pumpt
Leakt
88 12
9 85
0.5 0.1
0.05 0.4
Pumps/cellf
Antigen$
120 40
M L
*mmol/l cells; tK influx, mm&/l x hr, ouabain-inhibitable (pump) and ouabain-insensitive (leak): ~~3H]ouabain binding: sblood group antigen type.
K,, as a substrate. Glynn and Ellory (19’72) suggested that pumps of LK cells have a higher affinity for K as a dead-end inhibitor than for Na, as a substrate. They suggested further that stimulation was a consequence of altering the relative affinities for K, and Na,, perhaps by raising the affinity for Na, We re-examined the basis for this stimulation of the pump (Dunham and Anderson, 1987). We found that anti-L caused a two-fold increase of the number of functioning pump sites per cell from 41 to 85 pumps/cell (functioning pumps were determined as the number of ouabain molecules bound per cell necessary to cause 100% inhibition of the pump, measured at subsaturating ouabain concentrations, the method used by Lauf et al., 1970). The difference between our result and that of Joiner and Lauf (1978) was partially resolved (Dunham and Anderson, 1987). Joiner and Lauf (1978) used saturating ouabain concentrations, which may have counted some inactive pumps with low ouabain affinity. We also studied the kinetics of the pump flux by varying [Na], at three fixed [K],s. We made the surprising observation that K, is a noncompetitive inhibitor of the pump: raising [K], from 0.2 to 9 mM (near physiolo~cal [Xc],) reduced J,,, of the pump three-fold (Dunham and Anderson, 1987). Treatment with anti-L abolished this noncompetitive inhibition, but had no effect on the affinity for K, as a competitive inhibitor or on the affinity for Na, as a substrate. Thus, anti-L stimulates, in part, by a specific alteration in the kinetic properties of the pump. This is not the whole story because anti-L also stimulates with no K, present without causing further increase in the number of functioning pumps. Therefore, anti-l probably also raises the turnover number of the pumps. Since anti-L, acting extracellularly, inhibits binding of intracellular IS, we predicted that KCwould modify anti-L binding. We demonstrated modification (Farquharson and Dunham, 1986), but not in the direction expected: K, promoted binding of anti-L. The interactions between anti-L and K, can be understood in terms of a reversible membrane complex of pump and antigen, L-P, to which both KCand anti-L (A) bind in preference to the free pump and antigen, respectively, However, anti-L promotes dissociation Tabie 2. Stimulation of IGF-I of NajK pump in LK sheep red mlfs: effects of varying INa], and [ici,
lK1, n 30
Pump flux (qnol /I x hr) Control IGF-I Stimulation 44 29
62 j,
+1X +7
[IGF-II: 3 x IO-* M. Pump flux: ouabain-inhibitabie S6Rb influx. Media: 145mM NaCl, 5 mM KC1 ?r 0.1 mM ouabain. INaL: 30 mmolil cells; [Na], and w], varied -as wwdescribed before (Dunham and Anderson, 1987).
of the complex, thereby reducing K, binding, whereas KCstabilizes the complex, and this promotes antibody binding. This scheme is summarized as follows: A+L-P-K-A-L+P+K. In this way anti-L can inhibit the binding of K, while K, promotes the binding of anti-L. Stimulation of the pump by hormones The question arises, is there any general significance of stimulation of the pump by anti-L? It is of no obvious consequence to the sheep. There is no physiolo~cal signi~cance of stimulation of the pump by antibodies, but the phenomenon may relate to modulation of the pump by other ligands. Lytton (1985) reported stimulation by insulin of the Na/K pump in rat adipocytes. Insulin raised the affinity for Na, of one of the two isoforms of the pump. Though this is not how anti-L stimulates, this observation prompted us to test for stimulation of the pump by insulin and also insulin-iike growth factor I (IGF-I) in LK sheep cells. We observed stimulation by both hormones. Table 2 shows stimulation by IGF-I at a physiological concentration. At [K], of 30 mmol/l. there was less stimulation of the pump than in K-free cells, suggesting that relief of inhibition by KC is not the basis for stimuiation, unlike stimulation by antiL. On the other hand, stimulation was never seen in HK cells, suggesting that the presence of the L antigen is necessary for stimulation by the hormones. IGF-I may react with a different functionai consequence than anti-L or with a different region of the L antigen. Alternatively, the L antigen may promote interaction of IGF-I receptors (which are on red cells) with pumps. Insulin stimulated as well as IGF-I did, but concentrations -. IO-fold higher were required. Stimulation by the hormones was observed frequently in cells from a number of sheep, but unfortunately not in every experiment, even on cells from the same sheep. The hormones never inhibited transport, so the phenomenon is real enough, but it is difficult to work out in detail until the basis for the variability is determined (and corrected). K-Cl COTRANSPORT
The K-Cl cotransporter, found in a number of cell types (Dunham, 1990), mediates a net KC1 efllux from cells in an obligatorily coupled fashion when the transporter is activated. It can participate in transcellular salt transport in epithelia. It can also transport solute at a sufficient rate to promote osmotically obliged water efflux, and thereby participate in regulation of cell volume. It is responsible for the reduction in cell volume during the differentiation of reticulocytes to erythrocytes in red cells of sheep &auf and Bauer, 1983) and people (O’Neiil, 1989). LK sheep red cells provide a convenient system for study of K-Cl cotransport because it comprises the majority of K transport in these cells (Dunham and Ellory, 1981). To play a role in regulation of cell volume, K-Cl cotransport (or any solute transporter) must be associated with a sensor of cell volume. A small (N 10%) osmotically-induced swelling results in a
627
Ion transport in sheep red blood cells several-fold increase in K transport and shrinkage inhibits it (Dunham and Ellory, 1981). In our view, such a system requires the following: (1) a signal of the volume change, (2) a receptor for the signal, (3) a transducer of the signal to the transporter, and (4) changes in the transporter. It is possible that a single molecule or molecular complex serves more than one of these roles. None of these functions is well understood for the K-Cl cotransporter or for any other volume-sensitive transporter. However, we have made a little progress recently toward understanding all four of these features of the control of K-Cl cotransport. Kinetics of activation by swehng From measurements of steady state of K-Cl cotransport, we showed that both J,, and K,,, for I(, are increased by swelling; see Table 3 (Bergh et al., 1990). Furthermore, these two changes are separable in that changes in J,,,,, and K,,* can be induced independently. Reducing [Mg], caused an increase in J,,, without affecting K,,2. Swelling of low-Mg cells reduced K,,z without further effect on J,,,,, (Table 3). In cells swollen 50% (rather than 25% as in Table 3), the fluxes into control and low-Mg cells were the same, showing that the effects of swelling and reduced [Mgl, on J,,, are not additive. Since the changes in J,,, and Kli2 were separable, we undertook to determine if they occurred in sequence. Though swelling after a hypotonic challenge is complete in seconds, there is a delay of several minutes in the increase in K-Cl cotransport. This was first observed by Kregenow (197 1) in duck red cells, and was subsequently reported for red cells of pigs (Kim et ai., 1989) and sheep (Dunham, 1990). It was first studied in detail by Jennings and Al-Rohil(l990) in rabbit red cells. A two-state model based on chemical relaxation kinetics was applied. In this model, most of the transporters are in a slow state, A, in cells of normal volume. The fraction in a fast state, B, is increased in swollen cells: kl2 AB. kz1 The distribution between the two states is given by two first-order rate constants. After a volume change, the sum of the rate constants evaluated in the new steady state gives the rate of relaxation to that state. The reciprocal of the sum of the rate constants, (k,, + k,,)-‘, gives the time delay of the relaxation process. By fitting to the model results on changes in time course of K fluxes provoked by volume changes, tentative conclusions can be drawn about which rate constants are volume-~nsitive. The swelling-indu~d shift in the distribution of states of the transporter, increased B/A, was Table 3. Steady state kinetics of K-Cl cotransport in LK sheep red cells: effects of osmotic swelling and reduced [MgL
Control
Normal J mm K,,2 3.7 * 0.g 87 * 10 10.5* 2.8 85 f 44
Swollen J mm hi2 5.7 + 1.0 48+6 9.0 f 0.2 28_+2
Low lM& Cl-dependent K influxes in mmol/o~~naI 1 eelis; &, mM [K],. Swollen cells: 1.25 x volume of control ceils. Low (MA ~41s: 20 min preincubation, 10 PM A23187 (from Bergh er af., 1990).
swollen 50%
swollen 20%
/
.L Y
0
Iv
/
ficontro L
IO
1
20 minutes
I
30
Fig. I. K influxes in cells of constant volume or swollen at time zero. Curves were computer fits from mean constants calculated from results of eight experiments using the twostate model (Jennings and Al-Rohil, 1990). The mean constants were: (1) initial steady state influx in cells of constant volume, 4.3 * 0.5 pmol/l cells x min (control); (2) final steady state fluxes, 20% swelling, 16 f 3 pmol/original 1 cells, and 50% swelling, 27 f 4 pmol/original 1 cells; (3)
delays, 20% swelling, 27 & 8 min, and 50% swelling, 20 f 4 min. Curves are shown to 30 min; values were calculated from data collected to 90min.
envisioned to result either from increasing kll or decreasing k,, (or both). These possibilities can be distinguished by determining the relationship between extent of swelling and the length of the delay. If swelling increases k,z, the delay would decrease with increasing extents of swelling, because (k,, + kz,)-‘, the time delay, would progressively decrease as k,, increased. Conversely, if transport was activated by decreasing k,, , the delay would increase with increased swelling. We determined in a series of experiments the effect of the extent of swelling on the delay. The results are summarized in Fig. 1, showing mean curves calculated from eight experiments. The lower curve (control) shows the constant time course of unidirectional K influx into cells of constant volume in isotonic medium. The upper two curves show time courses of K influx into cells swollen 20 or 50% at time zero. By fitting the data to the two-state model, the delays in reaching the new steady state influxes were calculated. In some experiments, greater swelling increased the delay, and in others greater swelling decreased the delay, but the mean delays from all the experiments were similar (see legend, Fig. 1). Therefore both the forward and reverse reactions in the two-state model must be sensitive to changes in cell volume. Specifically, kt2 increases with swelling and k2, decreases, though to varying extents. Jennings and Al-Rohil(I990) arrived at a different conclusion about rabbit red cells. With increased extents of swelling, the delay of relaxation to the new steady state also increased, suggesting that k,, is decreased by swelling and that k,, need not change. When swollen rabbit cells were shrunken, there was no measurable delay, suggesting that shrinkage increases k,, , with no effect on k12. We compared delays of relaxation to a new steady state in sheep cells swollen 50% and in swollen cells shrunken to
628
PHILIPB. DUNHAM
normal volume at time zero. We found a delay of - 10 min when cells were swollen. In contrast to the results with rabbit cells, we observed a delay of the same duration when swollen cells were shrunken, supporting our conclusion that both rate constants are volume-sensitive. To determine whether the difference between results with sheep and rabbit cells is real, or a consequence of differences in techniques, we carried out the experiment just described on rabbit cells. Gratifyingly, we obtained the same results reported by Jennings and Al-Rohil (1990): a delay after swelling, but no delay when swollen cells are shrunken. Therefore rabbit and sheep cells are different: both k,, and k,, appear to be volume sensitive in sheep cells, but only k,, need be in rabbit cells.
Another level of complexity in the time course is expected from the results on steady state kinetics in sheep cells with low-Mg: lowering [Mg], increased -I,,, but not K,/2, while swelling low-Mg cells decreased KIj2 (Table 3). We looked at the time course of swelling-activation in low-Mg cells (Fig. 2). circles show influx in of normal of constant (solid circles) swollen at zero (open circles), with which there was activation with the usual delay. The solid triangles show K influx in low-Mg cells of unaltered volume. The flux is higher than in control cells, presumably due to an increase in J,,,. The open triangles show K influx into low-Mg cells swollen at time zero. The influx is increased further, presumably due to a decrease in and with no discernible delay. The results in K Fig.: 2 suggest that the delay after swelling is in the process whereby Jmaxis increased, and therefore this must precede the process of increase in K,,, which proceeds with little delay. It was important to look at the time course of sh~nkage-inactivation of the flux in swollen low-Mg cells. Interestingly, there was a in delay of - 10 min which was indistinguishable extent from the delay in swollen cells with normal [Mg] (results not shown). These various results with low-Mg cells indicate the necessity of a three-state model: ki2 k23 A-B-C. k;i ksz Most of the transporters in cells of normal volume and with normal [Mg] are in the A-state. In low-Mg cells of normal volume, the fraction of the transporters in the B-state is increased. In swollen cells, normal or low-Mg, the fraction of the transporters in the C-state is increased. We recently estimated that in cells swollen -5O%, about half of the transporters are in the C-state (Dunham and Logue, unpublished results). The rapid swelling-activation in low-Mg cells (Fig. 2) and the slow shrinkage-inactivation in these cells lead to the conclusion that both k,, and k,z increase with swelling, though to different extents, and both decrease with shrinkage. This conclusion is based on these two observations on time courses (B-C conversions in the three state model), and on the recent conclusion that k,, and k,, are approximately equal in low-Mg cells swollen SO%, a conclusion drawn from the estimate that approximately
minutes
Fig. 2. Effect of swelling on K influx in cells of normal Mg, (0, 0) and with M& reduced (A, A) (see Table 3 for method of reducing Mg,). Cells were either of constant volume (*, A) or swollen 20% at time zero (0, A). Lines were fitted by eye (redrawn from Dunham, 1990).
half of the transporters are in the C-state in this condition. The lower limit for k,, + k,,, the rate of activation by swelling of low-Mg cells, is about 1-l min [the delay, (k,, + k32)--L,would be 1 min, and this could have been resolved, so the rate is at least 1 min-‘1. Assuming this rate, kX3+ k,, = I min-’ and k 23= kjZ = 0.5 min-‘. The delay for the inactivation (C-B) was - 10 min, so k,, + k,, after shrinkage = -0.1 min-‘. Since 0.5 min-’ > 0.1 min-‘, both rate constants must decrease with shrinkage, even if k,, > k,,, and both must increase with swelling. Rate constants changing in the same direction is consistent with, but does not prove, a single catalytic process promoting Bw C conversions. The time courses observed after shrinking and swelling of nodal-Mg cells (A-C’) indicate that a rate constant for a forward reaction increase with swelling (Fig. 1); this must be k,,. In addition a rate constant for a reverse reaction must decrease with swelling, and this is k,, if kj2 increases with swelling, as just concluded. k,z need not be volume-sensitive. The same conclusions were reached about k,? and k,, by Jennings and Al-Rohil (1990) for rabbit cells and a two-state model. These conclusions are consistent with two catalytic processes responsible for the A+-+B conversions. Role of phosphate metabolism
The next step in the analysis of the transduction process is to attempt to assign the volume-sensitive rate constants to biochemical processes. Jennings and Schulz (1991) found that okadaic acid, a specific phosphatase inhibitor, inhibited swelling-activation of K influx in rabbit red cells, so protein dephosphorylation must be necessary for activation by sweiling. Similar results were reported for human red cells (Kaji and Tsukitani, 1991). It follows that the phosphatase is volume-insensitive (k,*) and that swellingactivation occurs by inhibition of a volume-sensitive protein kinase (k,, ). Jennings and Schulz (1991) were unable to assign this kinase to any of the known classes of protein kinases. Sheep and rabbit cells differ in that sheep cells appear to have a swelling-activated process (in the three-state model, the increases in k,, and k,, with
529
Ion transport in sheep red blood cells
swelling). Our working hypothesis about A-B conversions in sheep cells is that k12 is the rate constant for a volume-sensitive phosphatase and k,, is the rate constant for a protein kinase inhibited by swelling. We have shown in preliminary experiments that okadaic acid inhibits K-Cl cotransport and its activation in sheep red cells. We speculate below on a possible basis for the 3-C conversions,
In an attempt to gain further insight into the control of K-Cl cotransport, we employed a simplified experimental system, inside-out vesicles (IOVs) made from LK sheep red eel1 membranes. We demonstrated Cl-dependent K effluxes from these IOVs, and showed that the efflux was increased by swelling the vesicles and decreased by shrinking them (Kracke and Dunham, 1990). This is a convenient system for study of the cotransporter because there is essentiaiiy no cytoplasm, and yet at least some of the regulatory machinery remains associated with the membrane. Therefore, one can make measurements of the flux with simultaneous and continuous access to the cytoplasmic membrane surface, the presumed locus of the regulatory machinery. These results provide clues about the nature of the signal of volume changes. One can envision two classes of signals of cell swelling: (I) a mechanical change in the membrane, and (2) a decrease in the concentration of a critical cytoplasmic solute. The results with the IOVs rule out a change in concentration of a solute, and therefore indicate a mechanical change as the signal. The results also place limits on the nature of the m~hanical change which can serve as a signal. Swelling of both intact cells and IOVs increases the flux. Since the membranes of these two systems have opposite orientations, change in degree of curvature of the membrane is not likely to be the signal. Other possibilities are stretch, which is unlikely, and a change in relative orientation of integral and peripheral regions of the membrane, which we favor. In preliminary experiments, we have looked at the time course of swelling-activation of K influx in IOVs. There was no indication of a delay in the approximately six-fold increase of the flux after -40% swelling. Therefore, the vesicles behaved as though [Mg] was low even though it was 1 mM. We next looked at the dependence on [Mg] of K flux in the IOVs of unchanged or swollen volume, and found no effect when varying [Mgl from nominally zero to 1 mM. Apparently, in preparing the vesicles, there was a conversion of most of the transporters to the B-state, which have no delay in swelling-activation (expected from results on intact cells), and an insensitivity to Mg (not expected from intact cebs). This latter characteristic suggests an irreversible conversion of A to B, perhaps owing to a loss of constituent(s) of the membrane and/or cytoplasm. Multiple volume sensors
Jennings and Schulz (1990) presented evidence that the signal of swelling in rabbit red ceils is not a m~hanical change in the membrane, but rather a change in concentration of a critical cytoplasmic solute. Echinocytes and discocytes have the same rate
of K-Cl cotransport as the erythrocytes from which they were made. This and our results on IOVs raise the possibility of two types of signals of swelling. In terms of the three-state model, this could mean two signals operating in sequence, one promoting A -+ B and a second promoting B --f C. The first, a decrease in concentration of a ~ytoplasmic solute, would inhibit a volume-sensitive protein kinase (rate constant k,,) resulting in an increase in JmaX. Since Mg-ATP is a substrate for the kinase, a reduction in Mg activity caused by swelling could be the signal (if [Mg] is rate limiting). The resultant inhibition of the kinase would activate cotransport (since Mg may also be required by the phosphatase, the Mg activity need be limiting only for the kinase and not for the phosphatase). The argument for two signals is that reducing cell [Mg] does not fully activate cotransport; swelling is required. The results on IOVs indicate that the second signal is mechanical It was argued above that the Bc+C conversions are controlled by a single catalyst. A protein kinase is unlikely to be involved since neither Mg (nor ATP) is required. Indeed, the catalyst does not even need to be an enzyme. Thus, the reduction in K,,* for K associated with the B -+ C conversion could be due to a swelling-induced structural rearrangement in the membrane. Despite this speculation about the transduction mechanism, a dimly perceived picture of the regulation of K-Cl cotransport is beginning to emerge. Acknowiedgement-ale original work reported here was supported by NIH grant DK-33640. REFERENCES
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PHILIP B. IWiiH~t4
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Lytton J. (1985) Insulin affects the sodium affinity of the rat adipocyte (Na+, K+)-ATPase. J. biol. Chem. 260, 1~75-1~80.
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Tosteson D. C. and Hoffman J. F. (1960) Regulation of cell volume by active cation transport in high and low potassium sheep red cells. J. gen. Phsviol. 44, 1599194.
