Effect of changes in respiratory blood parameters on equine red blood cell K-Cl cotransporter

P. F. Speake1, C. A. Roberts2, and J. S. Gibson1

1 Department of Veterinary Preclinical Sciences, University of Liverpool, Liverpool L69 3BX; and 2 Centre for Equine Studies, Animal Health Trust, Newmarket, Suffolk CB8 7DW, United Kingdom

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

K influx into equine red blood cells (RBCs) was measured using 86Rb as a tracer for K under conditions designed to mimic the changes in respiratory blood parameters that occur in vivo during strenuous exercise. The effects on K influx of physiological changes in pH, cell volume, O2 tension (PO2), CO2 tension (PCO2), and bicarbonate and lactate concentrations were defined. Physiological PO2 exerted a dominant controlling influence on the H+-stimulated Cl-dependent K influx, consistent with effects on the K-Cl cotransporter; PO2 required for half-maximal activity was 37 ± 3 mmHg (4.9 kPa). Although RBCs were swollen at low pH, results showed explicitly that the volume change per se had little effect on K influx. Lactate had no effect on volume- or H+-stimulated K influxes, nor did bicarbonate or PCO2 affect the magnitude of K influxes after these stimuli or after treatment with protein kinase/phosphatase inhibitors. These results represent the first detailed report of O2 dependence of H+-stimulated K-Cl cotransport in RBCs from any mammalian species. They emphasize the importance of PO2 in control of RBC K-Cl cotransport.

oxygen; pH; bicarbonate; lactate

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

RED BLOOD CELLS (RBCs) from horses and ponies contain high intracellular levels of K, although K transport across their membranes is dominated by a high-capacity K-Cl cotransporter (7, 8). When active, this transporter will mediate net K-Cl efflux at high rates and cause the cells to shrink. Consequently, an understanding of both K balance and volume regulation in the equine RBC requires a thorough appreciation of the factors that affect the activity of this cotransporter.

In common with K-Cl cotransport in RBCs of other species (16), the equine system is sensitive to cell volume, pH, and urea (7, 8, 27). We have recently established that O2 represents a further modulatory factor (13). In the absence of an adequately high O2 tension (PO2), the cotransporter is inactive and refractory to stimuli such as cell volume and pH. The cotransporter is also unable to respond to low concentrations of urea, although at higher concentrations the activity of the cotransporter becomes independent of PO2 (26, 27).

A number of physiological variables that potentially affect the activity of RBC K-Cl cotransporter are altered by strenuous exercise (see Table 1). Perhaps the most important change is the drop in plasma pH on development of lactic acidosis (25). This fall in pH per se would stimulate K-Cl cotransport. Other exercise-induced changes in plasma variables that may affect the response of the cotransporter include PO2, CO2 tension (PCO2), and bicarbonate and lactate concentration. Although the transporter is inactive in the absence of PO2 (13), the effect of PO2 over the physiological range is not known and that of the other parameters has not been studied.

                              
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Table 1.   Effect of exercise on respiratory parameters in the equine circulation

In this study, we have investigated the extent to which these variables affect the equine RBC K-Cl cotransporter. Our results suggest that PO2, plasma pH, and cell volume are the most important determinants of cotransporter activity. We have characterized the O2 dependence of the cotransporter during stimulation by H+ over the complete physiological range of PO2. We found that changes in PCO2 and bicarbonate and lactate concentration had little effect. Our results are valuable in understanding the physiology of the equine RBC; they are also relevant to control of K-Cl cotransport in RBCs from other species.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials

Dimethyl sulfoxide (DMSO), Drabkin's reagent, 3-(N-morpholino)propanesulfonic acid (MOPS), N-ethylmaleimide (NEM), ouabain, lactic acid, KNO3, NaHCO3, NaNO3, trichloroacetic acid (TCA), and Triton X-100 were purchased from Sigma (Poole, Dorset, UK). Glucose, KCl, NaCl, NaOH, HNO3, and sucrose were purchased from Merck (Poole, Dorset, UK) and calyculin A from Calbiochem-Novabiochem (Nottingham, UK). N2, O2, and CO2 were purchased from BOC (Guildford, UK).

Salines

The standard MOPS-buffered saline (MBS) used for washing and storing the cells and for most experiments contained (in mM) 130 NaCl, 5 NaNO3, 5 glucose, and 20 MOPS (pH 7.4 at 37°C). The bicarbonate (HCO3)/CO2-buffered saline (BBS) contained (in mM) 130 NaCl, 25 NaHCO3, and 5 glucose, adjusted to the desired pH by varying PCO2. In experiments testing for Cl dependence of the K influx, Cl-containing salts were substituted with NO3-containing ones. For all salines, K (KCl or KNO3 as appropriate) was added with isotope to give a final K concentration ([K]) of 7.5 mM. Solutions had an osmolality of 291 ± 7 mosmol/kgH2O.

Stock solutions of calyculin A (10 µM) were prepared in 20% DMSO-80% MBS, stored at -20°C, and used at a final concentration of 100 nM. Stock solutions of ouabain (10 mM) were prepared with water, stored at 4°C, and used at a final concentration of 100 µM. Stock solutions of NEM (100 mM) were prepared daily in experimental saline and used at a final concentration of 1 mM.

Sample Collection and Handling

Blood samples for most experiments were obtained from Welsh mountain ponies or thoroughbred horses kept at the Department of Veterinary Clinical Sciences and Animal Husbandry, Leahurst, Neston, South Wirral, UK. All samples were taken by jugular venipuncture into heparinized vacutainers and kept on ice or refrigerated (4°C) until use (within 50 h of being taken). On experimental days, samples were centrifuged (Denley BR401 refrigerated centrifuge, Billingshurts, West Sussex, UK) at 10°C for 5 min at 1,500 g to remove the RBCs from the buffy coat and plasma. The RBCs were washed with the MBS three additional times (MBS volume was 20 times that of cells). Before experimentation, the cells were centrifuged as before and resuspended in the experimental saline at the requisite hematocrit. In some experiments, cells were either shrunken or swollen anisosmotically by addition of hypertonic sucrose or distilled water, respectively, to the experimental saline.

Hematocrit Determination

The hemoglobin content of the cell suspensions was measured as cyanomethemoglobin at 540 nM (UV-160A ultraviolet visible recording spectrophotometer; Shimadzu, Kyoto, Japan) in Drabkin's solution and converted to hematocrit by using a value for equine horse RBCs of 242 g hemoglobin/l (experimentally derived).

Tonometry

Before flux measurements, RBC suspensions were incubated at ~40% hematocrit in glass tonometers (Eschweiler, Keil, Germany). Gas mixtures with variable concentrations of O2, CO2, and N2 were made using a calibrated gas mixing pump (Wösthoff, Bochum, Germany), warmed to 37°C, and fully humidified through three humidifiers (Eschweiler) before delivery to the tonometers. Preliminary experiments showed that the RBCs equilibrated to the delivered gas concentrations in the tonometers within 10 min. Cells were then diluted (1/10) to a hematocrit of ~4% into saline preequilibrated at the required PO2 and PCO2 before measurement of K influx.

K Influx

Ouabain-insensitive K influx (with ouabain present at 100 µM) was measured by conventional radioactive tracer techniques, using 86Rb (DuPont NEN, Bad Homburg, Germany) as a tracer for K (7). 86Rb (0.02-0.05 MBq/ml final) was added to the cell suspension in 150 µM KCl or KNO3 (final [K] = 7.5 mM), and influx was measured over 10-15 min, during which time uptake was linear. Fluxes were terminated and samples then processed as described previously (7). Influxes are expressed as millimoles of K per liter of cells per hour. In experiments in which PO2 was varied, P50 for influx was estimated as the PO2 required to achieve 50% influx, measured at 150 mmHg (20 kPa) in MBS and 130 mmHg (17.3 kPa) in BBS [control experiments showed that influx peaked by 100 mmHg (13.3 kPa) and remained at this level at higher PO2 levels].

Measurement of Cell Water Content

Cell water content was determined in triplicate, using the wet weight/dry weight method (13). Cell water content was expressed as milliliters per gram of dry cell solids.

Measurement of Extracellular and Intracellular pH

Unless stated, pH refers to extracellular pH (pHe). pHe was measured directly in the presence of cells under the same experimental conditions as for flux measurement, using a Mettler Delta 340 meter (Mettler Toledo, Halstead, Essex, UK) in combination with a Mettler Inlab 423 micro pH probe. For the measurement of intracellular pH (pHi), 400 µl of relevant cell suspension were centrifuged through 500 µl dibutyl phthalate oil (10 s at 15,000 g in an Eppendorf 5415C centrifuge, Hamburg, Germany) to separate the RBCs from the incubation medium. Supernatant and excess oil were aspirated, and the cell pellet was subjected to two cycles of freeze-thawing. pHi was then measured directly in the cell lysates. Control experiments showed that the oil was not significantly permeable to O2 or CO2 over the time course of the pHi measurements.

Statistics

Unless otherwise stated, data are expressed as means ± SE for n animals. Data for some experiments are presented as means ± SD for quadruplicate measurements on one animal, and these were representative of similar experiments on at least two additional animals. Statistical analysis was performed using either Student's t-test or one-way analysis of variance followed by the Dunnett's post hoc test or Dunn's test for multiple comparisons based on descriptions from Zar (33). Differences were considered significant when P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Interaction of O2, H+, and Cell Volume on K-Cl Cotransport

Effect of pH on cell volume and K influx. During strenuous exercise in horses, plasma pH falls from the normal pH 7.4 to ~7.0, even in the arterial circulation, and remains low for many minutes (25). When measured in vitro in fully oxygenated cells over this pH range (from pH 7.4 to 7.0), RBC volume increased significantly from 1.63 ± 0.01 to 1.74 ± 0.01 mg/g dry cell solids (n = 4; P < 0.001, Student's t-test), while K influx increased from 0.222 ± 0.048 to 1.013 ± 0.407 mmol · l cells-1 · h-1 (n = 4; P < 0.05, Student's t-test). Thus acidosis was accompanied by cell swelling of the RBCs and marked stimulation of K influx.

H+-stimulated K-Cl cotransport and O2. We have previously shown that O2 has potent effects on the activity of the equine RBC K-Cl cotransporter (8). We have defined in detail the effect of PO2 on volume- and urea-stimulated K-Cl cotransport (27). H+-stimulated K influx has also been shown to be O2 dependent (13), but the relationship has not been characterized over physiological PO2 values.

We investigated the relationship between K influx and PO2 in Cl-containing and Cl-free salines (Cl substituted with NO3) at pH 7.0 over the physiological range of PO2 encountered in the circulation (Fig. 1). The relationship between Cl-dependent K influx and PO2 was sigmoidal, with a P50 of 37 ± 3 mmHg (n = 5). At low PO2 levels characteristic of those found in contracting muscle (<10 mmHg), H+ was unable to stimulate K influx; at mixed venous PO2 (~40 mmHg), K influx was about half-maximal, whereas arterial PO2 (between 70 and 100 mmHg) was required for full activation. K influxes in Cl-free media were low and unresponsive to changes in PO2 (Fig. 1), showing that H+ affected only the Cl-dependent component of K influx across the equine RBC membrane, often taken as representing K-Cl cotransport (7). Because K influxes in NO3 saline were unaffected by PO2, pH (Fig. 1), or volume (13), in all other experiments (for exceptions see Fig. 4B), the responses of the cells were only determined in Cl-containing media.


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Fig. 1.   Effect of oxygen tension (PO2, mmHg) on K influx (mmol · l cells-1 · h-1) in equine red blood cells (RBCs). K influx was measured at pH 7.0 in presence (open circle ) or absence (square ) of Cl (substituted with NO3). Difference between influx measured in the 2 salines is also given (bullet ). PO2 is indicated, and balance to atmospheric pressure was provided by N2. Symbols (means ± SD) represent quadruplicate determinations on a single sample and are representative of experiments from 4 additional animals.

Interaction of H+ and cell volume. High H+ concentration ([H+]) results in swelling of the RBC (see Interaction of O2, H+, and Cell Volume on K-Cl Cotransport) as Cl enters subsequent to titration of the negatively charged hemoglobin by protons (12). A component of the H+ stimulation of cotransport may be due to this H+-induced swelling of the RBCs. In this series of experiments, both K influx and cell volume were measured at two pH levels, 7.4 and 7.0, in cell samples in which volumes were also altered independently (Fig. 2). Cells were swollen or shrunken anisosmotically by addition of distilled water or hypertonic sucrose, respectively, to the normal saline while keeping ionic strength, Cl concentration, and [K] constant. In isotonic saline, at pH 7, K influx was stimulated by 0.772 ± 0.360 mmol · l cells-1 · h-1 (mean ± SD, n = 6) and caused the cells to swell by ~6% (both relative to cells in isosmotic saline at pH 7.4). This degree of cell swelling per se would stimulate influx at pH 7.4 by ~0.164 mmol · l cells-1 · h-1, only accounting for about one-fifth of the pH-stimulated influx (Fig. 2). Thus the major part of the H+-stimulated influx was not due to swelling: in effect, low pH sensitized the response of cells to volume increase. Figure 2 also shows that the volume of cells held at pH 7 must be decreased considerably (much more than 10%) to reduce K influx to that seen in isotonic saline at pH 7.4. 


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Fig. 2.   Effect of cell volume [ml/g dry cell solids (d.c.s.)] on K influx (mmol · l cells-1 · h-1) in equine RBCs. K influx was measured at pH 7.4 (open circle ) and 7.0 (square ). Cell volume was altered anisosmotically by addition of hypertonic sucrose or distilled water while ionic strength was kept constant. PO2 was 143 mmHg, and balance to atmospheric pressure was made up with N2. Arrows indicate cell volume and influx values in isotonic saline for the 2 pH levels. Symbols are means ± SE; n = 4 animals.

Effect of Changes in PCO2 and HCO3 Concentration on K-Cl Cotransport

It has been proposed recently that physiological HCO3 concentration ([HCO3]) or CO2 inhibits volume-stimulated K-Cl cotransport in human RBCs (J. C. Ellory and H. Godart, personal communication). In equine arterial plasma, [HCO3] and PCO2 are normally ~25 mM and 40 mmHg (5.3 kPa), respectively. During exercise, PCO2 initially rises but subsequently returns to ~40 mmHg as ventilation increases, while the [HCO3] falls to 10 mM or less (e.g., Refs. 1 and 31). We examined the effects of changes in PCO2 and [HCO3] on the equine RBC.

Intracellular pH and cell volume. In human RBCs, pHi, as opposed to pHe, is probably the more important determinant of pH-induced changes in K-Cl cotransport and cell volume (5). pHe was altered in MBS by the addition of NaOH or HNO3 and in BBS by varying PCO2 between 14 and 392 mmHg (1.9-52.3 kPa), while [HCO3] was kept nominally constant at 25 mM. pHe was measured after addition of RBCs, with stable pHe levels obtained within a few minutes in BBS, although a longer period of ~10 min was required in MBS. Varying pHe affected pHi (Fig. 3A) and cell volume to a similar degree in both salines (Fig. 3B).


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Fig. 3.   Effect of extracellular pH (pHe) on intracellular pH (pHi, A) and cell volume (ml/g dry cell solids, B) of equine RBCs in either bicarbonate (HCO3)- or MOPS-buffered saline (BBS or MBS, respectively). For MBS (square ), pH was adjusted by addition of KNO3 or NaOH; for BBS (open circle ), HCO3 concentration was kept nominally constant at 25 mM and pH varied by changing PCO2 from 14 to 392 mmHg (1.9 to 52.3 kPa). pHe was measured in presence of RBCs at experimental hematocrit. pHi was measured on cell lysates obtained by freeze-thawing aliquots centrifuged through dibutyl phthalate oil. Symbols are means ± SE; n = 4 different animals.

H+-sensitive K-Cl cotransport. Experiments were designed to compare H+-stimulated K influx, and its interaction with PO2, in cells held in either MBS or BBS (Fig. 4, A and B). As before, pHe of the MBS was altered by addition of NaOH or HNO3; for BBS, [HCO3] was kept nominally constant at 25 mM, and PCO2 was varied between 14 mmHg (1.9 kPa) and 392 mmHg (52.3 kPa). pHe levels in the range of 7.7-6.3 were allowed to stabilize before the measurement of K influxes. All pH levels were measured in the presence of RBCs at the experimental hematocrit. When pHe in [HCO3]/PCO2 saline was carefully controlled to match that seen in MBS saline, the K influxes observed were comparable (Fig. 4A).


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Fig. 4.   Effect of buffer composition on K influx (mmol · l cells-1 · h-1) in equine RBCs. A: K influx was measured in cells held in MBS (open circle ) and BBS (25 mM HCO3/CO2; square ) at 3 different pHe levels. For MBS, pH was adjusted by addition of KNO3 or NaOH; for BBS, HCO3 concentration was kept nominally constant at 25 mM and pH varied by changing PCO2 from 14 to 392 mmHg (1.9 to 52.3 kPa). pHe was measured in presence of RBCs at experimental hematocrit. PO2 was 143 mmHg, and balance to atmospheric pressure was provided by N2. Symbols are means ± SE; n = 4 different animals. B: effect of PO2 (mmHg) on Cl-dependent K influx in equine RBCs. BBS (open circle ) was buffered with 25 mM HCO3/107 mmHg (14.3 kPa) CO2 and MBS (square ) with 20 mM MOPS, both at pH 7. Cl-dependent influx was calculated as difference in influx measured in presence or absence of Cl (substituted with NO3) and expressed as a percentage of maximal values, which were 1.159 ± 0.102 and 1.098 ± 0.111 mmol · l cells-1 · h-1 in BBS and MBS, respectively (not significant, Student's t-test). In all cases, balance to atmospheric pressure (less PO2 and PCO2) was made up with N2. Symbols represent means ± SE; n = 4 different animals for BBS and 5 different animals for MBS.

The O2 dependence of H+-stimulated K influx in BBS was compared with that in MBS over the physiological range of PO2. K influx was measured at pH 7 in both Cl-containing and Cl-free media. The O2 dependence of the chloride-dependent K influx, shown in Fig. 4B, was comparable in BBS and MBS. The plots were sigmoidal, and the P50 was identical: 37 ± 3 in MBS (n = 5) and 37 ± 3 mmHg in BBS (n = 4) [not significant (NS), Student's t-test].

Similar results, with no significant difference between cells in MBS and BBS, were observed for volume-stimulated K influxes; thus volume-sensitive K influxes (defined as the difference between influx measured in cells swollen and shrunken anisosmotically by 10%) were 1.268 ± 0.302 in MBS and 1.113 ± 0.194 mmol · l cells-1 · h-1 in BBS (n = 4; NS, Student's t-test).

Protein phosphatase/kinase inhibitors and BBS. K influx in equine RBCs is modulated by protein phosphatase/kinase (PP/PK) inhibitors, and we determined the effect of these compounds in BBS compared with MBS. Cotransport is abolished by the specific serine-threonine inhibitor, calyculin A (8). Calyculin A also inhibits the stimulation caused by subsequent addition of NEM, a putative inhibitor of protein kinase. Combinations of NEM followed by calyculin will clamp flux at a level intermediate between zero (calyculin only) and maximal (NEM only) (see Ref. 13 for details). We determined the effect of MBS and BBS on the K influx measured after application of these drugs. With NEM only, K influxes were 5.910 ± 0.766 and 5.788 ± 0.688 mmol · l cells-1 · h-1 in MBS and BBS, respectively (n = 5; means ± SE; NS, Student's t-test). In cells in which K influx was clamped with combinations of NEM followed by calyculin, K influxes were 3.145 ± 0.448 and 3.078 ± 0.341 mmol · l cells-1 · h-1 in MBS and BBS, respectively (n = 5; means ± SE; NS, Student's t-test).

Effect of Lactic Acid on K-Cl Cotransport

The concentration of lactate in equine plasma rises from 1 mM at rest to >30 mM during exercise (e.g., Ref. 25). After allowing for equilibration to occur across the RBC, we investigated the effect of lactate on cell volume and volume and H+ sensitivity of K influxes. Cell volumes at pH 7.0 were 1.75 ± 0.01 and 1.77 ± 0.02 ml/g dry cell solids (n = 4, means ± SE) in the absence or presence of 30 mM lactate, respectively (NS, Student's t-test). Neither volume-sensitive influxes (Fig. 5A) (defined as the difference between cells anisosmotically shrunken and swollen by 10%) nor H+-stimulated influxes (Fig. 5B) (defined as the differences between influxes measured in pH 7.4 saline and pH 7.0 saline with or without lactate) were affected by lactate (NS, one-way analysis of variance followed by Dunn's test for multiple comparisons).


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Fig. 5.   Effect of lactate on pH- and volume-stimulated K influx (mmol · l cells-1 · h-1) in equine RBCs. A: K influx was measured at pH 7.4 in samples at normal cell volume and samples shrunken or swollen anisosmotically by addition of hypertonic sucrose or distilled water, respectively, in absence (open bars) or presence (hatched bars) of 30 mM lactate (bars represent means ± SE; n = 5 different animals). B: K influx was measured at pH 7.4 in absence of lactate and at pH 7.0 without lactate and with lactate present at 30 mM (bars represent means ± SE; n = 4 different animals). PO2 was 143 mmHg, and balance to atmospheric pressure was provided by N2.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this study, we have investigated the effect of changes in pH, cell volume, PO2, PCO2, HCO3, and lactate on the activity of the K-Cl cotransporter in the equine RBC. Our findings are important because they demonstrated a critical dependence of H+-stimulated K fluxes on PO2. Our results characterized the response of these fluxes to physiological PO2 and emphasize the preeminent role of PO2 in control of the K-Cl cotransporter. HCO3, lactate, and CO2 have not been studied previously with respect to their action on the equine K-Cl cotransporter. We showed that they had relatively little effect. Efforts aimed at understanding the role of the equine RBC cotransporter in vivo should be centered on PO2, H+ concentration, and cell volume and how they interact to regulate transporter activity.

Effect of Exercise in the Horse

Horses, along with dogs and some other species, such as the pronghorn antelope, are widely regarded as the supreme athletes among mammals (17, 30). It is less well recognized that the circulatory and respiratory stresses imposed by strenuous exercise in the horse result in profound changes in respiratory blood parameters (Table 1). Ventilation is inadequate to meet the huge increase in O2 demand from active muscle beds, and marked arterial hypoxemia and metabolic acidosis become evident (3, 31). In addition, in trained horses, the hematocrit will increase because of splenic contraction and expulsion of stored RBCs into the main circulation. This elevation in the circulating RBC number is necessary to maintain adequate O2 transport to the tissues in the face of profound hypoxemia. Polycythemia presents its own strains on the vasculature (29), however, through the rise in viscosity of the blood and hence vascular resistance. Because cardiac output has also increased to meet the metabolic demands, systemic and pulmonary blood pressures are highly elevated. It is not surprising that pulmonary capillaries may break under the strain, and exercise-induced pulmonary epistaxis is not uncommon (32).

Respiratory Blood Parameters and Modulation of the RBC K-Cl Cotransporter

The mature equine RBC has a very high capacity for K-Cl cotransport (7). In comparison with human RBCs, for example, the cotransport rate is ~10-fold higher in the horse (11). Regulation of the equine K-Cl cotransporter shares many features in common with that of other mammalian species (see Ref. 16 for review). We have shown previously that K influx across the equine RBC membrane is composed almost entirely of transport through the K-Cl cotransporter, which is stimulated by cell swelling and therefore provides a regulatory pathway to reduce volume (7). K-Cl cotransport activation is influenced by the protein phosphorylation status (8, 13-15), such that net dephosphorylation of the cotransporter or some regulatory protein activates and net phosphorylation inhibits. Physiologically, however, the main stimulus on circulating RBCs in vivo is likely to be plasma pH (5, 8). After strenuous exercise, the metabolic acidosis in the horse is marked and sustained, over some 30 min or so (25). Low pH per se causes cell swelling (Fig. 3B) and stimulation of the cotransporter (Fig. 4B), with the consequent loss of K-Cl, cell shrinkage, and a potential elevation in plasma K concentration. However, other plasma variables affected by exercise (especially PO2, PCO2, HCO3, and lactate) may all modulate the response of the equine RBC to pH and volume, and it was therefore important to test their effect on the activity of the cotransporter.

A Central Role for O2 in Control of K-Cl Cotransport

O2 modulates the response of the K-Cl cotransporter. We have shown previously that in the absence of O2, the cotransporter is inactive and refractory to stimuli such as cell swelling and changes in pH (8) but not to stimulation by urea (27). The latter may be important in stimulating cotransport as cells pass through the renal medulla, which physiologically is the only site at which RBCs will encounter significant concentrations of urea under normal conditions (19). We have fully characterized the O2 dependence of volume- and urea-activated fluxes (26). High concentrations of urea significantly altered the O2 dependence of K fluxes; swelling did not. To date, however, the response of H+-stimulated cotransport in mammalian red cells has not been investigated with regard to physiological changes in PO2.

Here we show that the P50 (the PO2 required for half-maximal activation of the transporter) is ~40 mmHg. In the arterial circulation, even during the hypoxemia seen during strenuous exercise, the cotransporter will be substantially or fully activated. In mixed venous blood, where PO2 levels of ~40 mmHg are seen, the transporter will be half activated. In muscle beds, with PO2 levels of <20 mmHg, the transporter will be turned off. For a substantial portion of their time during circulation, RBCs will encounter sufficiently high PO2 levels for prolonged or repeated short periods of acidosis to stimulate their K-Cl cotransporter. The precise effect, however, requires a knowledge of the kinetics of transporter activation and inactivation during cyclic changes in PO2, and this is a problem that is currently being addressed.

The mechanism by which O2 affects K-Cl cotransport is unknown, although it probably involves alteration in the relative activities of regulatory PP/PK enzymes (13). A number of other O2-sensitive systems are known and their transduction paths investigated (2); comparison with them may be helpful in establishing the O2-sensitive mechanism of the RBC K-Cl cotransporter.

K-Cl Cotransport, CO2, and HCO3

It has recently been proposed that the human RBC K-Cl cotransporter is inhibited by physiological concentrations of HCO3 and/or PCO2 (J. C. Ellory and H. Godart, personal communication). Thus volume-stimulated transport in saline buffered by artificial means, such as MOPS or tris(hydroxymethyl)aminomethane, may be substantially higher than that measured at the same pH in saline buffered with HCO3/CO2. Here we have investigated the effects of HCO3 on cell volume and on volume- and H+-activated K influxes in the equine RBC. We also investigated the effect of HCO3 on cells after pharmacological manipulation with PP/PK inhibitors, calyculin A and NEM. When PCO2 and PO2 were carefully controlled, using a gas mixing pump and tonometers, we found that CO2 and HCO3 had no significant effects on cell volume, pHi, or K influxes (Figs. 3 and 4). It remains to be established why the cotransporters in human and horse behave differently in BBS. The complex regulatory pathways in the RBC involving cascades of PP/PK enzymes may be subtly different between the two species. Such differences appear to exist [for example, in the effects of calyculin A on NEM-activated K fluxes in low-K-containing sheep RBCs (6) compared with those in trout (4) and horse (9)]. An alternative explanation may be that the inhibitory effects of anions on the cotransporters vary, perhaps because of differences in protein structure. The recent cloning of the K-Cl cotransporter from rat, rabbit, and human tissues (10) will enable a structure-function analysis across the different species to be carried out in due course.

K-Cl Cotransport and Lactic Acid

During strenuous exercise in the horse, lactic acidosis is profound. Lactate fluxes across the RBC membrane are very fast, especially in the dog and horse, in which the monocarboxylate transporter appears to operate at a higher maximal velocity than in less athletic species (23). It is possible that lactate affects the RBC cotransporter directly or indirectly via cell volume or pH. We have examined the effects of 30 mM lactate on the RBC after allowing for equilibration of lactate across the membrane (Fig. 5, A and B). Our results show, however, that under the conditions used here lactate had no significant effect on any of the RBC parameters examined. We are currently investigating the effects of transient changes in lactate concentration on the equine RBC.

Role of K-Cl Cotransport

The equine RBC K-Cl cotransporter may have several important roles. First, it will enable the cells to decrease their volume rapidly. Efficient osmoregulation will limit the occurrences when RBCs encounter an anisotonic environment, but they will still occur. In all mammals, anisotonicity is found in the renal medulla and certain other epithelial capillary beds, for example, those of the intestine during water absorption. In the horse, plasma osmolality fluctuates markedly after dehydration and subsequent rehydration in prolonged exercise or in desert conditions (22, 24). Equine sweat is notable in being hypertonic to plasma, unlike that of humans (22). Furthermore, low plasma pH, such as occurs during lactic acidosis, will cause significant red cell swelling and may necessitate efficient volume regulatory responses.

Second, the cotransporter may not be important for RBC volume regulation per se. A number of membrane transporters that participate in volume regulation are also involved in other functions, for example, pH regulation by the Na/H exchanger. In this context, the Na/H exchanger of trout red cells is especially noteworthy. This transporter responds to beta -adrenergic stimulation, rather than cell volume, and may be important in improving O2 carriage by RBC hemoglobin when PO2 in the environment is low (20). Low PO2 itself also stimulates the exchanger and may facilitate such a role (21). Efflux of K via the K-Cl cotransporter will elevate plasma K concentration and may contribute to the exercise-induced hyperkalemia. Modulation of the cotransporter by PO2 may be important in limiting K efflux to meet particular requirements; for example, during acidosis, K loss would be greater on the arterial side of the circulation and may participate in vasodilation of arterioles supplying active muscle beds. Conversely, further K loss from RBCs on the venous side of the circulation would be prevented. This would conserve RBC K and limit the elevation in plasma K that would otherwise occur.

Third, the equine RBC K-Cl cotransporter may be relevant pathologically. Excessive KCl efflux will cause RBC shrinkage, a rise in cytoplasmic viscosity, and hence increased vascular resistance (29). The rheological effect of shrunken RBCs may contribute to a number of peculiar equine disorders that involve ischemia or increased vascular resistance but for which etiologies remain obscure, for example, laminitis, rhabdomyolysis, and exercise-induced pulmonary epistaxis.

In conclusion, although the function of the K-Cl cotransporter in equine RBCs remains unclear, a thorough knowledge of the stimuli that regulate its activity will be required before a definitive role can be elucidated. Results contained in this paper clarify which plasma variables represent the most important determinants of K-Cl cotransport activity and, in particular, stress the importance of PO2.

    ACKNOWLEDGEMENTS

We thank Drs. J. E. Cox and M. Muzamba from Leahurst, Neston, South Wirral, UK, for assistance with blood samples.

    FOOTNOTES

This work was supported by the Wellcome Trust, the Nuffield Foundation, and the Physiological Society.

Some of these results have been published in abstract form (28).

Address for reprint requests: J. S. Gibson, Dept. of Veterinary Preclinical Sciences, Univ. of Liverpool, Brownlow Hill, Liverpool L69 3BX, UK.

Received 12 May 1997; accepted in final form 29 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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