J Mel
Cell
Cardiol
Endothelin
24, 1291-1305
Release
Friedrich
(1992)
during Ischaemia Perfused Rat Brunner*,
Eugene
and Reperfusion Hearts
F. du Toit
and
Lionel
of Isolated
H. Opie
MRC Ischaemic Heart Disease Research Unit, Department of Medicine, Groote Schuur Hospital and University of Cape Town, South Africa, and *Department of Pharmacology and Toxicology, University of Graz, Universitiitsplatz 2, 8010 Graz, Austria (Received 9
3~4 1991,
accepted in revised form 2 June 1992)
F. BRUNNER, E. F. DU TOIT AND L. H. OPIE. Endothelin Release during Ischaemia and Reperfusion of Isolated Perfused Rat Hearts. Journal of Molecular and Cellular Cardiology (1992) 24, 1291-1305. The hypothesis tested was that release of endogenous endothelin plays a role in events associated with or leading to myocardial ischaemia and/or post-ischaemic reperfusion damage. Release of endogenous endothelin into the coronary perfusate of isolated perfused rat hearts during ischaemia and reperfusion was measured with a sensitive radioimmunoassay using a polyclonal antibody with 100% cross-reactivity for all three endothelin isomers. Basal endothelin release was 0.69 f 0.02 pg/min/g wet heart weight (a= 35) and was constant up to 180 min. During low-flow hypoxic mmHg) and in the presence of 1% foetal calf serum, the release rate was ischaemia for 180 min (PO,%250 reduced to below 10% ofcontrols (P 0.05). Thereafter, release declined 0.15 pg/min/g (P~0.01, data not shown I. IXlOW
Effect of endothelin-2 on the coronacy perfusion pressure of non-ischaemic and ischaemzc isolated rat heart.r Bolus injections of endothelin-2 provoked dose-dependent increases in coronary perfusion pressure within the range of 1 ng to 1 ,~g
t0-'O
Change over control (-told i
0.73f0.07 0.92*0.10 o.f39* 0.07 0.66&0.04 0.85 f 0.07
Preischaemic control RP 0 -5 min RP IO--l5 min RP 2530 min RP 55-60 min
0
(RP) subsequent
ET release 1pg/mink~
period
lO-g Endotheltn-2
1297
Reperfusion
1.3210.17 1.04~tO.21 0.96 f 0.12 I .3 I f 0.30
per heart (Fig. 2, solid symbols). The vehicle for endothelin-2 had only a transient reversible effect. The perfusion pressure increased slowly and reached a maximum after l--3 min. This pressure was maintained for at least 5 min except when the highest dos.zs were administered. Coronary perfusion pressure increased from 65f 2.1 (baseline) to a maximum of 134+z 7.5 mmHg ( + 106% increase). The threshold dosr for the vasoconstrictive action was z 20 ng. and the EC,,, calculated
IO-*
IO-'
G6
(g 1
FIGURE 2. Dose response curves for the effect of endothelin-2 on coronary perfusion prcssurr in control and reperfused hearts. Endothelin was applied to hearts perfused at constant flow without (controls, filled symbols~ or subsequent to I h of low-flow ischaemia (reperfused hearts, open symbols). Data arr expressed as absolute pressure readings (mmHg) as a function of endothelin-2 dose (g per heart) applied cumulatively at 2.5 min intervals. Means* s.E.M., n= 3 each. The mean curve was fit by non-linear regression and the EC,, determined as 69 (r~ontrol) and 1.9 (reperfusicn) ng endothelin-2 per heart.
1298
F. Brunner
by non-linear regression analysis of pooled data was 69 ng per heart. Hearts made ischaemic through low-flow perfusion for 1 h reacted more readily to exogenous endothelin-2 (0.1 ng-1 p(g) than normoxic hearts (Fig. 2, open symbols): the baseline coronary perfusion pressure was 93 f 2.4 mmHg and increased to a maximum of 188 f 4.5 mmHg ( f 238%) at 1 pg endothelin-2. The threshold dose was below 1 ng, and the EC,, calculated by non-linear regression of pooled data was 1.9 ng per heart (Fig. 2). E$ects of endothelin-2 on mechanical function control and reperfused hearts
in
E$ect on systolic developed pressure and aortic
output Measurement of systolic developed pressure reflected the effect of endothelin-2 on the inotropic state of the heart. Under constant flow perfusion conditions (Langendorff mode, 1.25 mmol/l calcium), the baseline systolic developed pressure was 36f 2.3 mmHg, the threshold dose for the positive inotropic effect was x 50 ng, the maximum systolic developed pressure 65 f 1.8 mmHg ( + 81% increase), and the EC,, was 185 ng per heart (Fig. 3). In
Endothelm-2
et al.
the isolated working heart, the same dose of endothelin-2 (100 ng) had no significant influence on aortic output (inotropy) over 40 min post application (Table 4). Coronary flow was reduced, albeit not significantly (P>O.O5 at all time points). E$ect on post-ischaemic reperfusion function Since endothelin is a postive inotropic agent, the influence of endothelin on heart function during post-ischaemic reperfusion was studied. The influence of a 20 min global ischaemic period on reperfusion function in controls and hearts treated with 100 ng or 200 ng endothelin-2 at the onset of reperfusion is shown in Fig. 4 and Table 5. Aortic output was significantly lower between 10 and 40 min of reperfusion compared to pre-ischaemic controls (PO.O5).
Reference 10 min 20 min 30 min 40 min
TABLE
1300
F. Brunner
20 Reperfusion 0
et al.
time
t 100 ng ET-2
30 (min)
40 m
+ 200
ng ET-2
FIGURE 4. Influence of endothelin-2 (ET-2) on recovery of aortic output on reperfusion. The post-ischaemic aortic output recovery between 10 and 40min of reperfusion in the absence (control) and presence of 100 or 200 ng endothelin-2 given at the onset of rep&&ion was plotted. For pre-ischaemic reference values see Table 5. Meansfs.E.M., n= 7 (control and 100 ng endothelin-2) and 3 (200 ng endothelin-2). *P< 0.05 us. control. **P
Cell
Cardiol
Endothelin
24, 1291-1305
Release
Friedrich
(1992)
during Ischaemia Perfused Rat Brunner*,
Eugene
and Reperfusion Hearts
F. du Toit
and
Lionel
of Isolated
H. Opie
MRC Ischaemic Heart Disease Research Unit, Department of Medicine, Groote Schuur Hospital and University of Cape Town, South Africa, and *Department of Pharmacology and Toxicology, University of Graz, Universitiitsplatz 2, 8010 Graz, Austria (Received 9
3~4 1991,
accepted in revised form 2 June 1992)
F. BRUNNER, E. F. DU TOIT AND L. H. OPIE. Endothelin Release during Ischaemia and Reperfusion of Isolated Perfused Rat Hearts. Journal of Molecular and Cellular Cardiology (1992) 24, 1291-1305. The hypothesis tested was that release of endogenous endothelin plays a role in events associated with or leading to myocardial ischaemia and/or post-ischaemic reperfusion damage. Release of endogenous endothelin into the coronary perfusate of isolated perfused rat hearts during ischaemia and reperfusion was measured with a sensitive radioimmunoassay using a polyclonal antibody with 100% cross-reactivity for all three endothelin isomers. Basal endothelin release was 0.69 f 0.02 pg/min/g wet heart weight (a= 35) and was constant up to 180 min. During low-flow hypoxic mmHg) and in the presence of 1% foetal calf serum, the release rate was ischaemia for 180 min (PO,%250 reduced to below 10% ofcontrols (P 0.05). Thereafter, release declined 0.15 pg/min/g (P~0.01, data not shown I. IXlOW
Effect of endothelin-2 on the coronacy perfusion pressure of non-ischaemic and ischaemzc isolated rat heart.r Bolus injections of endothelin-2 provoked dose-dependent increases in coronary perfusion pressure within the range of 1 ng to 1 ,~g
t0-'O
Change over control (-told i
0.73f0.07 0.92*0.10 o.f39* 0.07 0.66&0.04 0.85 f 0.07
Preischaemic control RP 0 -5 min RP IO--l5 min RP 2530 min RP 55-60 min
0
(RP) subsequent
ET release 1pg/mink~
period
lO-g Endotheltn-2
1297
Reperfusion
1.3210.17 1.04~tO.21 0.96 f 0.12 I .3 I f 0.30
per heart (Fig. 2, solid symbols). The vehicle for endothelin-2 had only a transient reversible effect. The perfusion pressure increased slowly and reached a maximum after l--3 min. This pressure was maintained for at least 5 min except when the highest dos.zs were administered. Coronary perfusion pressure increased from 65f 2.1 (baseline) to a maximum of 134+z 7.5 mmHg ( + 106% increase). The threshold dosr for the vasoconstrictive action was z 20 ng. and the EC,,, calculated
IO-*
IO-'
G6
(g 1
FIGURE 2. Dose response curves for the effect of endothelin-2 on coronary perfusion prcssurr in control and reperfused hearts. Endothelin was applied to hearts perfused at constant flow without (controls, filled symbols~ or subsequent to I h of low-flow ischaemia (reperfused hearts, open symbols). Data arr expressed as absolute pressure readings (mmHg) as a function of endothelin-2 dose (g per heart) applied cumulatively at 2.5 min intervals. Means* s.E.M., n= 3 each. The mean curve was fit by non-linear regression and the EC,, determined as 69 (r~ontrol) and 1.9 (reperfusicn) ng endothelin-2 per heart.
1298
F. Brunner
by non-linear regression analysis of pooled data was 69 ng per heart. Hearts made ischaemic through low-flow perfusion for 1 h reacted more readily to exogenous endothelin-2 (0.1 ng-1 p(g) than normoxic hearts (Fig. 2, open symbols): the baseline coronary perfusion pressure was 93 f 2.4 mmHg and increased to a maximum of 188 f 4.5 mmHg ( f 238%) at 1 pg endothelin-2. The threshold dose was below 1 ng, and the EC,, calculated by non-linear regression of pooled data was 1.9 ng per heart (Fig. 2). E$ects of endothelin-2 on mechanical function control and reperfused hearts
in
E$ect on systolic developed pressure and aortic
output Measurement of systolic developed pressure reflected the effect of endothelin-2 on the inotropic state of the heart. Under constant flow perfusion conditions (Langendorff mode, 1.25 mmol/l calcium), the baseline systolic developed pressure was 36f 2.3 mmHg, the threshold dose for the positive inotropic effect was x 50 ng, the maximum systolic developed pressure 65 f 1.8 mmHg ( + 81% increase), and the EC,, was 185 ng per heart (Fig. 3). In
Endothelm-2
et al.
the isolated working heart, the same dose of endothelin-2 (100 ng) had no significant influence on aortic output (inotropy) over 40 min post application (Table 4). Coronary flow was reduced, albeit not significantly (P>O.O5 at all time points). E$ect on post-ischaemic reperfusion function Since endothelin is a postive inotropic agent, the influence of endothelin on heart function during post-ischaemic reperfusion was studied. The influence of a 20 min global ischaemic period on reperfusion function in controls and hearts treated with 100 ng or 200 ng endothelin-2 at the onset of reperfusion is shown in Fig. 4 and Table 5. Aortic output was significantly lower between 10 and 40 min of reperfusion compared to pre-ischaemic controls (PO.O5).
Reference 10 min 20 min 30 min 40 min
TABLE
1300
F. Brunner
20 Reperfusion 0
et al.
time
t 100 ng ET-2
30 (min)
40 m
+ 200
ng ET-2
FIGURE 4. Influence of endothelin-2 (ET-2) on recovery of aortic output on reperfusion. The post-ischaemic aortic output recovery between 10 and 40min of reperfusion in the absence (control) and presence of 100 or 200 ng endothelin-2 given at the onset of rep&&ion was plotted. For pre-ischaemic reference values see Table 5. Meansfs.E.M., n= 7 (control and 100 ng endothelin-2) and 3 (200 ng endothelin-2). *P< 0.05 us. control. **P
