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reperfusion injury in the rat lung: Modulation by K+ - channel activity
Session 9: Ischemia-Reperfusion
532
9:s
ISCHEMlAlREPERFUSlON INJURY IN THE RAT LUNG: MODULATION BY K+ - CHANNEL ACTIVITY. Aron B. Fisher and Abu Al-Mehdl. lnstltute for Envlronmental Medlclne, Unlverslty of Pennsylvanla School of Medlclne, Phlladelphla, PA 19104, U.SA
LEUKEMIA INHIBlTORY FACI’OR AN-D TUMOR NECROSIS FACTOR INDUCE MnSOD AND PRO!l’ECI’ RABBIT HEARTS FROM REPEXFUSION INJURY.
We have shown previously (Arch. Blochem. Blophys. 303:307, 1983) wlth the Isolated rat lung model that llpld peroxldatlon/proteln oxldatlon Is lnltlated durlng lung lschemla and Is not associated wlth 0, or substrate deprlvatlon. The failure to demonstrate a “metabolic” basls for lnltlatlon of lschemlc oxldatlve Injury led us to evaluate the posslblllty that alterations In lung flow and oxldatlve Injury are llnked through changes In membrane polarlxatlon. Isolated rat lungs ventilated with alr:S% CO, were subjected to global lschemla (I) followed by reperfuslon (R) wlth synthetic medlum. Wlth I (1 hr) t R (1 hr), lung homogenate thlobarblturlc acid reactive substance (TEARS) and conlugated dlenes (CD) Increased slgnlflcantly (control, 41 f 3.1 pmol/mg prot and 0.64 * 0.64 unlts, respectively; I/R, 174 * 9.3 and 1.65 * 0.11; n=4-7). Preperfuslon wlth cromakallm (10 rglml), an activator of K’ channels, reduced TBARS by 43% and CD by 34%. Lungs that were normally perfused for 2 hrs but wlth the addltlon of hlgh perfusate K’ (24 mM substltuted for equlmolar Na’) also showed elevation of TBARS (236 * 31 pmol/mg prot) and CD (1.52 * 0.30 unlts). The presence of the K channel antagonlst gllburlde (10 mM) or addltlon of Ba*’ (1mM) to the perfusate gave slmllar results. These results show lung llpld peroxldatlon wlth I/R, In the presence of elevated K , and by antagonlsts of K* channels; a K’ channel agonist lnhlblted I/R changes. The data suggest that depolarlzatlon of the endothellal cell membrane associated with the absence of flow serves as an lnltlator
Webb-Waring Institute for Biomedical Research, Denver, CO 80262 and ‘Genentech Inc., South San Francisco, CA 94080
of lung oxldatlve
g:7
rRsp-RMXC?S,S:
Injury.
Q,,ALITATX”B
Wilson Aondi,Jr, Jo& Rwpical
XVIUUATXOU
ds.Fellppe
Jr.
AT
BEDSIDE
$80 L-zopoldo,SZo P3ulo,SP,M93.4+KC-BK+AzIL
The increase in the free-radical generation 1s one of the intermediary mechanisms Of Several disoascs which affect the ctltlcally ill patients: SAW., low flow states, Infection, sepsis, trauma, niocardiaL infarct etc... The incrcasc in the generation of reactive oxygen toxic spocios (ROTS), affect vital structures Of cells and disturb several metabolic pathways. It is interesting to note that the ROTS react wi.Ch the componontr of the whole blood forming subp&dwts which provoke ro+phOlOgiC altoratiobr in the peripheral blood, which can bc seen under a oonmwiamicroscope. Such morphologic alterations ate dependant in one way of the quantity and kind or ROT.9 (0; OH+. o, singlet) end in anathor way of the atrer&b of the imune system. The quirIit.*tlVe evaluation of the ROTS ere made by tho N.L.B. blood tent, created by the reseerchcrs H&tan-La Garde-Uradford. M HOD 54 dry gleea Plato6 of 12 critically $11 pa ents(3 politrama, 3 pas-op. 1 unstable angina, +; I mioczkrdi& infarct, 1 aathm, 1 Va$culer hDnorragLccerebral accident, 1 hypertensive cfLsi5, 1 hepatic insufficiency. The number of blades vsricd from 3 to 8 by patient, heving been obt&wd on admission, on important event8 and upon discharge/ The reading was done by 2 indepondont obit. researchers not were of the clinical state of the petients. TECHNIQUE: h series of blood drops collected on a dry glass place, by one slight punction Of the fingertip until we g&t the desired decrease of the drop thickness. The preparation is Pried for abOut 10 minute+ end read (200X). The degree of morphologic alterations attxLbuted to ROTS can bc axbitrarily divided into A stages: 0 to 4, where 0 is normel. RBSOLTS: Thotc was a eignificant CO-XeLatiOn between mdagrca of the oxidative lesion and the degree of We patient's kolen, clinically and laboratory owluat6d. CDI?CLUSIO~:In only X0 minutes. the H.L.B. blood testgives the dlagno~ia of oxidative pathology and It mainlv wrnits the rnonitorization of the evolution snd the changes detcrmlned by the thetapcutica.
Sally K. Nelson,
Grace H.W. Wong’
9:6
and Joe M. McCord
Leukemia inhibitory factor (LIF) protects cells from radiation, hyperoxia and endotoxin shock. Tumor necrosis factor (TNF) induces MnSOD in vivo and in vitro. Therefore we examined the effects of these cytokines on reperfusion injury in isolated rabbit hearts. Rabbits were injected IV with 10 pg of either human TNF-cx or lymphotoxin (TNF-P), or murine TNF-IX or LIF and control animals were injected with an equal volume of saline. After 24 hr, hearts were isolated and perfused. After 15 min equilibration hearts were subjected to 1 hr ischemia and 1 hr of reperfusion. All treated groups showed significant increases in percent recovery of developed tension (O/o preischemic) when compared to saline treated control hearts. In addition, there were significant decreases in lactate dehydrogenase release, accumulation of thiobarbituric acid reactive substances, and accumulation of carbonyl proteins. These results correlate with increases in myocardial MnSOD activity and levels of hepatic M&SOD mRNA. Thus, pretreatment of animals with TNF-a, lymphotoxin, or LIF protects hearts from myocardial reperfusion injury by a mechanism that may involve the induction of MnSOD.
EVIDENCE OF FATTY ACID PEROXIDATION AND NA+K+-ATP-ASE INHIBITION BY ACTIVATED NEUTROPHILS INCUBATED WITH RENAL BASOLATERAL MEMBRANE. T Ringer, M Chobanian, J Lewandoski, C Julin, J Zimmerman. Divisions of Critical Care and Nephrology, University of Wisconsin Children’s Hospital, Madison, WI 53792. Neutrophil (PMN) influx into kidneys occurs in many critical illnesses. Subsequent renal tubular dysfunction may be partially mediated by PMN oxidant stress towards membrane proteins and phospholipid (PL). We previously reported inhibition of basolateral membrane (ELM) Na+-K+-ATPase activity by PMN oxyradicals. To assess one aspect of this enzyme inhibition, we examined peroxidation of the surrounding PL milieu. Canine renal BLM (0.2 mg protein) was incubated at 37” for 60 min alone, with 5 x lo6 resting PMNs, or with 5 x IO6 stimulated PMNs. Na+-K+-ATPase activity was then measured. Total BLM fatty acids (FA) were quantitated by gas chromatography/mass spectrometry (GC/MS). BLM lipid peroxidation products evaluated included FA hydroperoxides by GC/MS, phosphatidylcholine-specific conjugated dienes by high-pressure liquid chromatography (HPLC), and thiobarbituric reactive substance (TBARS) by spectrophotometry. The BLM antioxidant, a-tocopherol (aT) was assayed by HPLC. Results are expressed as mean f S.E. Inhibition (60.2 ?r 2.1%, n=14, pcO.0001) of BLM Na+K+-ATPase activity occurred only with coincubation with activated PMNs. BLM FA concentrations did not significantly change. However, hydroperoxides of linoleic acid (n=6, ~~0.05) and arachidonic acid (n=4, ~~0.05) increased 3-fold when incubated with activated PMNs. CD increases did not reach statistical significance with n=3. No alterations in TBARS were detected. BLM aT content decreased from 5.1 i 0.7 to 3.6 + 0.4 pg aT/mg PL when incubated with resting vs. stimulated PMNs (n=6, ~~0.05). Activated, but not quiescent, PMNs markedly inhibit BLM Na+-K+-ATPase with associated BLM FA peroxidation and aT consumption.
9:8
532
9:s
ISCHEMlAlREPERFUSlON INJURY IN THE RAT LUNG: MODULATION BY K+ - CHANNEL ACTIVITY. Aron B. Fisher and Abu Al-Mehdl. lnstltute for Envlronmental Medlclne, Unlverslty of Pennsylvanla School of Medlclne, Phlladelphla, PA 19104, U.SA
LEUKEMIA INHIBlTORY FACI’OR AN-D TUMOR NECROSIS FACTOR INDUCE MnSOD AND PRO!l’ECI’ RABBIT HEARTS FROM REPEXFUSION INJURY.
We have shown previously (Arch. Blochem. Blophys. 303:307, 1983) wlth the Isolated rat lung model that llpld peroxldatlon/proteln oxldatlon Is lnltlated durlng lung lschemla and Is not associated wlth 0, or substrate deprlvatlon. The failure to demonstrate a “metabolic” basls for lnltlatlon of lschemlc oxldatlve Injury led us to evaluate the posslblllty that alterations In lung flow and oxldatlve Injury are llnked through changes In membrane polarlxatlon. Isolated rat lungs ventilated with alr:S% CO, were subjected to global lschemla (I) followed by reperfuslon (R) wlth synthetic medlum. Wlth I (1 hr) t R (1 hr), lung homogenate thlobarblturlc acid reactive substance (TEARS) and conlugated dlenes (CD) Increased slgnlflcantly (control, 41 f 3.1 pmol/mg prot and 0.64 * 0.64 unlts, respectively; I/R, 174 * 9.3 and 1.65 * 0.11; n=4-7). Preperfuslon wlth cromakallm (10 rglml), an activator of K’ channels, reduced TBARS by 43% and CD by 34%. Lungs that were normally perfused for 2 hrs but wlth the addltlon of hlgh perfusate K’ (24 mM substltuted for equlmolar Na’) also showed elevation of TBARS (236 * 31 pmol/mg prot) and CD (1.52 * 0.30 unlts). The presence of the K channel antagonlst gllburlde (10 mM) or addltlon of Ba*’ (1mM) to the perfusate gave slmllar results. These results show lung llpld peroxldatlon wlth I/R, In the presence of elevated K , and by antagonlsts of K* channels; a K’ channel agonist lnhlblted I/R changes. The data suggest that depolarlzatlon of the endothellal cell membrane associated with the absence of flow serves as an lnltlator
Webb-Waring Institute for Biomedical Research, Denver, CO 80262 and ‘Genentech Inc., South San Francisco, CA 94080
of lung oxldatlve
g:7
rRsp-RMXC?S,S:
Injury.
Q,,ALITATX”B
Wilson Aondi,Jr, Jo& Rwpical
XVIUUATXOU
ds.Fellppe
Jr.
AT
BEDSIDE
$80 L-zopoldo,SZo P3ulo,SP,M93.4+KC-BK+AzIL
The increase in the free-radical generation 1s one of the intermediary mechanisms Of Several disoascs which affect the ctltlcally ill patients: SAW., low flow states, Infection, sepsis, trauma, niocardiaL infarct etc... The incrcasc in the generation of reactive oxygen toxic spocios (ROTS), affect vital structures Of cells and disturb several metabolic pathways. It is interesting to note that the ROTS react wi.Ch the componontr of the whole blood forming subp&dwts which provoke ro+phOlOgiC altoratiobr in the peripheral blood, which can bc seen under a oonmwiamicroscope. Such morphologic alterations ate dependant in one way of the quantity and kind or ROT.9 (0; OH+. o, singlet) end in anathor way of the atrer&b of the imune system. The quirIit.*tlVe evaluation of the ROTS ere made by tho N.L.B. blood tent, created by the reseerchcrs H&tan-La Garde-Uradford. M HOD 54 dry gleea Plato6 of 12 critically $11 pa ents(3 politrama, 3 pas-op. 1 unstable angina, +; I mioczkrdi& infarct, 1 aathm, 1 Va$culer hDnorragLccerebral accident, 1 hypertensive cfLsi5, 1 hepatic insufficiency. The number of blades vsricd from 3 to 8 by patient, heving been obt&wd on admission, on important event8 and upon discharge/ The reading was done by 2 indepondont obit. researchers not were of the clinical state of the petients. TECHNIQUE: h series of blood drops collected on a dry glass place, by one slight punction Of the fingertip until we g&t the desired decrease of the drop thickness. The preparation is Pried for abOut 10 minute+ end read (200X). The degree of morphologic alterations attxLbuted to ROTS can bc axbitrarily divided into A stages: 0 to 4, where 0 is normel. RBSOLTS: Thotc was a eignificant CO-XeLatiOn between mdagrca of the oxidative lesion and the degree of We patient's kolen, clinically and laboratory owluat6d. CDI?CLUSIO~:In only X0 minutes. the H.L.B. blood testgives the dlagno~ia of oxidative pathology and It mainlv wrnits the rnonitorization of the evolution snd the changes detcrmlned by the thetapcutica.
Sally K. Nelson,
Grace H.W. Wong’
9:6
and Joe M. McCord
Leukemia inhibitory factor (LIF) protects cells from radiation, hyperoxia and endotoxin shock. Tumor necrosis factor (TNF) induces MnSOD in vivo and in vitro. Therefore we examined the effects of these cytokines on reperfusion injury in isolated rabbit hearts. Rabbits were injected IV with 10 pg of either human TNF-cx or lymphotoxin (TNF-P), or murine TNF-IX or LIF and control animals were injected with an equal volume of saline. After 24 hr, hearts were isolated and perfused. After 15 min equilibration hearts were subjected to 1 hr ischemia and 1 hr of reperfusion. All treated groups showed significant increases in percent recovery of developed tension (O/o preischemic) when compared to saline treated control hearts. In addition, there were significant decreases in lactate dehydrogenase release, accumulation of thiobarbituric acid reactive substances, and accumulation of carbonyl proteins. These results correlate with increases in myocardial MnSOD activity and levels of hepatic M&SOD mRNA. Thus, pretreatment of animals with TNF-a, lymphotoxin, or LIF protects hearts from myocardial reperfusion injury by a mechanism that may involve the induction of MnSOD.
EVIDENCE OF FATTY ACID PEROXIDATION AND NA+K+-ATP-ASE INHIBITION BY ACTIVATED NEUTROPHILS INCUBATED WITH RENAL BASOLATERAL MEMBRANE. T Ringer, M Chobanian, J Lewandoski, C Julin, J Zimmerman. Divisions of Critical Care and Nephrology, University of Wisconsin Children’s Hospital, Madison, WI 53792. Neutrophil (PMN) influx into kidneys occurs in many critical illnesses. Subsequent renal tubular dysfunction may be partially mediated by PMN oxidant stress towards membrane proteins and phospholipid (PL). We previously reported inhibition of basolateral membrane (ELM) Na+-K+-ATPase activity by PMN oxyradicals. To assess one aspect of this enzyme inhibition, we examined peroxidation of the surrounding PL milieu. Canine renal BLM (0.2 mg protein) was incubated at 37” for 60 min alone, with 5 x lo6 resting PMNs, or with 5 x IO6 stimulated PMNs. Na+-K+-ATPase activity was then measured. Total BLM fatty acids (FA) were quantitated by gas chromatography/mass spectrometry (GC/MS). BLM lipid peroxidation products evaluated included FA hydroperoxides by GC/MS, phosphatidylcholine-specific conjugated dienes by high-pressure liquid chromatography (HPLC), and thiobarbituric reactive substance (TBARS) by spectrophotometry. The BLM antioxidant, a-tocopherol (aT) was assayed by HPLC. Results are expressed as mean f S.E. Inhibition (60.2 ?r 2.1%, n=14, pcO.0001) of BLM Na+K+-ATPase activity occurred only with coincubation with activated PMNs. BLM FA concentrations did not significantly change. However, hydroperoxides of linoleic acid (n=6, ~~0.05) and arachidonic acid (n=4, ~~0.05) increased 3-fold when incubated with activated PMNs. CD increases did not reach statistical significance with n=3. No alterations in TBARS were detected. BLM aT content decreased from 5.1 i 0.7 to 3.6 + 0.4 pg aT/mg PL when incubated with resting vs. stimulated PMNs (n=6, ~~0.05). Activated, but not quiescent, PMNs markedly inhibit BLM Na+-K+-ATPase with associated BLM FA peroxidation and aT consumption.
9:8