1 Laboratory of Pathobiochemistry, School of Veterinary Medicine and 2 High Tech Research Center, Institute of Biosciences, Azabu University, Fuchinobe, Sagamihara, Kanagawa 229, Japan; and Departments of 3 Pharmacology and Toxicology and 4 Physiology and Biophysics, School of Medicine, Wright State University, Dayton, Ohio 45435
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ABSTRACT |
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Thiol reagents activate K-Cl cotransport (K-Cl COT), the Cl-dependent and Na-independent ouabain-resistant K flux, in red blood cells (RBCs) of several species, upon depletion of cellular glutathione (GSH). K-Cl COT is physiologically active in high potassium (HK), high GSH (HG) dog RBCs. In this unique model, we studied whether the same inverse relationship exists between GSH levels and K-Cl COT activity found in other species. The effects of GSH depletion by three different chemical reactions [nitrite (NO2)-mediated oxidation, diazene dicarboxylic acid bis-N,N-dimethylamide (diamide)-induced dithiol formation, and glutathione S-transferase (GST)-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB)] were tested on K-Cl COT and regulatory volume decrease (RVD). After 85% GSH depletion, all three interventions stimulated K-Cl COT half-maximally with the following order of potency: diamide > NO2 > CDNB. Repletion of GSH reversed K-Cl COT stimulation by 50%. Cl-dependent RVD accompanied K-Cl COT activation by NO2 and diamide. K-Cl COT activation at concentration ratios of oxidant/GSH greater than unity was irreversible, suggesting either nitrosothiolation, heterodithiol formation, or GST-mediated dinitrophenylation of protein thiols. The data support the hypothesis that an intact redox system, rather than the absolute GSH levels, protects K-Cl COT activity and cell volume regulation from thiol modification.
potassium-chloride cotransport; glutathione; high-potassium dog erythrocytes; volume regulation
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INTRODUCTION |
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IN RED BLOOD CELLS (RBCs) of several vertebrate families, K-Cl cotransport (K-Cl COT, defined as the ouabain-resistant, Na-independent, and Cl-dependent K flux) is responsible for electroneutral outward flux of K and Cl with obligatory water loss, and hence, regulatory volume decrease (RVD) (reviewed in Refs. 29 and 32). Recently, the K-Cl cotransporter was cloned and found in four isoforms, KCC1 to KCC4, in many organ systems of several animal species (16, 19, 20, 37, 39-41, 43). The ubiquitous KCC1 isoform, maintaining cellular homeostasis (17), was cloned from dog erythroid progenitor cells (13), and, together with KCC3, was detected immunochemically in sheep RBCs (35).
A variety of interventions such as cell swelling (8, 32), reduction of cellular Mg (7, 28), kinase inhibitors (5, 9), oxygen (15), hydroxylamine (26, 44), vasodilators (3), and treatment with thiol reagents (1, 23-25, 27-30, 33) stimulate K-Cl COT in RBCs, and the cellular redox system has been implicated in its regulation (37). When glutathione (GSH) is reduced in low-K (LK) sheep RBCs by alkylation with N-ethylmaleimide (NEM) (33), alkanethiol formation with methylmethane thiolsulfonate (27), and oxidation with diazene dicarboxylic acid bis-N,N-dimethylamide (diamide), K-Cl COT is stimulated but fully reversibly only in the case of diamide (1, 25). Through direct or indirect reactions, these reagents are thought to modify the thiol group(s) of at least one kinase that inhibits K-Cl COT upstream within the regulatory cascade (21, 30). Nonoxidative depletion of cellular GSH level by treatment with 1-chloro-2,4-dinitrobenzene (CDNB) revealed effects both upstream and downstream of the putative regulation cascade or the transporter itself (30). Exposure of human RBCs to H2O2 also stimulates K-Cl COT (6), an effect ascribed to modification of a protein phosphatase (PP1) or of a regulatory protein.
In this report, high-K and high-GSH (HK/HG) dog RBCs of variants found only in Japan and Korea (10, 11, 14) were studied to test whether the same inverse relationship exists between GSH levels and K-Cl COT activity found in other species. Unlike the ubiquitous normal LK dog RBCs (38), their HK/HG counterparts possess Na/K pumps (36). Because of the 10-fold higher outwardly oriented driving forces for K-Cl COT, HK dog RBCs also have higher K-Cl COT activity than LK dog RBCs, which offers the additional opportunity to study RVD under physiological conditions (10, 14). The effects of GSH depletion by three different chemical reactions [nitrite (NO2)-mediated oxidation, diamide-induced dithiol formation, and glutathione S-transferase (GST)-catalyzed conjugation of GSH with CDNB] were tested on K-Cl COT and on RVD. After 85% depletion of GSH, all three interventions stimulated K-Cl COT half-maximally with the following order of potency: diamide > NO2 > CDNB. Although the effects of diamide and CDNB on GSH and K-Cl COT were similar to those found in LK sheep RBCs (1, 25, 30), NO2 concentrations 10-fold lower than in sheep RBCs (2) reversibly decreased GSH and activated K-Cl COT in HK/HG dog RBCs. Furthermore, a Cl-dependent RVD accompanied K-Cl COT activation by diamide and NO2 but not by CDNB treatment, suggesting modification of the RVD mechanism. At concentration ratios of oxidant/GSH greater than unity, K-Cl COT was irreversibly activated due to either nitrosothiolation, heterodithiol formation, or GST-mediated dinitrophenylation of protein thiols. Our data support the hypothesis that an intact redox system, most likely, rather than the absolute GSH levels, protects physiological K-Cl COT activity and cell volume regulation against thiol modification.
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MATERIALS AND METHODS |
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Erythrocytes. Blood was obtained by venipuncture from five variant dogs with HK/HG RBCs and also from normal dogs with LK RBCs as control. The blood was centrifuged at 10,000 rpm in a Sakuma RB7 centrifuge within 30 min to separate RBCs from plasma. The GSH concentrations were 7.6-8.8 mmol/l of original cells (LOC) in HK/HG and 1.5-2.7 mmol/LOC in LK dog RBCs, respectively. HK/HG dog RBCs had 115 ± 8 mmol of K/LOC and 8.5 ± 3.2 mmol of Na/LOC (n = 4), and LK dog RBCs had 9.1 ± 3.1 mmol of K/LOC and 106 ± 12 mmol of Na/LOC (n = 5), respectively.
Chemicals. Diamide and CDNB were purchased from Sigma (St. Louis, MO). Stock solutions of NaNO2 were prepared in distilled water and used for 1 wk. Diamide in DMSO and CDNB in ethanol were dissolved before the experiment. All reagents were of analytic grade.
Determination of cell volume, GSH, and methemoglobin. Cells were washed with washing medium and then incubated in Rb influx media containing the respective chemicals. Aliquots of cell suspensions were removed at several time points for measurement of cell volume with a Coulter counter (Sysmex 100). During or after incubation with the respective chemicals, cells were removed and assayed for GSH with 5,5'-dithiobis (2-nitrobenzoic acid) (4). Methemoglobin was determined spectrophotometrically at 632 nm (18).
Measurement of ouabain-resistant Rb influx. To measure K-Cl COT, ouabain-resistant, Cl-dependent Rb influx was measured as reported elsewhere (10, 23). Cells were washed three times in an isosmotic solution containing (in mM) 150 NaCl (or NaNO3) and 10 Tris-MOPS, 295 ± 5 mOsm, pH 7.4, at 4°C. For Rb uptake, cells were suspended in media containing (in mM) 30 RbCl (or RbNO3), 115 NaCl (or NaNO3), 10 Tris-MOPS, 5 glucose, and 0.1 ouabain, 295 ± 5 mOsm, pH 7.4, at 37°C. At time 0, cells were added to the flux media to yield a final hematocrit of 4%. At 20-min intervals, 0.7-ml aliquots were transferred into isosmotic 110 mM MgCl2 washing solution, buffered to pH 7.5 with 10 mM Tris · HCl, and the cells were washed three times with this solution. After the initial velocity of Rb uptake for each chemical was determined, Rb uptake was measured at two time points (20 and 60 min), and the influx was calculated from the slope. For determination of Rb content, cells were hemolyzed in a solution containing Triton X-100, NH4OH, and CsCl. Rb was measured by flame photometry at 780 nm, and the Rb concentrations, [Rb]c in mmol/LOC, were calculated on the basis of hemoglobin measured by spectrophotometry at 527 nm. Cl-dependent influx or K-Cl COT (mmol of Rb/LOC × h) was calculated by subtraction of the influx in NaNO3 from that in NaCl media (12, 23).
Pretreatment. To study the effect of the various chemicals on ouabain-resistant Rb influx, cellular GSH, and volume, cells were preincubated for 50 min with NaNO2, diamide, and CDNB in flux medium without Rb, washed twice with washing medium, and then incubated without the reagents but with 5 mM glucose. The GSH concentration was then measured at several time points. At time 0 and 50 min, fresh flux media containing 60 mM RbCl or NO3 were added to yield 30 mM final Rb concentrations.
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RESULTS |
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To deplete cellular GSH and/or oxidize cellular components,
NaNO2, diamide, and CDNB were employed. Figure
1 shows, in one of three experiments, the
cellular GSH levels in HK/HG dog RBCs as a function of the
incubation time in NO2 (Fig. 1A), diamide (Fig.
1B), and CDNB (Fig. 1C). Without any treatment
(open circles), GSH concentrations were 7.6-8.8 mmol/LOC and
remained constant up to 80 min. However, at 0.2 mM (filled circles) and
0.5 mM (open squares) NO2, GSH fell after 20 min by ~30
and 60%, respectively, remaining constant thereafter. At 1 mM (filled
squares) and 2 mM (open triangles) NO2, GSH decreased to
0.8 and 0.3 mmol/LOC, respectively, i.e., >90% (Fig. 1A).
Figure 1B shows that diamide at all concentrations reduced
GSH by >90% (to ~0.2-0.7 mmol/LOC at 20 min). With 0.2 mM
(filled circles) and 0.5 mM (open squares) diamide, GSH was restored by
60 and 17% (4.5 and 1.3 mM) at 80 min. Figure 1C shows that
CDNB at 0.2 (filled circles), 0.5 (open squares), 1 (filled squares),
and 2 mM (open triangles) reduced GSH by 38, 62, 78, and 85% after 20 min, respectively. Thus the minimum effective concentration to decrease
GSH levels differed among the three thiol reagents, and the order of
their potency was diamide > NO2 > CDNB.
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Figure 2 shows the dose dependency of the
effect of NO2, diamide, and CDNB on ouabain-resistant Rb
influx in HK/HG dog RBCs. The Rb influxes were stimulated sigmoidally
with increasing NO2 in Cl (circles), saturated at 3 mM
NO2, and reached values eightfold above controls (Fig.
2A). However, Rb influxes in NO3 (squares) increased linearly with the oxidant concentration. Thus
ouabain-resistant Cl-dependent Rb influx and hence K-Cl COT rose
ninefold at 3 mM NO2, with a half-maximal activation
concentration (AC50) of ~0.8 mM NO2.
Figure 2B shows a similar response of Rb influx to
increasing diamide concentrations. The Cl-dependent Rb influx was
sigmoidally increased eightfold at 2 mM diamide with an
AC50 ~0.5 mM. Although the stimulation by CDNB was
smaller compared with that attained with NO2 and diamide,
ouabain-resistant, Cl-dependent Rb influx increased sigmoidally
fourfold at 2 mM CDNB with an AC50 of ~0.7 mM.
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Figure 3 shows the response of HK/HG dog
RBC volumes to NO2 (A), diamide (B),
and CDNB (C). Incubation with 2 mM NO2 or
diamide for 2 h in Cl and NO3 (Fig. 3, A
and B, filled circles) decreased HK/HG dog RBC volumes by 4 and 3%, respectively, without a change in controls (open circles). In
contrast, incubation in NO3 increased the cell volumes both
in the presence (filled squares) and absence (open squares) of
NO2 (A) or diamide (B), although the
effect was larger in the presence of the chemicals. This cell swelling is most likely due to a greater entry of Na in NO3, which
may affect Na influx directly or through membrane potential changes. It
is known that NO3 is less rate limiting than Cl for K
fluxes in human RBCs (22). Therefore, NO2 and
diamide stimulated K-Cl COT and Cl-dependent RVD. In contrast, 3 mM
CDNB (Fig. 3C) did not affect the cell volumes in Cl
(circles) but, rather, caused cell swelling in NO3
(squares), most likely due to Na entry.
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Figure 4 shows the effect of pretreatment
with and removal of NO2, diamide, and CDNB on cellular GSH
concentration in HK/HG dog RBCs. Cells were pretreated for 50 min with
the reagents, washed twice to remove the drugs, and then incubated
without the agents but in the presence of glucose. No change in GSH
occurred in untreated cells (diamonds) during the incubation period.
Although the GSH levels decreased to ~0.6 mM (>90%) after
pretreatment with 2 and 5 mM NO2, they quickly recovered up
to 70 and 60%, respectively, after removal of NO2
(circles; see time 0 at abscissa) and by 90% 30 min after
being washed. After pretreatment with 2 and 5 mM diamide (squares), GSH
decreased to <0.2 mM and gradually recovered (by 75 and 50%,
respectively) during 60 min of incubation. A tendency of slower and
lower recovery was observed in 5 mM compared with 2 mM diamide. After
being exposed to 3 mM CDNB, GSH (triangles) decreased to ~1 mM (by
86%) and failed to recover at 120 min, even with medium glucose. Thus
NO2 and diamide, but not CDNB, reversibly lowered cellular
GSH.
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Figure 5 shows the effects of
pretreatment with NO2, diamide, and CDNB on
ouabain-resistant, Cl-dependent Rb influx in HK/HG dog RBCs.
NO2 and diamide enhanced Rb influxes approximately sixfold. However, 50 min after washing, Rb influxes remained elevated 2.5-fold despite the recovery of cellular GSH by 90% (cf. Fig. 4). After pretreatment with CDNB, Rb influx was not significantly stimulated with
respect to the control and disappeared 50 min after washout.
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Figure 6 shows the effects of
NO2, diamide, and CDNB on methemoglobin formation in HK/HG
dog RBCs, which increased by up to 85% during 50 min of incubation
with 2 mM NO2 (Fig. 6A) and was reversed by only
24% 3 h after washing. In contrast, 2 mM diamide (Fig.
6B) augmented methemoglobin formation by 5.5% after 50 min, decreasing to ~2% within 2 h after washout, whereas 2 mM CDNB (Fig. 6C) increased methemoglobin to 16% after 50 min and
to 37% of total hemoglobin 3 h after washout.
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DISCUSSION |
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In a variety of RBCs, K-Cl COT activity is inversely related to cellular GSH levels when lowered by chemical interventions with thiol-alkylating or -oxidizing compounds (1, 25, 30). This paradigm does not seem to apply to HK/HG dog RBCs that, compared with LK dog RBCs, have much higher GSH levels and K-Cl COT activity, even under physiological conditions (10, 14). By virtue of a 10-fold higher outward gradient for the transported species' K and Cl, HK/HG dog RBCs are also capable of RVD (10, 14). However, the results of this study have shown a similar inverse association between GSH levels and K-Cl COT, and, in addition, Cl-dependent RVD, suggesting that the overall redox potential, rather than the absolute GSH levels, protect the physiological K-Cl COT activity, and, therefore, cell volume regulation, from thiol modification in all cellular models studied. To measure RVD in LK dog RBCs, one would have to alter intracellular K by an ionophore or study maturational RVD, as in sheep and pig reticulocytes (31, 34).
Reduction of cellular GSH by three different chemical interventions, treatment with NO2, diamide, and CDNB (Fig. 1, A-C), was accompanied by stimulation of K-Cl COT as measured by Cl-dependent Rb influxes in HK/HG dog RBCs (Fig. 2, A-C). The dose dependence of K-Cl COT stimulation by all three compounds was similar, although the maximum effects achieved with NO2 and diamide were 2-3 times greater than with CDNB (Fig. 2, A-C). The effects of NO2 and diamide on cellular GSH (Fig. 4) and K-Cl COT (Fig. 5) were largely reversible, and both reagents caused significant RVD in Cl media (Fig. 3). Furthermore, methemoglobin formation was gradually (NO2) and partially (diamide) reversed. In contrast, the action of CDNB on the three parameters was clearly irreversible, and RVD was not elicited (Fig. 3).
The decrease in GSH was maximal (>95%) after a 20-min exposure to 2 mM NO2 (Fig. 1A) and was reversed within 30 min (Fig. 4). Maximum activation of K-Cl COT occurred at 3 mM NO2 (Fig. 2A) and was 60% reversed at 50 min after chemical removal (Fig. 5). These data suggest that oxidation of GSH to glutathione disulfide (GSSG) by NO2 occurred with a stoichiometry of unity and was reversed by GSH reductase upon removal of NO2 and incubation with glucose. Because K-Cl COT was half-maximally activated at 0.8 mM NO2, it is possible that the 60% reconstitution of its normal activity after NO2 treatment is related to partial reduction of GSSG to GSH. However, the fact that higher NO2 concentrations were required for maximum but irreversible stimulation of K-Cl COT suggests that these NO2 concentrations modified the regulatory machinery of K-Cl COT, perhaps through nitrosothiol formation (2). Once activated, K-Cl COT elicited RVD (Fig. 3), indicating that NO2 did not interfere with the basic homeostatic function of the transporter. It is interesting to note that the concentrations of NO2 required to cause K-Cl COT stimulation were significantly lower in dog RBCs than in LK sheep RBCs (2). This might be due to the differences in mechanisms involved in detoxification of NO2 (i.e., hemoglobin and methemoglobin reductase) or to differences in the accessibility of the target thiols to form nitrosothiols. Only partial reversal (reduction) of methemoglobin occurred by washing RBCs after 50 min of incubation with NO2. Hemoglobin may have been oxidized or further modified, preventing reduction by NADH/NADPH-linked methemoglobin-reducing systems (45) that, in turn, also may have been altered. Thus recovery of the redox system may have been insufficient to reverse oxidation.
Depletion of some 7 mmol of GSH/LOC was complete after 20 min of incubation in 1 mM diamide (equivalent to 10 mM/LOC), well within the known stoichiometry of 2 mol GSH/mol diamide (25). Lower diamide concentrations (0.2 mM) lead to its elimination from the cells (see in Ref. 25) and thus partial restoration of GSH (Fig. 1B). Half-maximal K-Cl COT stimulation occurred at ~0.5 mM (~5 mmol/LOC) diamide, consistent with its effect on K-Cl COT in LK sheep and human RBCs (1, 25). The maximum activation of K-Cl COT occurred at 2 mM diamide (20 mmol/LOC; Fig. 2B). More than 60% of GSH (Fig. 4) and of the original K-Cl COT rate (Fig. 5) were restored after removal of diamide, suggesting that at least a major fraction of K-Cl COT depends on the GSH/GSSG redox system. Although methemoglobin formation by diamide was only 5.5% (Fig. 6B), a fraction of the K-Cl COT activity was apparently irreversibly oxidized once GSH was depleted (Fig. 5). These findings contrast with the observation made in sheep RBCs in which diamide activation of K-Cl COT was fully reversible (12, 25), suggesting species differences in the oxidative susceptibility of the K-Cl COT mechanism. Nevertheless, as with NO2, the diamide-activated K-Cl COT was accompanied by Cl-dependent RVD (Fig. 3B), indicating that this mechanism remained intact.
The depletion of GSH by CDNB (Fig. 1C) was not as complete as in the case of the previous reagents and was maximal after 20 min at 2 mM CDNB (equivalent to 20 mmol/LOC), a concentration saturating the GST activity, presumably present in these cells (30). At 2 mM CDNB, K-Cl COT was maximally stimulated (Fig. 2C), although at lower levels than those elicited by NO2 and diamide. Attempts to restore GSH as well as K-Cl COT to the original levels failed (Figs. 4 and 5), suggesting irreversible modification of both the redox system as well as the transport mechanism. It is possible that the GSH ether of CDNB, S-[2,4-dinitrophenyl]-glutathione, remained intracellularly and by inhibiting GSH reductase (discussed in Ref. 30), contributed to low GSH/GSSG ratios. Further supporting this conclusion is the fact that methemoglobin continued to rise even though external CDNB was removed by washout (Fig. 6C). Furthermore, in contrast to NO2 and diamide, CDNB-treated HK/HG dog RBCs did not show a significant RVD (Fig. 3C), commensurate with the small stimulatory effect of CDNB on K-Cl COT. Alternatively, CDNB exerted additional, irreversible effects with an apparent dissociation of RVD from the GSH and K flux parameters. In LK sheep RBCs, CDNB "freezes" volume-activated K-Cl COT by rendering the hypothetical regulator insensitive to osmotic changes (30). At high (2 mM) concentrations and at 37°C, NEM inhibits irreversibly K-Cl COT of LK sheep RBCs activated by swelling, Mg removal, staurosporine, or hydroxylamine, possibly through "deoccluded" inhibitory thiol groups (27). It remains to be seen whether the action of CDNB involves a similar mechanism in HK/HG dog RBCs.
The effects of NO2, diamide, and CDNB to elicit RVD may have been underestimated since in NO3, all three chemicals markedly increased cell volume. This is probably due to a Donnan-driven net gain of NaNO3 and obligatory water that, in all cases, exceeded the swelling trends in NO3 in controls (Fig. 3, A-C). Indeed, thiol reagents are long known to alter Na permeability (42) as well, and the rate of swelling may be greater in NO3 due to direct effects or changes in membrane potential.
In conclusion, this study suggests that GSH depletion by three chemically different reactions revealed that not the absolute GSH levels but the redox state of the cell determines K-Cl COT activity and RVD in HK/HG dog RBCs. Partially irreversible K-Cl COT inhibition occurring at reagent concentrations in excess of those depleting GSH may be explained by direct modification of the transport mechanism by nitrosothiolation, diamide-mediated irreversible heterodithiol formation between proteins, or GST-mediated dinitrophenylation of protein thiols. Of the three compounds tested, CDNB appeared to exert the most irreversible effect on K-Cl COT and appeared to uncouple RVD from the latter.
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ACKNOWLEDGEMENTS |
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This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (09660331), a Foundation of Japanese Private School Promotion (1998) grant to Azabu University Graduate School, Grant 0050451N from the American Heart Association, and a travel grant from Wright State University.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. K. Lauf, Dept. Physiology and Biophysics, Wright State Univ., School of Medicine, Dayton, OH 45435 (E-mail: Peter.Lauf{at}wright.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 30 March 2001; accepted in final form 23 August 2001.
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