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Kidney International, Vol. 51 (1997), pp. 1529—1534

Effects of blockers on hypoxic injury in rat proximal tubules

W. BRIAN REEVES

Division of Nephrology, University of Arkansas for Medical Sciences and J.L. McClellan Memorial Veterans Hospital, Little Rock, Arkansas, USA

Effects of chloride channel blockers on hypoxic injury in rat proximal that C1 channels are present in the basolateral membrane of tubules. These studies examined the pathways and consequences ofproximal tubule cells where they may participate in either net CL chloride uptake into proximal tubule cells during in vitro hypoxia. The transport or cell volume regulation [11-441. The effect of inhibi- chloride diphenylamine-2-carboxylate (DPC) markedly reduced the degree of hypoxia-induced membrane damage as measured tion of CL uptake by proximal tubules cells on the subsequent by the release of lactate dehydrogenase (LDH). DPC reduced the release development of ischemic injury has not been examined directly. of LDH from hypoxic tubules from 38 2.7% to 16 1.7% after 30The demonstration of CL channels in proximal tubules, along minutes of hypoxia (P < 0.001, N =16)and also reduced 36C1 uptake by with the availability of several classes of agents known to inhibit hypoxic tubules. The reduction in LDH release was not associated with better preservation of cell ATP content or with protection againstCL channels [151 prompted the present studies, which tested the hypoxia-induced DNA damage. Other C1 channel blockers, such as niflumic effect of CL channel blockers on hypoxic proximal tubule cell acid, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 2-[(2-cyclopen- injury. The results indicate that several CL channel blockers tyl-6,7-dichloro-2,3-dihyrdo-2-methyl-1-oxo- IH-inden-5-yl)oxy] acetic acid (IAA-94) provided even greater protection than DPC and were asprovided marked cytoprotection against the hypoxa-induced re- effective as 2 mrt glycine. The CI channel blockers appear to act late in lease of lactate dehydrogenase (LDH), a marker of lethal cell the course of hypoxic injury since DNA damage, an early manifestation of injury. These results are consistent with the view that during injury, is not prevented by the blockers and since addition of the C1 hypoxia, and perhaps other forms of injury associated with ATP channel blocker after the hypoxic injury has begun reduces furtherdepletion, CL uptake occurs via membrane CL channels. More- membrane damage. These results support the conclusion that transport over, the transport of CL or other solutes through these channels through C1 channels contributes to hypoxic cell injury in proximal tubular cells. contributes to cell injury. Methods Preparation of tubule suspensions The mechanisms leading to proximal tubular cell injury during Rat proximal tubules were isolated by collagenase digestion and renal ischemia remain incompletely defined. It has been suspected Percoll gradient ceritrifugation [16, 17]. The isolated proximal for some time, however, that the uptake of solute, mainly sodiumtubules were resuspended at a concentration of —0.5 to I mg and chloride, with subsequent cell swelling contributes to ischemic protein/ml and equilibrated for 30 minutes at 37°C in a solution cell injury. In support of this view, first articulated by Leaf [1], are both in vivo [2] and in vitro studies [3, 4] that demonstrate ancontaining (in mM): 103 NaCl, 5 KCI, 2 NaI-T2P04, 1 MgSO4, I increase in cell chloride content and cell volume following isch-CaCl2, 5 glucose, 5 malate, 5 glutamate, 4 lactate, 1 alanine, I valerate, 0.2 dextran-40, 20 HEPES, and 20 NaHCO3 under a emia or 'chemical anoxia.' In addition, impermeant solutes such as mannitol or sucrose are effective in ameliorating ischemic acute95% 02-5%C02 atmosphere prior to experiments. renal failure in vivo [5—7] and reduce cell injury in in vitro models Protocol for in vitro hypoxia of proximal tubule ATP depletion [8—10]. Following the 30-minute equilibration period, the tubules were In normal renal epithelial cells, the negative membrane poten- pelleted by centrifugation and resuspended in the same solution at tial keeps the intracellular Cl activity in the range of 20 to 25 mst a protein concentration of 0.5 to 1.0 mg/mI. Aliquots of tubules (6 [Ii]. In energy-deprived cells, depolarization of the membraneto 10 ml) were dispensed into small Erlenmeyer flasks and potential due to cellular potassium leak and inhibition of Na,K- incubated in a rotary shaking water bath at 37°C. Throughout the ATPase activity allows CL to enter the cell. While an increase in cell chloride content during renal ischemia has been demon-incubation, the flasks were gassed continuously with either 95% strated, the pathways responsible for the entry of chloride have02:5% CO2 (normoxia) or 95% N2:5% CO2 (hypoxia). At the indicated times, usually 30 minutes, aliquots of tubules were not been determined, Several recent studies support the notion removed for measurement of lactate dehydrogenase (LDH) re- lease or double-stranded DNA (DS-DNA) content. Unless indi- cated otherwise, test agents were added at the beginning of the Received for publication December 24, 1994 incubation. Diphenylamine-2-carboxylate (DPC) and 5-nitro-2-(3- and in revised form November 19, 1996 phenylpropylamino)-benzoate (NPPB) were added from stock Accepted for publication December 5, 1996 solutions in DMSO, 2-[(2-cyclopentyl-6,7-dichloro-2,3-dihyrdo-2- © 1997 by the International Society of Nephrology methyl-1-oxo-IH-inden-5-yl)oxy] acetic acid (IAA-94) was added

1529 1530 Reeves: Cytoprotective effects of C1 channel blockers

from a stock solution in ethanol. and 4-acetamido- 80 4'-isothiocyanato-stilbene-2,2'-disulphonic acid (SITS) were added from aqueous stock solutions. The vehicles alone had no effect on hypoxia-induced LDH release. 60 LDH release (1) A I ml aliquot of tubule suspension was centrifuged through a ci) 40 2:1 mixture (vol/vol) of dibutyl phthalate and dioctyl phthalate to ci) separate the tubules from the supernatant fluid. A second aliquot aI of tubules was lysed with 0.2% Triton X-100. The LDH activities -J 20 in the supernatant and in the tubule lysate were determined spectrophotometrically from the oxidation of NADH [18].LDH release into the supernatant was expressed as a percentage of the 0 total LDH. None of the test agents, or vehicles, interfered with the 0 30 60 90 assay for LDH. Time,minutes DNA damage Fig. 1. Time course of the release of LDH by proximal tubules. Suspensions DNA damage was determined using the alkaline unwindingof rat proximal tubules were gassed continuously with either 95% °2or assay [19, 20] as reported previously from this laboratory [16]. In95% N2 (hypoxia) as described in Methods for 90 minutes. Samples were this assay, the rate of DNA unwinding under mild alkalineremoved at the indicated times for measurement of LDH release. P < 0.001 versus normoxia; P < 0.01 versus hypoxia; N =3.Symbols are: (•) conditions is increased by the presence of either single or double- + DPC. strand DNA breaks. The amount of residual double stranded°2;(U) N2; (A) N2 DNA remaining at the end of the alkaline treatment is deter- mined from the fluorescence of ethidium bromide (tube P). The fluorescence from non-DNA components in the sample wasthe tubule pellets were dissolved in 1 N NaOH. The 36CL content determined from a second tube (tube B) that was sonicated priorof the tubule lysate, determined by liquid scintillation counting, to the alkaline incubation in order to introduce a large number ofwas expressed relative to the protein content of the tubule pellet. strand breaks so that there was complete unwinding of the DNA.Within an experiment, the 36CL content of the hypoxic tubules The total fluorescence (tube T) was determined from DNA not(either with or without DPC) was normalized to the 36CL content exposed to alkaline conditions (pH < 11) so that unwinding wasof normoxic tubules from the same tubule preparation. All minimized. The amount of residual double-stranded DNA in tubeexperiments were performed in triplicate. P was expressed as a fraction of the total DNA in the sample using the equation: % residual DS-DNA =(PB)I(T —B)>< 100 [19]. Materials DPC was obtained from Fluka Chemical Co. NPPB was ob- ATP content tained from Biomol Research Laboratory, Inc. IAA-94 was a gift The effects of hypoxia on the ATP content of the tubules wasof Dr. Donald Landry (Columbia University, NY, USA). SITS determined from aliquots of tubule suspension removed at thewas obtained from Molecular Probes, Inc. end of the 30 minutes incubation. Tubules were pelleted by centrifugation at 1000 g X one minute. The supernatant was Results removed quickly and the tubule pellet was extracted into 200 jxl of The role of CL channels in hypoxic injury to proximal tubules 10% perchloric acid. The perchloric acid extract was neutralizedwas examined by testing the effects of the CL channel blocker, with KOH and then diluted 500- to 1000-fold with a 10 mivi TrisDPC, on the release of LDH during in vitro hypoxia. LDH release, (pH 7.5) buffer. The ATP content of the diluted sample wassignifying a loss of membrane integrity to macromolecules, is determined by mixing 20 xl of sample with 100 .d of a 10 mg/mIgenerally considered to represent severe, and likely irreversible, solution of luciferin-luciferase (Sigma Chemical Co.) while mea-cell damage. The results indicated that 1 mrvi DPC significantly suring the luminescence in a Turner Model TD-20e luminometer.reduced the release of LDH during hypoxia. Figure 1 shows the The ATP content was expressed relative to the protein content oftime course of the release of LDH from suspensions of proximal the tubules determined from the perchloric acid pellet. tubules maintained under either normoxic or hypoxic conditions. The release of LDH by tubules maintained under oxygen re- 36C/ uptake mained low, between 5 and 10%, throughout the 90 minutes Six milliliter aliquots of proximal tubule suspensions wereincubation. In contrast, there was a progressive rise in the release incubated under normoxic or hypoxic conditions for 20 minutes.of LDH from hypoxic tubules, beginning at 15 minutes and At 20 minutes, I Ci/ml of 36Cl -wasadded to each flask. At theexceeding 70% after 90 minutes of hypoxia. Notably, 1 mri DPC same time, 1 m DPC was added to one of the hypoxic flasks. Atprovided significant protection against the hypoxia-induced re- 25 minutes, that is, after five minutes of uptake, 1 ml aliquots oflease of LDH throughout the entire 90 minutes incubation. the suspensions were removed from each flask and immediately A time point of 30 minutes was selected for further evaluation spun through a cushion of phthalate as described above for LDHof the cytoprotective effects of DPC. Figure 2 presents summary release. The supernatants were discarded and the walls of thedata for all experiments in which tubules were exposed to 30 tubes were washed three times with water to remove any remain-minutes of normoxia or hypoxia in the presence or absence of 1 ing traces of 36C1 .Finally,the phthalate layer was aspirated andmM DPC. The LDU release from normoxic tubules was 60.7%. Reeves: Cytoprotective effects of C1 channel blockers 1531

50 100

40 0z 0(I) 0 U) :: 0 Normoxia Hypoxia Hypoxia + Hypoxia + 1 mM DPC 2 mM glycine 10 Fig. 4. Effect of DPC and 2 msi glycine on DNA damage during hypoxia. Residual double strand DNA content of tubules at the end of a 30 minute incubation was determined using the alkaline unwinding assay (Methods). 0 *P < 0.01 versus normoxia. N =3to 6 experiments in each group. Normoxia Hypoxia Hypoxia + 1 mM DPC Fig. 2. Effect of DPC on the release of LDI-l during hypoxia. The release of tubule suspensions, the results for hypoxic tubules are presented LDH was determined after a 30 minute incubation under either normoxic relative to the normoxie controls from the same tubule suspension or hypoxic conditions. *p < 0.001 versus hypoxia; N =16. (N =3).The C1 uptake by hypoxic tubules was increased (158.5 7%) when compared to normoxic tubules. One m DPC reduced the rate of 36CL uptake in the hypoxic tubules to 114 200 - 5% of control (P =0.035vs. hypoxia, N =3).In these experi- ments, the Cl channel blocker was present only during the five + minute uptake period to minimize indirect effects of the agent due to improved cell viability. 150 - The ATP content of tubules exposed to hypoxia (1.1 0.2 nmol/mg protein) was significantly lower than that of the nor- moxie tubules (5.3 0.8 nmol/mg protein, N =9,P < 0.0001). 8 The presence of DPC during hypoxia did not affect the ATP content of tubules (0.90.1 nmol/mg protein, N =4,P> 0.2 vs. . 100 1 hypoxia), indicating that the reduction in cell injury with DPC was ('5 a. not due to better preservation of ATP levels. Thus, 1 m'vi DPC reduced LDH release during hypoxia, and reduced C1 uptake 0 during hypoxia, but did not affect cell ATP content. 50 The effect of DPC on the extent of hypoxia-induced DNA damage in proximal tubule suspensions was also examined (Fig. 4). Tubules incubated in hypoxic solutions for 30 minutes sus- tained marked DNA damage (50 5% residual DS-DNA vs. 90 2.1% residual DS-DNA in normoxic tubules, P =0.0002).In 0 contrast to the marked protection provided by DPC against LDH Normoxia Hypoxia Hypoxia + release, DPC did not significantly reduce the extent of DNA 1mM DPC damage during hypoxia (616.8%, N =5,P> 0.2 vs. hypoxia). Fig.3. Effect of hypoxia and DPC on 36C1 uptake by proximal tubules. The Similarly, 2 miu glycine, another very effective cytoproteetant (Fig. results for hypoxic tubules, with and without DPC, are normalized to the 5) [21], did not protect against DNA damage during hypoxia uptake by normoxic tubules from the same tubule preparation. *P < 0.05 4.7% residual DS-DNA, N = Since DNA damage has versus hypoxia; p < 0.0001 versus normoxia; N =3. (51 3). been shown to be an early manifestation of hypoxia in proximal tubules [16, 22], these results are consistent with the view that DPC, and perhaps glycine, act on later steps in the injury process. Thirty minutes of hypoxia resulted in an increase in LDH release In addition to DPC, several other chloride channel blockers to 38 2.7%.DPC(1 mM) reduced the LDH release fromalso provided significant protection against hypoxia-induced LDH hypoxic tubules at 30 minutes to only 141.7% (P < 0.001, N = release (Fig. 5). The effects of 2 mrvi glycine are also provided for 16).Associated with the decrease in LDH release due to DPC wascomparison. In the absence of any test agents, 30 minutes of a decrease in the uptake of Cl. Figure 3 shows the five minuteshypoxia increased LDH release from 8 1% to 402.4% (P < uptake of C1 after 20 minutes of normoxia or hypoxia. Because0.0001). DPC (1 mM), as noted in Figures 1 and 2, and 2 m of day-to-day variation in the basal rate of Cl uptake betweenglycine reduced LDH release during hypoxia to 14 1.7 and 9 1532 Reeves:Cytoprotective effects of C1 channel blockers

50 60

50 • 40 40 a) U) 0) 30 Ca a) 30 a) a) + a) 20 I20 I -Ja + -Ja 10 10 + 0 I 0 02 t=0 t=10 t=20 None GLY DPC NFA NPPB IAA SITS DNDS I 100uMNFA Fig. 5. Cytoprotective effects of C1 channel blockers. LDH release was Hypoxia measured after 30 minutes of normoxia ()orhypoxia (U).Theconcen- trations were: glycine (GLY) 2 mtvi,DPC1 mM, and 100 /LM for the Fig. 7. Effect of delaying the addition of nifiumic acid on LDH release. remaining agents. Data are mean SE. The number of experiments for Tubules were incubated under either 02 or N2 (hypoxia) for 30 minutes. each agent is given at the top of the bar. NFA is niflumic acid; other Niflumic acid (NFA) was added at the beginning of the incubation, t =0, abbreviations are given in the text. *P < 0.001 versus hypoxia alone. or after 10 or 20 minutes of hypoxia. The solid bar represents tubules incubated in the absence of niflumic acid throughout the incubation. Data are mean SE, N =3.*p < 0.001 vs. none; *p < 0.005 vs. 02.

niflumic acid, which are required to inhibit certain ligand-gated 100 Ci channels [28, 291. C The effect of delaying the addition of the chloride channel 0 blocker on subsequent hypoxic injury was also examined. In these C-) a) experiments, 100 j.LM niflumic acid was added to the tubule 0 °- suspensions either at the initiation of the hypoxia, or 10 or 20 50 minutes after the onset of hypoxia. Tubules were sampled after 30 minutes of hypoxia for LDH release. The results (Fig. 7) show that the cytoprotective effect of the chloride channel blocker is com- pletely maintained with a 10 minute delay after the onset of hypoxia. Even after 20 minutes of hypoxia, the subsequent addi- 0 tion of niflumic acid still reduced LDH release measured at 30 0 100 200 300 400 500 minutes from 49 1.3% to 21 1.0% (P < 0.0001, N 3). Niflumic acid, pM Discussion Fig. 6. Concentration dependence of the protection against hypoxia-induced LDH release by nifiumic acid. The solid line is the best fit of the data to the Previous studies have demonstrated an increase in the CL Michaelis-Menton relation: % protection =V[niflumic acidl/(kd + content of proximal tubule cells during ischemia either in vivo [2] [nifiumic acidi). Data are mean SE for 3 to 11 experiments at each or in Vitro[3]and in chemically-induced ATP depletion in vitro [4]. concentration. It has also been proposed that the increase in cell chloride content, with its attendant increase in cell volume, might contrib- ute to ischemic cell injury [1]. In this regard, perfusion of kidneys with solutions containing impermeant solutes such as mannitol or 1.3%, respectively. Clearly, at concentrations of 100 /.LM, a number sucrose, reduces the degree of injury of kidneys subjected to warm of channel blockers such as niflumic acid (NFA) and NPPB wereand cold ischemia [30]. Likewise, substitution of chloride by also very effective, providing virtually complete protection againstimpermeant anions effectively prolongs the viability of kidneys in LDH release (10 1.1% and 11 2.5%, respectively). IAA-94,cold storage [311. The pathways for CL influx into proximal a blocker of outward rectifying CL channels [231 and of certaintubule cells during ischemia have not been identified. Likewise, CL channels in the renal cortex [24, 251, also was highly effectivethe effects of inhibiting Cl uptake into hypoxic proximal tubules in preventing hypoxia-induced LDH release. 1-lowever, not all Cl on the development of hypoxic injury have not been examined. channel blockers were cytoprotective. The stilbene compoundsAccordingly, given the recent electrophysiologic demonstration of SITS and DNDS, which block outward rectifying Cl channels atplasma membrane CL channels in proximal tubule cells, the mieromolar concentrations [23, 26, 27], had little effect on LDHeffects of Cl channel blockers on the development of hypoxic release. injury in proximal tubule suspensions were examined. The results Figure 6 shows the concentration dependence of the cytopro-indicate that certain blockers of CL channels have potent cyto- tective effects of niflumic acid. The K112 for the cytoprotectiveprotective effects against hypoxia-induced injury to proximal effect (48 fLM) of niflumie acid is similar to the concentrations oftubules in vitro. These agents were as effective as 2 mrvt glycine in Reeves: C'ytoprotective effects of Cl channel blockers 1533

the same system (94.5 4.5% protection, N =7)[21]. The Since these experiments were completed, two other studies cytoprotective effect persisted throughout 90 minutes of hypoxia,have examined the effects of CL channel blockers on renal and could be observed even when the blocker was added afterepithelial cell injury [36, 37]. In agreement with the present hypoxia was well established. results, Waters and Schnellmann [36] found that NPPB, IAA-94, This laboratory has demonstrated recently that the activation ofand niflumic acid reduced both LDH release and CL uptake in potassium channels occurs as an early response to hypoxia inrabbit proximal tubules treated with antimycin A. Venkatachalam proximal tubules and contributes to cellular damage [16]. Specif-et al [37] also found that certain CL channel blockers reduced ically, the activation of potassium channels appears to be aLDH release from MDCK cells treated with the mitochondrial proximate event to the initiation of DNA damage during hypoxia.uncoupler, CCCP. However, in that study, it was necessary to Blockers of potassium channels provided modest protectionreduce the extracellular free calcium concentration to 100 flM.tO against hypoxia-induced LDH release and almost complete pro-observe the effects of CL channel blockers on LDH release. tection against hypoxia-induced DNA damage [161. The protec- The identity of the CL channel responsible for the CL entry tive effects of Cl channel blockers seen in the present studyduring hypoxia is unknown. The pattern of cytoprotection by the differed from those seen with blockers in twovarious CL channel blockers, however, resembles the pattern of respects. First, the Cl -channelblockers, while providing markedinhibition of the CFTR CL channel by these same blockers. A protection against LDH release, had no effect on hypoxia-inducedCFTR-like channel has been identified [38] in the basolateral DNA damage (Fig. 4). Second, the time course of protectionmembrane of proximal tubule cells. This channel, however, is not differed. As shown in Figure 7, the addition of niflumic acid as late volume-sensitive, and requires ATP for function, so its activity as 20 minutes into the hypoxia still provided significant protectionwould likely be low during ischemia. A volume-sensitive anion against LDH release. In the case of potassium channel blockers,channel has been characterized in glial cells, hepatocytes and the blockers needed to be present within the first 10 minutes toerythrocytes [reviewed in 39]. This channel, which also appears to prevent DNA damage [16]. Since DNA damage can be demon-transport small organic osmolytes, is inhibited by NPPB and strated within the first five minutes of hypoxia [22], while LDHniflumic acid but is only moderately sensitive to stilbene com- release is a marker of advanced cellular injury, the present resultspounds. It is possible that this channel, or a similar channel, is are consistent with the view that CL channel blockers affect anpresent in the renal proximal tubule and accounts for the cyto- event late in the cascade of hypoxic injury, but one that precedesprotective effects of the CL channel blockers reported here. the loss of membrane integrity. Whether or not CL channelMiller and Schnellmann have suggested that a ligand-gated CL blockers, by blocking a late event in the injury process but notchannel similar to the glycine-gated CL channel in the CNS may preserving cellular ATP content or preventing early damage tomediate CL influx during ATP depletion and also account for the DNA (Fig. 4) can actually prevent hypoxic cell death rather thancytoprotective effects of glycine [4]. They demonstrated that just delay its onset remains to be determined. strychnine, an antagonist to the CNS glycine-gated CL channel, Most CL channel blockers inhibit CL channels with relativelywas cytoprotective against metabolic inhibitors in rabbit proximal low affinity and often have effects on other transport systems ortubule suspensions, albeit at much higher concentrations than are [15]. For example, niflumic acid, in addition to blockingrequired to inhibit the CNS glycine-gated Cl channel. Moreover, several known chloride channels including the CFTR CL chan-components of the CNS glycine have been identified in nel, glycine-gated CL channels, and calcium-activated CL chan-renal cortex by Western blot analysis [40] and RT-PCR (W.B. nels [29, 32, 33], is an inhibitor of cyclooxygenase [34]. It isReeves, unpublished results), Venkatachalam et al [37] also found noteworthy, then, that cytoprotection against hypoxic injury wasthat strychnine and ligands of GABA-gated channels were cyto- provided by several different CL channel blockers belonging toprotective in MDCK cells exposed to metabolic inhibitors. In the separate chemical classes. In addition, the concentrations oflatter study, CL influx was not measured but was felt to be niflumic acid required to prevent LDH release are similar to thoseunrelated to the cytoprotective effects of the glycine and GABA required to block CL channels [29, 33], but are 100-fold higherreceptor ligands [37]. Nonetheless, there are as yet no direct data than those required to inhibit cyclooxygenase [35]. Thus, whileto indicate that a glycine or GABA-gated CL channel is involved none of these agents is entirely specific for CL channels, the in hypoxic renal injury. Clearly, further electrophysiologic studies similar effects of several different CL channel blockers supportwill be required to identify the channel responsible for the C1 the conclusion that the cytoprotection observed in this study wasinflux during hypoxia. due to inhibition of Cl channels rather than an interaction with In summary, a number of different CL channel blockers were other pathways. Moreover, at least one of these CL channelfound to have marked cytoprotective efficacy in an in vitro system blockers, DPC, reduced the uptake of CL into hypoxic tubules. Inof hypoxia to rat proximal tubules. These observations are con- addition, substitution of CL by impermeant anions dramaticallysistent with the view that Cl uptake, mediated via CL channels, reduced oxidant-induced LDH release from cultured renal epi-is an important step in the development of irreversible hypoxic thelial cells (W.B. Reeves, manuscript in preparation). Takencell injury. This finding may have clinical implications as more together, these observations are consistent with the view that CL potent, less toxic, CL channel blockers are developed. entry, via the opening of CL channels, is increased during hypoxia and contributes to cell injury and the loss of membrane integrity Acknowledgments in that setting. Alternatively, it is possible that the cytoprotective effects of the CL channel blockers were not due to inhibition of This work was supported by the Extramural Grant Program of Baxter Healthcare, Inc. and by the Veterans Administration Merit Review CL uptake per se, but rather, to other effects such as membraneprogram. The author is an Established Investigator of the American Heart hyperpolarization or inhibition of the flux of other solutes throughAssociation. I thank Linda Traylor, Ying Si, and Dainette Powell for these channels. excellent technical assistance. 1534 Reeves: Cytoprotective effects of Cl channel blockers

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