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Effects of hypochlorous acid and chloramines on vascular resistance, cell integrity, and biliary glutathione disulfide in the perfused rat liver: modulation by glutathione
Effects of hypochlorous acid and chloramines on vascuiar resistance, cell integrity, and biliary glutatr,lone disulfide in the perfused rat liver: modulation by glutathione
The accumulation of oolymorphonuclear leucocytes (PMN) may pl?y an important role in liver injury by twins and ischemtaireperfusion. Upan activation there cells generate hypochlorcus acid
of 2.lyM
resulted in a marked increase in the perfusion pressure atd the xlease
of LDH
concentration of extracellular glutathione in the perfusate Lcrphysiological levels. NH&l (15 PM) and TauNHCl (65 PM) mcreased the perfusion pressure only slightly, but resulted in significant increases in the biliary excretioa nf glutathioce disultide, indicntiq tha: chloranincs are reduced intrzcellularly by glutathione. The increment in biliary glutathtrme disulfide depended on the amount of chloramme taken up by the liver. The extraction of NH&2 averaged 98% compared to 13% for TauiJHCI. The preswt data indicates that intra- and extracellular glutathione plays an important role not only in the detoxiiication of Oi” and H202 generated by activated PMN but also in the protection a@xt the cytotoxic effects of products nf myeloperoxidare rclra.rd by P‘rlN upon activation. associated wth a decrease
in bile flaw. These effects were abolished by increasing&e
Neutrophils contribute tc organ damage in d number of direase wocw?s (l-4). In the liver, infiltration by polymorpbonuclrai cc,!< lic hepatitis, nnd PM% may ‘ontribstc 10 hepadc dun..gc in acu:e and chronic hepatitis and wrhosis. Experimzntdl data moreover mdicates that the invasion of neutroghils rortributes to cellular damage produced by acetammophen, galactosaminelendotoxin, and ischemiairctlow (S-S). Activation of PMN results in the stzztion of supcioxide anion radicals (Oz.*) and of cytoplasmic granule components including myeloparoxidase int@ the extracel-
lular space. Dismutation of 0~~’ yields hydrogeo perox;!e (H,O,). The myeloper~xidaae-chtalyzed reaction of HaQ2 with chloride results in the formatian 01 the potent oxidant hypocnlorous acid (HOCI) which can react with endogenous ni:rogen compounds (9,lO) to generate longlived oxidants such as monochloramine (NH&l) and taurinechloramine (TauNHCI). Glutathione (GSH) which is present intracellulorly in high concentrations nxq play an imponant mle in the detoxification of these microbicids oxidants. In the present study the effects of HOCI, NH,CI and TauNHCl on the function of the per;~sed a I~verRI.LI t!k modification by GSH were invertidated.
85
Dawtey rzts weighing 225 + 25 g were
by measunng the hrmzt,t;on of the coojugate kct~e-;,, tchloro-2.4.mn,?robe~lene and GSH at W-4(,. r,.n after addition ?f GSH S-transferase (13). This assm me~su:e~ only tte redwed Corm of glutathione. Gtxathione di-
purchased from Interfauna, Tottlingen, F.R.G., and hwzred in P climatized room with a 12 h light/dark cyde. The animals !tad free access to chow and water op :P !br tone of the experiments. Under pentotarbital anesthea (50 mgikg i.p.) the portal vein was cannulated and the livers were perfused with oxygenated Krehs-Henseleit
sulfiJe (GSSG! m bile was measured by 1’5reaction with N \DPH catalyzed by glutathione redoaase (14). GSSG concentrations are expressed in GSH-equivalents. LDH in the perfcsate ‘was assayed on a COBAS analyzer using routine procedures. Chlorammes in the perfusate were assayed spectrophotometrically hy followmg the oxida-
buffcr at a flow rate of 3.8+0.6 mlimin per ,j ‘iver in a II XIrecirculating fashion (11).
tion of 5.thio-2.,litrobenzoic acid at 412 nm (IS). The uptake of chloramines by the perfused bver VW catcula!zd from the difference berwcen inflow and uurtlor coocentrafion~ and the rate of perfusion. T!te uptake of HOC1 could not be detcrmioed because even the inflow coocentration of c3~hi was near the detection limi’ of the assay.
Male Sprague
The livers were equilibrated for 50 min whereupon chloramines or hypochlorous acid, :erpenively, were infused at various concentrations for 7 to 25 min. Portal pressure. LDH release, bile flow, and the biliary excretion of glutathione dtruifide (GSSG). ar. index of oxidant stress, were monitored throughout rhe experiment. Bile was coiiectrd iii:o prweighed polyet$lene tubes cootaining 0.1 ml 4% sulfosalicylic acid to provent oxidatioo
Results arc indicated a~ mean F S.E. Group means wxc compared using the non-parametric test at ‘+Vilcoxon.
of GSH ex viva In order lo elucidate
the role of extracellular
GSH in
the modulation of the effects of HOC1 on the perfused liver, HOCI was infused with and without GSH. GSY was infused at a tate of 0.64~molhnin for 46 &I. IXean minutes after staniog the infwiocionof GSH, HOCI was mfosed at rates of 0.10 and 0.25pmollmm. respecttvety, for 6 min each. Five minutes aft?7 stopping the infusion of GSH. !he infusion of HOC1 at a rate of 0.25~moUmbt ws repeated. In addirional experiments HZOZ was infused at a rate of 0.3 fimot/min between 50 and 65 min and at a rate of 0.6 pmot/mio between 65 and 80 min.
Chloramines were synthesired by adding NH,CI and taurine, respectively, m Z-fold molar excess to 0.1 M NsC)CI (12). The formation of chloranines was followed spectropbotometrically.
GSU (10 mh:, corresponding
IO tbe intracellular
con-
centration of the sulfhydryl) was incubated in 0.1 M pbosphate buffer (pH 7.4) containing 150 PM NADPH and glotathione redoctase, and the consumption of NADPH was followed spectrophotome:rintty (W-400 nm). After2 min, NH,Cl, tinri wncrntration 12 i#M, was added to the reaction mixture.
Tne cwcenrt?wn
of GSH in the perfusatr
~zs z;sayed
ReWRS HOC1 8’ i. concentration of i.lpM in the per&ate rcsulted it, a prompt ixrease in perfusion prersurr, reflectiog a marked increase in resisexce stxe the flow rate was kept constant (Fig. tj. This increase in pertusmn prerwr~ was assn*i?!~I with a rl”erease in bile flw aad a decrease in the biliary excretion of GSH and GSSG which did not recover III the 45 min following the short infusion of HOC1 (Figs. 1 nod 21. In contrast. the release of LDH markedly increased d&g the infusion of HOCI wherecpon it returned towards baseline (Fig. 1). In order to demonstrate a possible role of extracellular glutathione in the modulation of these effects of YOGI no the perfused liver, HOC! was infuaad xitb and without erng~nnnlc GSH. Due to the r;l&c!y high tT3w a;cs cmnloued in the perfain? 3: ia livers with a solution with r--ibmited oxygen carrying capacity, the coocentratioo of GSH in :he hepatic effluent is below the physiological conccntratian. With the infusion of exogenous gluta:bi;ne the ccrxemmtion in the eftluent corresponded to physiological concentrations (16). A rapre;entazive experiment is shown in Fig. 3. During the info&an of GSH rhe concentration of GSH in the keoadc emucot rose from an average d 3@, so 17 ,d,, which w?spr,ds iu the concrntration of GSH measured in: the hepatic win in viva (16). torusion a: HOC1 (calcu!ated final conceotradons 2.’ and o.dpM a! the lower and higher infusmn rate, reapectiveiy) resulted in a approximately stoichiomwic
_
decrease of GS!t in the hepatic eXlwrnt witiut s:fecting bile flow, portal pressure, 01 LDH release (Fig. 3). When the infxsicn of GSH was stopped, i’le concentration of GSH in the effluent decreased to baseline velues. The infusion of HOC1 at i final concentration of 6.8ptvl at this tine again readted in a decreased eftlux of GSA from the liver but this time it was asscciated with a marked increase in porta! pressure, a waked, reversible increase in the efflux of LDH. and a virtual abolishment of bile tlow. The chloramines -ere much less potent than HOC, and did not increase :bc :etezc of LDH into the perfusafe (cumclatiw release of 186 f 44, II7 f 28, and 144 + 45 mu/g per SO min following NH,CI, ‘TauNHCl and in control perfusions, respectivAy). NH&I periused at a con-
GLuTKHIGNE
AND CHLORAMINES
ceorration of 15yM
increased the perfusion prtwure
0.06pUmin pcrgfollowingNH~CI,O.58
+ 0.17pllmin
Hpl
from
5.8 f 0.2 cmH,O (n = 6) prior fo starting the infusion of NHzCl to 7.5 + 0.6 cmHzO 5 min following the onset of tb,: infusion of the chloramine @<0.05). Although perfured at much higher concentrations (65 ,uM), TaoN’iCI had no effect on perfusion pressure in five experlmrnts (6.0 f 0.2 cmH,O vs. 6.1 ??0.2 cmH,O peek pressure). A: the end of the experiments bile flow averaged 0.63 +
per
gfollowrng TauNHCI,
and0.70 + 0.09fillmin per g in control experiments. NH&I resulted in a marked increase in the biliary excretion of GSSG and the ratio of GSSGESH in bile (Figs. 4 axd Sj. TauNk:CI had a similar, albeit quantitatively Lessereffect on biliary GSSG (Fig. 2). Differences in hrpatic uptake could explain the quantitatively differem effects of the two chloramines which ex-, bibit different physicwchemical properties. Therefore, the uptake o:chloramines by the perfused liver was determined. The uptake of both compounds remained constant dming the 25 min of infusion (data nor shown). In the case
Fig. 5. Ratio of GSSG:GSH in bile duringtheperfusionoi NHQ a”*NH,C*.
(slj.
of NH&i 93.4 f I .6% of the infused amount was taken up With?auNHCI. however, the extraction amounted to onp 12.8 f 1.5% (mean i S.IJ.. n = S! When the increment in the biliary excretian nf GSSG was expressed as a fxxtix~ of r!x actual amount of chloramjne taken up hy the liver the effects of the two chloramines on biliary CSSG were similar (Fig. 6). Approximately
0.7% of the
calwlated amount of chtoramines taken up by the liver appeared in bile in form of GSSG. In order to csmpare the known affects of H20z on bilir< GSSC wirh the effects of cixiau!inca, i-izOL 7:~s hfwed at a rate of 0.3 and 0.6 ~motP.nin, respectively. The biliary 0.W.l
excretion f
of GSSG
0.015 nmolknm
increased
by an average of
pei g (n = 4) an3 Xi4
“moUrnin per g (n = 5) at the bwer
+ 0 ilt:
aEd the higher perfu-
sion mtc. rcspcctiwly.
Assuming lhot H,O,
tnken up by the liver, :hc inmmtent
mdy
for
given amwnt
is quantita-
in bilinry
GSSG
taken up reflcctcd by the slope ol the corresponding correlation in Kg. 6 amounted to spprorimately half of what was ob.wved with the chloramincs. In vitro. NH&!] at a concentration of l:!.SrM reacted promptly and qrrantitativcly with GSH (Ifl mM, corresponding to the intrnccllulsr concentration of the sutthy dryl) to form FSSG (Fig. 7). 2
of H,O,
HOC, at low conccntmtions hod dramatic effect- on the vascular ,esis,ancc and on bePatocyte integrity ii, the perfused rat liver (Fig. 1). Activated PMN relax HOCt not only into phagocytic vacuoles but also into the exhacellolx space (10.15.17). Approximately one third of the H,O, generated by PMN is halogenated to HOC], Dot the concentration of HOC1 in the liver upon activation of PMN that have accumulated in the organ is oat known. Local concentrations of HOC] have heen estimated to reach the 1COpM range in the immediate vicinity of activated PMN (15.18), but blood flow will of course rapidly dilute the oxidant. Although it is obviously difficult to mimick the localized release of HOC1 by activated PMN in the liver, regional increases in vascular resistance and ceil damage due to HOC! stmikn to what WC observed in our model ryrtem may occur a. sites of neutrophil
accu-
mulat*on in wvo. The effeeta of HOC1 most liks!y result from the oxida-
tion of critical sulfiydryl grwps. At concentrations of lo-20 PM. HOCI causes the oxidation of plasma membrane sultltydryls and impairs various plasma membrane functions in cultured tumor cells without loss of cell viability (19). Our ooserwion that extracellular GSH at physiological concentrations praented the toxic effects of HOC1 is mnsistent with such a mode of action. Since the liver is the najor source of circulating GSH (l6), the highest ~xtrac:~Utar concentrations of GSH i? the body probably exist ire bepatic sinusoids. There. KOC, relsrsed by PMN in vh 3 can radily rext .rith GSEl prior to kteracting with cri:b:a, ao!%ydry,s on plasma membraxr. The extracellular xxtion
of oxidants with GSH released by
the toxic effects of neutrophils.
Besides sulfhydryls, HOC, readily texts with amines to form
chloraminer.
chtotamines
Although
have potentially
less reactive deleterious
than
axidative
HOC,. ptop-
(23). Compared to HOCI. NH&I and TaauNHCI affected perfusion pressure. LDH leakage and bile flow much less. Both, however, markedly increased the biliary excretion of GSSG and the GSSG:GSH ratio i’? bile. Thus, chloramines taken up by the liver react with intutcellular GSH a~ they do in vitro (Fig. 7) to form GSSG which is in part excreted in bile (24,25). The different effects of the two chtoraminea on the bilet&s
intg twretion of GSSG is best cnp!ained hy diffcrenc~a in the extraction of the two compounds. NH&, is much more lipophilic than TauNHCt and therefore essentially quantitatively extracted by the liver at the soncentrations used in out experiments. In contrast, only a small fraction of the Perfused TauNHCl was taken up by the liver. The uptake of HOC1 could not he detettnined because the concentration used was new the limit of detection of our assay. Once taken up by the liver the effects of chloraminec appesr to be identical. The same anaunt of GSSG is fonned for a given amount ofchloramine taken up. Only a &action of the generated GSSG, however, will be excreted into bile rather than reduced back to GSH by glutathione reductase and NADPH (24-26). Considering the slope of the regression for chloramines shown in Fig. bit is not surprising that HOC, which was infused at a rate avercging 13 nmollmin per g liver ~antwt he expected to result in a measurable increase in biliary GSSG even if it is taken up quantitatively. The fraction of H202 metabolized to GSSG (a8 reflected by the slope of the tzgres,iun shown in Fig. 6) was approximately half of what was seen with the chloramines. This agrees with as&r findings that a substantial fraction of the intracellular HzO, is metabolized via catalase rather than via GSH peroxidase (26) and, therefore. does not result in the formation of GSSG. During the perfusion of organic hydroperoxides which are
quaotitativcly metabolized via the glutathione peroxidase system approximately twice as much GSSG appears in hi,e per moi peroxide taken up by the liver than with H20z (26). The 2-fold difference in slope of the recrcssion lines shown in Fig. 6 tnerefore suggests that the chloramines are quatdtatively reduced by GSH intrace,!tt!arly. In wnciusion, our findings demonstrate that cblor-
hepatocytes may, thus, represent an important mechanism of protedicn of the liver and other organs from oxidative damage. Therefore, subjean with decreased circolating concentrations of GSH such as alcoholics (20), pattents wdh the acquired knmune deficiency syndrrrw !?I) and patients whew the efflux of GSH from the liver is de-
amines aw rftkientiy detoxified in the liver by intracellu-
creased such Uscirrhotics (22) may be n:ore susceptible to
zayeloperaxidasereleased by activated PMN.
lar GSH. lttteractions of the much mote toxic HOC1 with hepatic cells are orevented bv extracellulx~ GSA. Thus. gb:tathione plays an important role not only in the detoxification of Oz.’ and H201 generated hy PMN hut also in the :r@tcction against the cyiotoxic effects of products of
Deut%he
*c*nowkdgnlents
lance
Supported lional
by grar*
Foundation
No. 3.812-0.87 for
Scientific
from
rbe Swiss Na-
Research
and
by
sinuroidal cndothcos, dls of lY0.s.tiwr ..h its modulation in mule phase. J Hepatoll988: 7: 239-49. i Grirham MB, Hsmander LA. Crqer DN. Xanthme sxwlarc and “euw.phi, inmtmion in inrcidna, ir,lemia. Al” I Physio, 1986;251: G567-74. 8 GlUespie MN, Kojima 5, Kunitomo M, Jay M. Coronary and mvocardial eflec,r of activa,lxl nrutroohils in oerfued ,.3bb”
Fnrrchungsgemeinachaft.
of Ms.
Xk”.XVlZdg.Sd
E. Junker
and Mr.
The
c.
acsisis gra*efuOy
technica,
Schraoz
The accumulation of oolymorphonuclear leucocytes (PMN) may pl?y an important role in liver injury by twins and ischemtaireperfusion. Upan activation there cells generate hypochlorcus acid
of 2.lyM
resulted in a marked increase in the perfusion pressure atd the xlease
of LDH
concentration of extracellular glutathione in the perfusate Lcrphysiological levels. NH&l (15 PM) and TauNHCl (65 PM) mcreased the perfusion pressure only slightly, but resulted in significant increases in the biliary excretioa nf glutathioce disultide, indicntiq tha: chloranincs are reduced intrzcellularly by glutathione. The increment in biliary glutathtrme disulfide depended on the amount of chloramme taken up by the liver. The extraction of NH&2 averaged 98% compared to 13% for TauiJHCI. The preswt data indicates that intra- and extracellular glutathione plays an important role not only in the detoxiiication of Oi” and H202 generated by activated PMN but also in the protection a@xt the cytotoxic effects of products nf myeloperoxidare rclra.rd by P‘rlN upon activation. associated wth a decrease
in bile flaw. These effects were abolished by increasing&e
Neutrophils contribute tc organ damage in d number of direase wocw?s (l-4). In the liver, infiltration by polymorpbonuclrai cc,!< lic hepatitis, nnd PM% may ‘ontribstc 10 hepadc dun..gc in acu:e and chronic hepatitis and wrhosis. Experimzntdl data moreover mdicates that the invasion of neutroghils rortributes to cellular damage produced by acetammophen, galactosaminelendotoxin, and ischemiairctlow (S-S). Activation of PMN results in the stzztion of supcioxide anion radicals (Oz.*) and of cytoplasmic granule components including myeloparoxidase int@ the extracel-
lular space. Dismutation of 0~~’ yields hydrogeo perox;!e (H,O,). The myeloper~xidaae-chtalyzed reaction of HaQ2 with chloride results in the formatian 01 the potent oxidant hypocnlorous acid (HOCI) which can react with endogenous ni:rogen compounds (9,lO) to generate longlived oxidants such as monochloramine (NH&l) and taurinechloramine (TauNHCI). Glutathione (GSH) which is present intracellulorly in high concentrations nxq play an imponant mle in the detoxification of these microbicids oxidants. In the present study the effects of HOCI, NH,CI and TauNHCl on the function of the per;~sed a I~verRI.LI t!k modification by GSH were invertidated.
85
Dawtey rzts weighing 225 + 25 g were
by measunng the hrmzt,t;on of the coojugate kct~e-;,, tchloro-2.4.mn,?robe~lene and GSH at W-4(,. r,.n after addition ?f GSH S-transferase (13). This assm me~su:e~ only tte redwed Corm of glutathione. Gtxathione di-
purchased from Interfauna, Tottlingen, F.R.G., and hwzred in P climatized room with a 12 h light/dark cyde. The animals !tad free access to chow and water op :P !br tone of the experiments. Under pentotarbital anesthea (50 mgikg i.p.) the portal vein was cannulated and the livers were perfused with oxygenated Krehs-Henseleit
sulfiJe (GSSG! m bile was measured by 1’5reaction with N \DPH catalyzed by glutathione redoaase (14). GSSG concentrations are expressed in GSH-equivalents. LDH in the perfcsate ‘was assayed on a COBAS analyzer using routine procedures. Chlorammes in the perfusate were assayed spectrophotometrically hy followmg the oxida-
buffcr at a flow rate of 3.8+0.6 mlimin per ,j ‘iver in a II XIrecirculating fashion (11).
tion of 5.thio-2.,litrobenzoic acid at 412 nm (IS). The uptake of chloramines by the perfused bver VW catcula!zd from the difference berwcen inflow and uurtlor coocentrafion~ and the rate of perfusion. T!te uptake of HOC1 could not be detcrmioed because even the inflow coocentration of c3~hi was near the detection limi’ of the assay.
Male Sprague
The livers were equilibrated for 50 min whereupon chloramines or hypochlorous acid, :erpenively, were infused at various concentrations for 7 to 25 min. Portal pressure. LDH release, bile flow, and the biliary excretion of glutathione dtruifide (GSSG). ar. index of oxidant stress, were monitored throughout rhe experiment. Bile was coiiectrd iii:o prweighed polyet$lene tubes cootaining 0.1 ml 4% sulfosalicylic acid to provent oxidatioo
Results arc indicated a~ mean F S.E. Group means wxc compared using the non-parametric test at ‘+Vilcoxon.
of GSH ex viva In order lo elucidate
the role of extracellular
GSH in
the modulation of the effects of HOC1 on the perfused liver, HOCI was infused with and without GSH. GSY was infused at a tate of 0.64~molhnin for 46 &I. IXean minutes after staniog the infwiocionof GSH, HOCI was mfosed at rates of 0.10 and 0.25pmollmm. respecttvety, for 6 min each. Five minutes aft?7 stopping the infusion of GSH. !he infusion of HOC1 at a rate of 0.25~moUmbt ws repeated. In addirional experiments HZOZ was infused at a rate of 0.3 fimot/min between 50 and 65 min and at a rate of 0.6 pmot/mio between 65 and 80 min.
Chloramines were synthesired by adding NH,CI and taurine, respectively, m Z-fold molar excess to 0.1 M NsC)CI (12). The formation of chloranines was followed spectropbotometrically.
GSU (10 mh:, corresponding
IO tbe intracellular
con-
centration of the sulfhydryl) was incubated in 0.1 M pbosphate buffer (pH 7.4) containing 150 PM NADPH and glotathione redoctase, and the consumption of NADPH was followed spectrophotome:rintty (W-400 nm). After2 min, NH,Cl, tinri wncrntration 12 i#M, was added to the reaction mixture.
Tne cwcenrt?wn
of GSH in the perfusatr
~zs z;sayed
ReWRS HOC1 8’ i. concentration of i.lpM in the per&ate rcsulted it, a prompt ixrease in perfusion prersurr, reflectiog a marked increase in resisexce stxe the flow rate was kept constant (Fig. tj. This increase in pertusmn prerwr~ was assn*i?!~I with a rl”erease in bile flw aad a decrease in the biliary excretion of GSH and GSSG which did not recover III the 45 min following the short infusion of HOC1 (Figs. 1 nod 21. In contrast. the release of LDH markedly increased d&g the infusion of HOCI wherecpon it returned towards baseline (Fig. 1). In order to demonstrate a possible role of extracellular glutathione in the modulation of these effects of YOGI no the perfused liver, HOC! was infuaad xitb and without erng~nnnlc GSH. Due to the r;l&c!y high tT3w a;cs cmnloued in the perfain? 3: ia livers with a solution with r--ibmited oxygen carrying capacity, the coocentratioo of GSH in :he hepatic effluent is below the physiological conccntratian. With the infusion of exogenous gluta:bi;ne the ccrxemmtion in the eftluent corresponded to physiological concentrations (16). A rapre;entazive experiment is shown in Fig. 3. During the info&an of GSH rhe concentration of GSH in the keoadc emucot rose from an average d 3@, so 17 ,d,, which w?spr,ds iu the concrntration of GSH measured in: the hepatic win in viva (16). torusion a: HOC1 (calcu!ated final conceotradons 2.’ and o.dpM a! the lower and higher infusmn rate, reapectiveiy) resulted in a approximately stoichiomwic
_
decrease of GS!t in the hepatic eXlwrnt witiut s:fecting bile flow, portal pressure, 01 LDH release (Fig. 3). When the infxsicn of GSH was stopped, i’le concentration of GSH in the effluent decreased to baseline velues. The infusion of HOC1 at i final concentration of 6.8ptvl at this tine again readted in a decreased eftlux of GSA from the liver but this time it was asscciated with a marked increase in porta! pressure, a waked, reversible increase in the efflux of LDH. and a virtual abolishment of bile tlow. The chloramines -ere much less potent than HOC, and did not increase :bc :etezc of LDH into the perfusafe (cumclatiw release of 186 f 44, II7 f 28, and 144 + 45 mu/g per SO min following NH,CI, ‘TauNHCl and in control perfusions, respectivAy). NH&I periused at a con-
GLuTKHIGNE
AND CHLORAMINES
ceorration of 15yM
increased the perfusion prtwure
0.06pUmin pcrgfollowingNH~CI,O.58
+ 0.17pllmin
Hpl
from
5.8 f 0.2 cmH,O (n = 6) prior fo starting the infusion of NHzCl to 7.5 + 0.6 cmHzO 5 min following the onset of tb,: infusion of the chloramine @<0.05). Although perfured at much higher concentrations (65 ,uM), TaoN’iCI had no effect on perfusion pressure in five experlmrnts (6.0 f 0.2 cmH,O vs. 6.1 ??0.2 cmH,O peek pressure). A: the end of the experiments bile flow averaged 0.63 +
per
gfollowrng TauNHCI,
and0.70 + 0.09fillmin per g in control experiments. NH&I resulted in a marked increase in the biliary excretion of GSSG and the ratio of GSSGESH in bile (Figs. 4 axd Sj. TauNk:CI had a similar, albeit quantitatively Lessereffect on biliary GSSG (Fig. 2). Differences in hrpatic uptake could explain the quantitatively differem effects of the two chloramines which ex-, bibit different physicwchemical properties. Therefore, the uptake o:chloramines by the perfused liver was determined. The uptake of both compounds remained constant dming the 25 min of infusion (data nor shown). In the case
Fig. 5. Ratio of GSSG:GSH in bile duringtheperfusionoi NHQ a”*NH,C*.
(slj.
of NH&i 93.4 f I .6% of the infused amount was taken up With?auNHCI. however, the extraction amounted to onp 12.8 f 1.5% (mean i S.IJ.. n = S! When the increment in the biliary excretian nf GSSG was expressed as a fxxtix~ of r!x actual amount of chloramjne taken up hy the liver the effects of the two chloramines on biliary CSSG were similar (Fig. 6). Approximately
0.7% of the
calwlated amount of chtoramines taken up by the liver appeared in bile in form of GSSG. In order to csmpare the known affects of H20z on bilir< GSSC wirh the effects of cixiau!inca, i-izOL 7:~s hfwed at a rate of 0.3 and 0.6 ~motP.nin, respectively. The biliary 0.W.l
excretion f
of GSSG
0.015 nmolknm
increased
by an average of
pei g (n = 4) an3 Xi4
“moUrnin per g (n = 5) at the bwer
+ 0 ilt:
aEd the higher perfu-
sion mtc. rcspcctiwly.
Assuming lhot H,O,
tnken up by the liver, :hc inmmtent
mdy
for
given amwnt
is quantita-
in bilinry
GSSG
taken up reflcctcd by the slope ol the corresponding correlation in Kg. 6 amounted to spprorimately half of what was ob.wved with the chloramincs. In vitro. NH&!] at a concentration of l:!.SrM reacted promptly and qrrantitativcly with GSH (Ifl mM, corresponding to the intrnccllulsr concentration of the sutthy dryl) to form FSSG (Fig. 7). 2
of H,O,
HOC, at low conccntmtions hod dramatic effect- on the vascular ,esis,ancc and on bePatocyte integrity ii, the perfused rat liver (Fig. 1). Activated PMN relax HOCt not only into phagocytic vacuoles but also into the exhacellolx space (10.15.17). Approximately one third of the H,O, generated by PMN is halogenated to HOC], Dot the concentration of HOC1 in the liver upon activation of PMN that have accumulated in the organ is oat known. Local concentrations of HOC] have heen estimated to reach the 1COpM range in the immediate vicinity of activated PMN (15.18), but blood flow will of course rapidly dilute the oxidant. Although it is obviously difficult to mimick the localized release of HOC1 by activated PMN in the liver, regional increases in vascular resistance and ceil damage due to HOC! stmikn to what WC observed in our model ryrtem may occur a. sites of neutrophil
accu-
mulat*on in wvo. The effeeta of HOC1 most liks!y result from the oxida-
tion of critical sulfiydryl grwps. At concentrations of lo-20 PM. HOCI causes the oxidation of plasma membrane sultltydryls and impairs various plasma membrane functions in cultured tumor cells without loss of cell viability (19). Our ooserwion that extracellular GSH at physiological concentrations praented the toxic effects of HOC1 is mnsistent with such a mode of action. Since the liver is the najor source of circulating GSH (l6), the highest ~xtrac:~Utar concentrations of GSH i? the body probably exist ire bepatic sinusoids. There. KOC, relsrsed by PMN in vh 3 can radily rext .rith GSEl prior to kteracting with cri:b:a, ao!%ydry,s on plasma membraxr. The extracellular xxtion
of oxidants with GSH released by
the toxic effects of neutrophils.
Besides sulfhydryls, HOC, readily texts with amines to form
chloraminer.
chtotamines
Although
have potentially
less reactive deleterious
than
axidative
HOC,. ptop-
(23). Compared to HOCI. NH&I and TaauNHCI affected perfusion pressure. LDH leakage and bile flow much less. Both, however, markedly increased the biliary excretion of GSSG and the GSSG:GSH ratio i’? bile. Thus, chloramines taken up by the liver react with intutcellular GSH a~ they do in vitro (Fig. 7) to form GSSG which is in part excreted in bile (24,25). The different effects of the two chtoraminea on the bilet&s
intg twretion of GSSG is best cnp!ained hy diffcrenc~a in the extraction of the two compounds. NH&, is much more lipophilic than TauNHCt and therefore essentially quantitatively extracted by the liver at the soncentrations used in out experiments. In contrast, only a small fraction of the Perfused TauNHCl was taken up by the liver. The uptake of HOC1 could not he detettnined because the concentration used was new the limit of detection of our assay. Once taken up by the liver the effects of chloraminec appesr to be identical. The same anaunt of GSSG is fonned for a given amount ofchloramine taken up. Only a &action of the generated GSSG, however, will be excreted into bile rather than reduced back to GSH by glutathione reductase and NADPH (24-26). Considering the slope of the regression for chloramines shown in Fig. bit is not surprising that HOC, which was infused at a rate avercging 13 nmollmin per g liver ~antwt he expected to result in a measurable increase in biliary GSSG even if it is taken up quantitatively. The fraction of H202 metabolized to GSSG (a8 reflected by the slope of the tzgres,iun shown in Fig. 6) was approximately half of what was seen with the chloramines. This agrees with as&r findings that a substantial fraction of the intracellular HzO, is metabolized via catalase rather than via GSH peroxidase (26) and, therefore. does not result in the formation of GSSG. During the perfusion of organic hydroperoxides which are
quaotitativcly metabolized via the glutathione peroxidase system approximately twice as much GSSG appears in hi,e per moi peroxide taken up by the liver than with H20z (26). The 2-fold difference in slope of the recrcssion lines shown in Fig. 6 tnerefore suggests that the chloramines are quatdtatively reduced by GSH intrace,!tt!arly. In wnciusion, our findings demonstrate that cblor-
hepatocytes may, thus, represent an important mechanism of protedicn of the liver and other organs from oxidative damage. Therefore, subjean with decreased circolating concentrations of GSH such as alcoholics (20), pattents wdh the acquired knmune deficiency syndrrrw !?I) and patients whew the efflux of GSH from the liver is de-
amines aw rftkientiy detoxified in the liver by intracellu-
creased such Uscirrhotics (22) may be n:ore susceptible to
zayeloperaxidasereleased by activated PMN.
lar GSH. lttteractions of the much mote toxic HOC1 with hepatic cells are orevented bv extracellulx~ GSA. Thus. gb:tathione plays an important role not only in the detoxification of Oz.’ and H201 generated hy PMN hut also in the :r@tcction against the cyiotoxic effects of products of
Deut%he
*c*nowkdgnlents
lance
Supported lional
by grar*
Foundation
No. 3.812-0.87 for
Scientific
from
rbe Swiss Na-
Research
and
by
sinuroidal cndothcos, dls of lY0.s.tiwr ..h its modulation in mule phase. J Hepatoll988: 7: 239-49. i Grirham MB, Hsmander LA. Crqer DN. Xanthme sxwlarc and “euw.phi, inmtmion in inrcidna, ir,lemia. Al” I Physio, 1986;251: G567-74. 8 GlUespie MN, Kojima 5, Kunitomo M, Jay M. Coronary and mvocardial eflec,r of activa,lxl nrutroohils in oerfued ,.3bb”
Fnrrchungsgemeinachaft.
of Ms.
Xk”.XVlZdg.Sd
E. Junker
and Mr.
The
c.
acsisis gra*efuOy
technica,
Schraoz