European Journal of Pharmacology, 213 (1992) 171-181

171

© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

EJP 52367

Cardioprotective effect of pindolol in ischemic-reperfused isolated rat hearts Y o s h i h i s a Nasa, A.N. E h s a n u l H o q u e , K a z u o I c h i h a r a a n d Yasushi A b i k o Department of Pharmacology, Asahikawa Medical College, Asahikawa 078, Japan Received 1 August 1991, revised MS received 15 November 1991, accepted 7 January 1992

The effects of pindolol and timolol on ischemia reperfusion damage were studied in isolated working rat hearts. Ischemia (15 min) decreased the mechanical function and the energy state, and increased the tissue levels of free fatty acids (FFA). During reperfusion (20 min), the mechanical function did not recover, but the energy state recovered incompletely, whereas FFA increased further. Pindolol (50 /zM) accelerated recovery of the mechanical function and the energy state that had been decreased by ischemia during reperfusion, and inhibited the accumulation of FFA during ischemia and reperfusion, especially when it was applied during the whole period of reperfusion. Timolol (50 /zM), however, did not accelerate recovery of the mechanical function and the energy state during reperfusion, although it attenuated FFA accumulation during reperfusion. The pindolol-induced recovery of the mechanical function during reperfusion was reduced by timolol. The results suggest that the intrinsic sympathomimetic activity of pindolol may play an important role, at least in part, in producing the cardioprotective effect, especially during reperfusion. Pindolol; Ischemia; Reperfusion; Cardioprotection; Sympathomimetic activity (intrinsic); (Rat)

1. Introduction Pindolol is a non-selective potent /3-adrenoceptor antagonist possessing intrinsic sympathomimetic activity on both /3 t- and /32-adrenoceptors (Clark et al., 1982; McDevitt, 1983; Abrahamsson, 1986) and weak membrane stabilizing actions, and has been used for the treatment of patients with arrhythmias and hypertension (McDevitt, 1983; Wood and Tenn, 1984; Tamargo and Delpon, 1990). It is accepted that /3adrenoceptor antagonists have a cardioprotective effect on the ischemic myocardium, because they decrease myocardial oxygen demand by reducing cardiac contractile force and heart rate in therapeutic doses (Reimer et al., 1973; Rasmussen et al., 1977). If the cardiac depression induced by /3-adrenoceptor antagonists is the only mechanism responsible for their cardioprotective effect on the ischemic myocardium, /3-adrenoceptor antagonists with intrinsic sympathomimetic activity (such as pindolol) may not be very effective in protecting the myocardium from ischemic injury. Pindolol blocks /3-adrenoceptors when sympathetic nerves are stimulated, but the decrease in myocardial contractile force and heart rate is less than that in-

Correspondence to: Y. Nasa, Department of Pharmacology, Asahikawa Medical College, Asahikawa 078, Japan. Tel. 81.166.65 2111 ext. 2362, fax 81.166.65 5016.

duced by other /3-adrenoceptor antagonists without intrinsic sympathomimetic activity (Aellig, 1983; McDevitt, 1983). During ischemia, the sympathetic nerves in the myocardium are stimulated, and therefore pindolol may be useful for the treatment of ischemic heart disease. In fact, pindolol has been used for the treatment of ischemic heart disease in some cases (Frishman et al., 1979). It is possible to use /3-adrenoceptor antagonists with intrinsic sympathomimetic activity for the treatment of ischemic heart disease associated with mild cardiac failure (Aellig, 1983; McDevitt, 1983), because these agents block /3adrenoceptors but do not markedly decrease myocardial contractile force. In addition, /3-adrenoceptor antagonists with intrinsic sympathomimetic activity seem beneficial for postischemic functional recovery, because they stimulate cardiac /3-adrenoceptors, leading to an increase in contractile force and heart rate during reperfusion. The contribution of the intrinsic sympathomimetic activity of pindolol to its cardioprotective effect, however, has never been elucidated. The present study, therefore, was designed to examine whether pindolol has a direct effect on the isolated working rat heart to protect against ischemia- and reperfusion-induced damage. As indicators of ischemia reperfusion damage, we used changes in the mechanical function of the heart and changes in the myocardial levels of metabolites such as high-energy phosphates

172 and free fatty acids (FFA) (Nasa et al., 1990b). The fact that FFA accumulate during both ischemia and reperfusion suggests that FFA accumulation during ischemia can be used as an indicator of ischemia-induced damage and during reperfusion as an indicator of reperfusion-induced damage (Nasa et al., 1990b). We compared the effect of pindolol with that of timolol, which has neither intrinsic sympathomimetic activity nor membrane stabilizing activity but has non-selective /3-adrenoceptor antagonistic actions, being similar in potency to pindolol (McDevitt, 1983; Wood and Tenn, 1984).

2. Materials and methods

2.1. Heart perfusion Male Sprague-Dawley rats (280-340 g) were anesthetized with sodium pentobarbital (50 mg/kg i.p.). Hearts were quickly removed and then perfused according to the Langendorff technique followed by the working heart technique. The solution for perfusion was a modified Krebs-Henseleit bicarbonate buffer containing (mM) 11 glucose, 2.9 CaC12 and 0.5 EDTA-2Na (37°C), equilibrated with a gas mixture of 95% 0 2 and 5% CO 2. Aortic pressure and heart rate were monitored with a pressure transducer placed in the aortic cannula. Cardiac mechanical function was estimated as a rate-pressure product: peak aortic pressure multiplied by heart rate. Hearts were initially perfused with the Langendorff method at a constant pressure of 90 cm H 2 0 for 10 min, and then perfused with the working heart method at a left atrial filling pressure of 12.5 cm H 2 0 and an afterload pressure of 90 cm H 2 0 for 15 min (Nasa et al., 1990b). Ischemia was induced by removing the afterload pressure (i.e. no-afterload ischemia; Ichihara and Abiko, 1983), resuiting in 0 cm H 2 0 , and reperfusion was induced by returning it to its initial level. Ischemia was performed for 15 min and reperfusion for 20 min. During reperfusion, the heart was perfused with the Langendorff method for the first 5 min, and then perfused with the working heart method.

reperfusion following ischemia, respectively. In the pindolol-treated group, the reperfused group was divided into two groups according to the period of drug administration; pindolol : RA and pindolol : RB groups. In the pindolol : RA group, pindolol (50/zM) was administered to the perfusion solution during a period beginning 5 min before the onset of ischemia and lasting for the first 10 min of reperfusion. In the pindolol:RB group, pindolol in the same concentration was administered 5 min before the onset of ischemia until the end of reperfusion. Timolol (50 tzM) was administered to the perfusion solution during a period lasting from 5 min before the onset of ischemia until the end of reperfusion (timolol:RB). There was no timolol:RA group. There were 10 groups in all; control:Nl, control : I, control : R, pindolol : NI, pindolol : I, pindolol : RA, pindolol : RB, timolol : NI, timolol : I, and timolol : RB groups. In the additional experiment, both pindolol (50 ~M) and timolol (50 /xm) were administered to the perfusion solution 5 min before the onset of ischemia until the end of reperfusion.

2.3. Determination of tissue metabolites Hearts were freeze-clamped with aluminum Wollenberger clamps, which had been cooled to the temperature of liquid nitrogen (-195°C). The freeze-clamped hearts were stored in liquid nitrogen until biochemical analysis.

2.3.1. Assay of the tissue high-energy phosphates A part of the frozen cardiac tissue sample (about 0.8 g) was purverized in a mortar and a pestle cooled to the temperature of liquid nitrogen. High-energy phosphates (ATP, ADP, AMP and creatine phosphate) and lactate were extracted from the pulverized tissue sample with perchloric acid, and the extract was then neutralized with KOH. These metabolites were assayed by standard enzymatic methods (Gutmann and Wahlefeid, 1974; Lamprecht et al., 1974; Lamprecht and Trautschold, 1974), using a spectrophotometer (Gilford system 2600). Energy charge potential was calculated according to the following formula: energy charge potential = (ATP + 0.5ADP)/(ATP + ADP + AMP).

2.2. Experimental protocol and groups of hearts All the hearts (except for the hearts in an additional experiment) were divided into three groups: control (no drug, n = 23), pindolol-treated (n = 25) and timolol-treated (n = 15) groups. In each group, the hearts were divided further into small subgroups; non-ischemic (preischemic; NI), ischemic (I) and reperfused (R) groups, in which the hearts were freeze-clamped immediately before the onset of ischemia, immediately after the end of ischemia, and immediately after the end of

2.3.2. Assay of tissue FFA The levels of tissue FFA were measured according to the method described in our previous report (Nasa et al., 1990b). Briefly, tissue FFA were extracted from pulverized tissue with chloroform/methanol (2 : 1), and then converted to their fluorescent derivatives by adding 9-anthryldiazomethane in methanol at room temperature for 1 h. The fluorescent derivatives of FFA were filtered through a milliporeTM filter (FH 0.5 /xm; Nihhon Millipore Kogyo K.K., Yonezawa, Japan)

173

and injected onto a reverse-phase high-performance liquid chromatography system with a Zorbax-ODS column (0.46 × 25 cm; Dupont, Philadelphia, PA). Methanol/distilled water (1000:80) was used as mobile phase. The level of individual FFA was determined by comparing the peak height of the FA with that of a known amount of heptadecanoic acid (an internal standard).

2. 4. Drugs Pindolol (base) was supplied by Sandoz Pharmaceutical, Ltd., Tokyo, and timolol maleate was purchased from Sigma Chemical Company, St. Louis, MO. Pindolol was dissolved in the perfusate as a salt with hydrochloric acid, the concentrations of pindolol and hydrochloric acid being equimolar. Timolol was dissolved in the solution for perfusion. Biochemicals, reagents, enzymes and authentic FFA were purchased from Sigma Chemical Company, St. Louis, MO.

2.5. Statistical analysis All data are expressed as means + S.E.M. Statistical analysis of results was done with an analysis of variance followed by a Duncan multiple range test. Differences between means were considered significant at P < 0.05.

3. Results

3.1. Cardiac mechanical function The effect of treatment with pindolol or timolol for 5 rain on cardiac mechanical function in the working rat heart is shown in table 1. In the non-ischemic heart, pindolol increased peak aortic pressure slightly though non-significantly and decreased heart rate markedly, resulting in a decrease in the rate-pressure product. Timolol also decreased the rate pressure-product be-

cause it decreased both peak aortic pressure and heart rate. The decrease in mechanical function induced by timolol was less than that induced by pindolol (P < 0.05), however. Changes in the rate-pressure product during nonischemia, ischemia and reperfusion are shown in fig. 1 (pindolol) and fig. 2 (timolol). After the onset of ischemia, coronary flow became almost 0 ml/min and aortic pressure became 0 mm Hg, and therefore the rate-pressure product became 0 mm Hg/min. In the control heart, cardiac mechanical function that had been decreased by ischemia did not recover but remained at the lowest level during the entire period of reperfusion, indicating that the heart was irreversibly damaged in terms of mechanical function within 20 min of the start of reperfusion. In the pindolol-treated group, ischemia decreased the rate-pressure product to 0 mm H g / m i n as in the control group. In the pindolol:RA and pindolol:RB groups, there was a considerable improvement of cardiac mechanical function during reperfusion. Recovery of postischemic mechanical function in the pindolol:RB group was slightly less than that in the pindolol: RA group (fig. 1), but the difference was not significant (P > 0.05). In our preliminary experiment, in which pindolol (10/xM) was given during a preischemic period for 5 min, the mechanical function that had been decreased by ischemia did not recover substantially even after 20 min of reperfusion (data not shown). In the timolol: RB group, cardiac mechanical function did not recover even after 20 min of reperfusion. These results indicate that pindolol but not timolol accelerates the postischemic recovery of cardiac mechanical function during reperfusion.

3.2. High-energy phosphates Changes in the levels of ATP, ADP, AMP and total adenine nucleotides after ischemia and reperfusion in the presence or absence of pindolol or timolol are

TABLE 1 Peak aortic pressure, heart rate and rate-pressure product before and after 5 min of treatment with pindolol (50 g M ) or timolol (50 p.M) in non-ischemic hearts. Hearts were perfused by working heart method and pindolol or timolol was applied to the heart after 10 min of working heart perfusion. Data are expressed as means_+ S.E.M. ( ): change in percent, n: n u m b e r of experiments. Group

n

Peak aortic pressure ( m m Hg)

Heart rate (beats/min)

Control

23

Pindolol

25

110.3 5= 1.2

Before

After

Before

After

Before

After

104.9+ 1.4

103.3_+ 1.6 ( - 1.5%) 113.5 -+ 1.8 ( + 2.9%) 111.5 + 1.9 ( - 3.2%)

349+ 8

353+ 9 ( + 0.9%) 213 + 12 ( - 38.2%) 264 5= 11 ( - 19.0%)

36563_+853

36286+ 867 ( - 1.3%) 23 276 _+ 1609 (-38.9%) 29 276 _+951 ( - 21.7%)

Timolol

15

115.3 -+ 1.9

347 -+ 6 325 _+ 10

Rate-pressure product (mm Hg/min)

38 361 -+ 764 37 360 -+ 937

174

xlO00

40

E

30

Q.

20 o o. 10 t~ n"

0

-5

I

5

10

15

20

25

30

35

"on-"c'em'a I 'sc"em'a J •

I

Working

45

"ooe"o"o° •

Lan~endorff

4'0

I

50

(min)

I

Working

Lan~endorf!

I.......-.......'...v.........-.......v,v.v,'...'.-.'.'.'.'.:.:.:..l

t

RA

Pindolol

Fig. 1. Experimental protocol and effect of pindolol on the rate-pressure product (heart rate times peak aortic pressure) during ischemia and reperfusion. Pindolol was applied to the heart either 5 min before the onset of isichemia until the first 10 min of reperfusion (pindolol: RA) or 5 min before the onset of ischemia until the end of reperfusion for 20 min (pindolol: RB). All data are expressed as means _+S.E.M. (©) Control: R group, (e) pindolol : RA group, ( • ) pindolol: RB group. * Significantly different from the value in the control group (P < 0.05).

summarized in table 2. In the control group, the tissue level of ATP markedly decreased after ischemia, but it recovered after reperfusion, though incompletely; the level of ATP in the control : R group was significantly higher than that in the control : I group but was significantly lower than that in the control:NI group. The level of ADP decreased during both ischemia and reperfusion. The level of AMP increased significantly after ischemia (about 10 times the preischemic level) and decreased after reperfusion toward the preis-

chemic level. The level of total adenine nucleotides decreased markedly after ischemia and decreased further after reperfusion in the control group. Pindolol or timolol did not affect the basal levels of these adenine nucleotides in the non-ischemic heart. The pattern of changes in the levels of ATP and AMP after ischemia and reperfusion in the pindolol- and timolol-treated groups was essentially the same as that in the control group. The levels of ATP and ADP in the pindolol:I group were significantly higher than

xlO00 40

0

~

30

"~-

~

~ EE2 o Q)

10 t~ tr

j

|

J

•

i

-5

0

5

10

15

i

•

J

a

kangendorff

w 20

i

!

i

i

i

25

30

35

40

45

, ,.c-,.

Working

i

el

°eoo.--o

J

Working

50

(rain)

I

i

L.angendorff ¢.'. "-".".'. ".";".".";";';'; ";'.';';';" ;';';';';" ;'; ";'; ";': ":': ":":":': ":.""."-"-'-"" ".".'. '.'J Timolol

•

RB

Fig. 2. Effect of timolol on the rate-pressure product during ischemia and reperfusion. Timolol was applied to the heart 5 min before the onset of ischemia until the end of reperfusion (timolol : RB). All data are expressed as means _+S.E.M. (©) Control : R group, ( • ) timolol : RB group.

175 TABLE 2 Effects of pindolol and timolol on the tissue levels of ATP, ADP, AMP and total adenine nucleotides in ischemic-reperfused rat hearts. Data are expressed as means _+S.E.M. ( # m o l / g dry weight). Total adenine nucleotides: sum of the levels of ATP, ADP and AMP. NI: non-ischemic group, I: ischemic group, R: reperfused group, RA: one of the reperfused groups in which a drug was applied until 10 min after reperfusion (ref. fig. 1), RB: one of the reperfused groups in which a drug was applied until the end of reperfusion (ref. fig. 1). n: number of experiments. Group

n

ATP

ADP

AMP

Total adenine nucleotides

Control : NI Control : I Control: R

6 9 8

20.94 _+0.32 5.00 + 0.62 a 8.51_+0.52 a,b

5.22 _+0.13 4.37 ± 0.32 3.40_+0.14 a,b

1.00 + 0.07 9 . 8 9 -{- 0 . 5 0 a

3.31_+0.28 a,b

27.16 + 0.38 19.18 + 0.54 a 15.22_+0.57 ,,b

Pindolol Pindolol Pindolol Pindolol

5 7 7 6

19.25 _+0.38 7.26 _+0.89 a,c 12.26 _+0.82 a,b,c,d 14.20 _+0.64 a,b,c,d,e

5.49 _+0.17 5.82 _+0.47 c,d 3.96 -+0.09 a,b,c 3.37 _+0.11 a,b,d.e

1.01 _+0.07 7.32 + 0.84 a,c,d 1.59 -+ 0.18 b,c,d 0.99 _+0.15 b,c,d

25.75 _+0.44 20.40 _+0.57 a 17.81 -+ 0.67 a,b,c 18.57 _+0.63 a,c

5 5 5

19.71 _+0.56 5.51 -+0.88 a 10.29 -+0.79 a,b

5.68 _+0.25 4.39 -+ 0.33 a 3.98 _+0.28 ~,c

0.85 _+0.06 9.65 _+0.64 a 3.19 -+ 0.42 a,b

26.24 _-t-0.74 19.55 _+0.72 a 17.47 -+ 0.92 a,c

: NI :I : RA : RB

Timolol : NI Timolol : I Timolol : RB

a Significantly different from the values in the corresponding non-ischemic group (P < 0.05). b Significantly different from the values in the corresponding ischemic group (P < 0.05). c Significantly different from the values in the corresponding control group (P < 0.05). d Significantly different from the values in the corresponding timolol group (P < 0.05). e Signifiicantly different from the values in the pindolol: RA group

(P < 0.05).

those in the control : I group, and the level of AMP in the pindolol:I group was significantly lower than that in the control : I group, whereas the levels of A T P and AMP in the timolol:I group were similar to those in the c o n t r o l : I group. These results indicate that the ATP depletion and AMP elevation induced by ischemia were attenuated by pindolol but not by timolol. It must be noted, however, that the levels of total adenine nucleotides in the pindolol:I and timolol:I groups were similar to those in the c o n t r o l : I group, suggesting that neither pindolol nor timolol prevented the loss of adenine nucleotides from the myocardium during ischemia. The level of ATP in both p i n d o l o l : R A and pindolol: RB groups was significantly higher than that in the c o n t r o l : R group, and the level of AMP in the pindolol : R A and pindolol : RB groups was significantly lower than that in the c o n t r o l : R group. The level of A T P in the timolol : RB group was slightly higher than that in the c o n t r o l : R group, although the difference was not significant. The level of total adenine nucleotides in both timolol:RB, pindolol:RA, and pind o l o l : R B groups was significantly higher than that in the c o n t r o l : R group, indicating that both drugs prevented the loss of adenine nucleotides during reperfusion. These results suggest that pindolol has a beneficial effect in terms of preservation of high-energy phosphates in the ischemic-reprefused heart, and that the beneficial effect of pindolol is more prominent when pindolol is applied to the heart until the end of reperfusion. In contrast, timolol seems not so effective in preserving high-energy phosphates in the isichemic-reperfused heart.

1.0

0.5 Energy

charge

potential

NI

R

NI

I RA RB

NI

t RB

*¢ 30

2o

E

Creatine phosphate

I0

0

NI

I

R

NI

I RA RB

NI

I RB

tO0

9O

8O

~

20 Lactate

E

0

NI

I

R

Control

NI

I RARB

Pindolol

NI

I RB

Timolol

Fig. 3. Effects of pindolol and timolol on the levels of energy-charge potential, creatine phosphate and lactate after ischemia and reperfusion. All data are expressed as means _+S.E.M. NI: non-ischemic group, I: ischemic group, R: reperfused group, RA: reperfused RA group, RB: reperfused RB group. * Significantly different from the values in the control group (P < 0.05). * Significantly different from the values in the pindolol : RA group (P < 0.05).

176 The effects of pindolol and timolol on energy-charge potential are shown in fig. 3. The energy-charge potential decreased significantly after ischemia and then recovered incompletely after reperfusion in the control group. The energy-charge potential in the pindolol:I group, but not in the timolol : I group, was significantly higher than that in the control : I group. Moreover, the energy-charge potential in both pindolol: RA and pindolol: RB groups was significantly higher than that in the timilol:RB and c o n t r o l : R groups. The energycharge potential in the pindolol : RB group was similar to that in the pindolol:NI group, indicating that the energy-charge potential recovered almost completely when pindolol was applied to the heart during the entire reprefusion period. Figure 3 also shows the effects of pindolol and timolol on the tissue level of creatine phosphate during ischemia and reperfusion. The level of creatine phosphate in the control group decreased markedly after ischemia and recovered completely after reperfusion. Neither pindolol nor timolol affected the non-ischemic level of creatine phosphate and attenuated the decrease in creatine phosphate caused by ischemia. The level of creatine phosphate in the p i n d o l o l : R B group was significantly higher than that in both c o n t r o l : R and p i n d o l o l : R A groups. The level of creatine phosphate in the t i m o l o l : R B group, however, was significantly lower than that in the control : R group.

3.3. Lactate

Changes in the level of lactate during ischemia and reperfusion are shown in fig. 3. The level of lactate, a sensitive marker of ischemia in tissues, increased significantly after ischemia. In the control group, the level of lactate decreased markedly after reperfusion, although the lactate level in the control : R group was still higher than that in the control:NI group. Neither pindolol nor timolol attenuated the elevation of lactate caused by ischemia. The level of lactate in the timolol:RB group was significantly higher than that in the c o n t r o l : R group, whereas the level of lactate in the p i n n d l o l : R A group or in the pindolol:RB group was not different from that in the control: R group. 3.4. FFA

Figure 4 shows the changes in the levels of linoleic and arachidonic acids. In the control group, the level of linoleic acid increased significantly after 15 min of ischemia. The level of arachidonic acid also increased after ischemia (about 4.3 times the non-ischemic level) although the difference was not statistically significant. In the pindolol-treated group, neither linoleic nor arachidonic acid levels increased substantially after ischemia, whereas in the timolol-treated group, the level of arachidonic acid increased significantly after

200

16:0

100

80

0

18:2

40

150 o OI 100 18:0

"6

80 50

0

Oj

40

100 18:1 50

NI

I R

Control

0

NI I

R

Control

NI I RA RB PindoIol

.j NI I RA RB PindoIoI

20:4

NI I RB Timolol

NI I RB Timolol

Fig. 4. Effects of pindolol and timolol on the myocardial levels of palmitic acid (16:0), stearic acid (18:0), oleic acid (18: 1), linoleic acid (18:2) and arachidonic acid (20:4) in hearts after ischemia and reperfusion. All data are expressed as means_+S.E.M. Abbreviations are those of fig. 3. * Significantlydifferent from the values in the control group (P < 0.05).

177

ischemia. Furthermore, the levels of linoleic and arachidonic acids increased significantly after reperfusion in the pindolol : R A and in the timolol : RB groups, when compared with their levels in the pindolol:NI and timolol:NI groups, respectively, but these levels were significantly lower than those in the c o n t r o l : R group. The levels of linoleic and arachidonic acids in the pindolol:RB group were similar to those in the pindolol:I group, indicating that pindolol prevented the reperfusion-induced accumulation of linoleic and arachidonic acids completely, when it was applied to the heart until the end of reperfusion. These results also indicate that timolol has an inhibitory effect on the reperfusion-induced accumulation of tissue linoleic and arachidonic acids, although the inhibitory effect of timolol was less than that of pindolol. The effects of pindolol and timolol on the levels of palmitic, stearic and oleic acids are shown in fig. 4. In the control group, these FFA did not increase after ischemia but increased significantly after reperfusion. The levels of stearic acid in both p i n d o l o l : R A and p i n d o l o l : R B groups and of oleic acid in the pindolol : RB groups were significantly lower than those in the control : R group. The level of oleic acid in the pindolol : RB group was significantly lower than that in the t i m o l o l : R B group. The results suggest that pindolol inhibited, but timolol did not affect, the accumulation of tissue palmitic, stearic and oleic acids induced by ischemia and reperfusion. The effects of pindolol and timolol on the level of total FFA, including lauric, myristic, palmitic, stearic, palmitoleic, oleic, linoleic and arachidonic acids, after ischemia and reperfusion are shown in table 3. Neither pindolol nor timolol affected the levels of total FFA in the non-ischemic and ischemic groups. Reperfusion resulted in the accumulation of total FFA in the control : R, pindolol : RA and timolol : RB groups. The level of total tissue FFA in the pindolol : RB group was not greatly different from that in the pindolol:NI and pindolol:I groups, but it was significantly lower than that in the c o n t r o l : R and timolol:RB groups. These results indicate that pindolol prevented the accumulation of FFA in the reperfused myocardium effectively, when it was applied to the heart until the end of reperfusion.

TABLE 3 Effect of pindolol and timolol on the tissue level of total free fatty acids (FFA) in the isichemic-reperfused rat heart. Data are expressed as m e a n s + S.E.M. ( n m o l / g dry weight). Total FFA: sum of the levels of each FFA including lauric, myristic, palmitic, stearic, palmitoleic, oleic, linoleic and arachidonic acids. Abbreviations are those of table 2. n: number of experiments. Group

n

Total FFA

Control : NI Control : I Control : R

6 9 8

322.7 + 20.3 403.5 _+35.0 620.5 _+46.2 a,b

Pindolol : NI Pindolol : I Pindolol : RA Pindolol : RB

5 7 7 6

294.6 311.0 463.6 420.4

Timolol" NI Timolol : I Timolol : RB

5 5 5

301.8 + 14.4 366.1 + 50.8 557.1 _+ 12.5 a,b

+ 34.0 _+30.1 _+46.1 a,b,c _+54.3 c,d

Significantly different from the values in the corresponding nonischemic group (P < 0.05). b Significantly different from the values in the corresponding ischemic group (P < 0.05). c Significantly different from the values in the corresponding control group (P < 0.05). ° Significantly different from the values in the corresponding timolol group (P < 0.05).

rate-pressure product) in the preischemic heart from 39208 _+ 837 to 16090 _+ 4247 mm H g / m i n (n = 4). Ischemia made the rate-pressure product 0 mm Hg, and reperfusion increased it slightly. Figure 5 shows a comparison of the recovery of mechanical function after 20 rain of reperfusion in the heart treated with pindolol, the heart treated with timolol, and the heart treated with pindolol + timolol. The result with pindolol + timolol was obtained in the additional experiment. The rate-pressure product in the pindolol + timolol group was significantly lower than that in the p i n d o l o l : R A

xlO00

20,

° 3E ~

E E

m'

3.5. Additional experiment 0

If the functional recovery induced by pindolol is due to its intrinsic sympathomimetic activity, the recovery should be reduced by a /3-adrenoceptor antagonist. Therefore, in the additional experiment, both pindolol and timolol were administered to the perfusion solution before 5 rain of ischemia until the end of reperfusion. Administration of both pinolol and timolol reduced cardiac mechanical function (expressed as a

Control

PIndolol

Pindolol

Timolol

RA

RB

RB

Pindolol 4Timolol

Fig. 5. Comparison of the rate-pressure product after 20 min of reperfusion in the control, pindolol: RA, pindolol: RB, timolol : RB and pindolol + timolol groups. The values of the control, pindolol : RA and pindolol:RB groups are those in fig. 1, and the value in the timolol : RB group is that in fig. 2. The value in the pindolol + timolol group is that in the additional experiment. All data are expressed as m e a n s + S.E.M.

178 group (P < 0.05) and pindolol:RB group (P < 0.05), and was slightly but non-significantly higher than that in the timolol:RB group (P > 0.05). This result indicates that the intrinsic sympathomimetic activity of pindolol contributes to an acceleration of recovery of mechanical function during reperfusion.

4. Discussion

Our previous studies have demonstrated that noafterload ischemia for 10 min followed by reperfusion for 20 min reduces the ability of the heart to recover mechanical cardiac function, and that no-afterload ischemia for more than 20 min results in no recovery of mechanical function during reperfusion (Ichihara and Abiko, 1983; Hara et al., 1990). Therefore, no-afterload ischemia for 15 min was enough to irreversibly damage mechanical function. It should be pointed out that no-afterload ischemia in the perfused working rat heart produces severer ischemic damage than no-flow ischemia in the Langendorff perfused heart (Nasa et al., 1990a). In the ischemic heart, there were decreases in the levels of ATP, ADP, total adenine nucleotides, creatine phosphate and energy-charge potential, and increases in the levels of AMP, lactate and FFA. The levels of these metabolites that had been altered by ischemia returned toward their preischemic levels during reperfusion, except for the levels of FFA, which increased further during reperfusion. The observation that FFA accumulated during both ischemia and reperfusion reflects that there is both ischemia-induced and reperfusion-induced damage, because the accumulation of FFA in the ischemic-reperfused myocardium is considered to be due to an abnormal degradation of membrane phospholipids. According to Van Bilsen et al. (1989), there is a close relation between the level of FFA and the amount of tissue lactate dehydrogenase released during reperfusion. Our previous study also demonstrated the accumulation of FFA during ischemia and during reperfusion, although the increase in arachidonic acid during ischemia in the previous study was more prominent than in the present study because of a longer ischemic period (20 min) (Nasa et al., 1990b). During reperfusion, however, the level of arachidonic acid increased more in the present study, suggesting that the experimental protocol of the present study (i.e. ischemia for 15 min followed by reperfusion for 20 min) is suitable to evaluate reperfusion-induced damage. There are reports showing that both pindolol and timolol have a cardioprotective action in the ischemic myocardium in vivo (Lefer et al., 1977; Grover et al., 1988). Nevertheless, the results of the present study with the drugs clearly demonstrated that pindolol but

not timolol protected the heart from ischemic damage in terms of cardiac mechanical function; mechanical function recovered, albeit incompletely, in the presence of pindolol but not in the presence of timolol. Metabolic data for high-energy phosphates (ATP, ADP, AMP and energy-charge potential) support the beneficial effect of pindolol on the ischemic myocardium; pindolol attenuated the changes in high-energy phosphates induced by ischemia. Timolol, however, did not attenuate the metabolic changes caused by ischemia. Since the energy demand of the heart before induction of ischemia is an important factor in determining the degree of protection of the heart against ischemic damage, the degree of cardiodepression induced by pindolol before ischemia may be responsible for its cardioprotective effect against ischemia. According to Lefer et al. (1977), the mechanism of the protective effect of timolol appears to be via a reduction in myocardial oxygen demand, by reducing heart rate in the cat ischemic myocardium. In the present study, timolol did not decrease mechanical function markedly. This is probably one of the reasons for the ineffectiveness of timolol on ischemia reperfusion damage of mechanical function. It is of interest that pindolol, but not timolol, prevented the accumulation of FFA during ischemia. Because the ischemia-induced accumulation of tissue FFA is related to the level of ATP and AMP (Van Bilsen et al., 1989; Hara et al., 1990), it is likely that pindolol attenuated the ischemia-induced changes in the levels of ATP and AMP and hence prevented the accumulation of FFA during ischemia. After reperfusion, the levels of tissue metabolites, except for FFA, that had been altered by ischemia returned toward the preischemic levels. The results of the present study suggest that the reperfusion-induced recovery of these metabolites was accelerated by pindolol, and the pindolol-induced acceleration was more prominent when pindolol was applied to the heart during the entire period of reperfusion. The presence of pindolol in the perfusing solution during both preischemia and reperfusion seems important to produce the cardioprotection more effectively, because our preliminary experiment failed to demonstrate the protective action of pindolol when it was applied to the heart only during the preischemic period. Changes in the level of lactate caused by ischemia and reperfusion were not affected by pindolol or timolol, except for the change during reperfusion with timolol; the level of lactate after reperfusion was significantly high in the timolol : RB group. The high level of lactate could produce tissue acidosis and hence a reduction in myocardial contractility. This may explain why timolol did not accelerate the recovery of cardiac mechanical function during reperfusion. The low level of creatine phosphate in the timolol:RB group may also explain the reason for the smaller extent of recov-

179 ery of mechanical function during reperfusion with timolol. The results also demonstrated that timolol as well as pindolol prevented the reperfusion-induced accumulation of FFA such as linoleic and arachidonic acids, indicating that not only pindolol but also timolol may have a membrane-protective action against reperfusion-induced membrane damage, although the latter is less potent. Interestingly, timolol as well as pindolol prevented the loss of adenine nucleotides during reperfusion, but not during ischemia. This preservation in the level of total adenine nucleotides might be due to inhibition of membrane damage induced by the drug during reperfusion. The effect of timolol on myocardial levels of FFA and total adenine nucleotides during reperfusion, however, may not contribute to the cardioprotective effect, because timolol did not cause recovery of cardiac mechanical function during reperfusion. It is suggested, therefore, that the inhibitory effect of pindolol against both ischemia- and reperfusion-induced metabolic damage may not be a primary cause of the cardioprotective effect of pindolol. Accordingly, there should be another effect by which pindolol produces the cardioprotective effect on the ischemic-reperfused heart. Acceleration of the recovery of myocardial high-energy phosphates such as ATP, energycharge potential and creatine phosphate by pindolol during reperfusion seems more important for producing the cardioprotective effect of pindolol on the ischemic-reperfused heart. One of the possible mechanisms by which pindolol produces the cardioprotective effect is its /3-adrenoceptor antagonistic action. The /3-adrenoceptor antagonistic action of pindolol, however, is not likely to contribute to the cardioprotective effect, because pindolol protected but timolol did not protect the ischemic-reperfused heart in terms of mechanical function. Pindolol and timolol both have similar/3-adrenoceptor antagonistic action on a molar basis (6 times the potency of propranolol), 50 #M being enough to produce the /3-adrenoceptor antagonistic action for both drugs (Kaumann and Blinks, 1980; McDevitt, 1983; Wood and Tenn, 1984; Abrahamsson, 1986). However, the inhibitory effect of pindolol on the tissue accumulation of FFA during reperfusion may be related to its /3-adrenoceptor antagonistic action, because timolol also inhibited the reperfusion-induced accumulation of FFA. The second possibility is the membrane stabilizing action of pindolol. It has been suggested that the cardioprotective effects of/3-adrenoceptor antagonists are related to the /3-adrenoceptor antagonistic activity a n d / o r membrane stabilizing action (Marie et al., 1989; Takeo et al., 1990). Nevertheless, the membrane stabilizing action of pindolol is weak, and therefore it seems doubtful that this action of pindolol contributes to its cardioprotective effect.

Pindolol has a potent intrinsic sympathomimetic activity, but timolol does not. A contribution of intrinsic sympathomimetic activity to the cardioprotective effect, however, has never been demonstrated (Lange et al., 1984). Marie et al. (1989) failed to demonstrated a favorable effect of pindolol on the heart subjected to anoxia followed by reoxygenation. We cannot explain the difference between the results of Marie et al. (1989) and ours at presents, but it may be due to a difference in the concentration of pindolol; Marie et al. (1989) used 0.5 /~M pindolol, which was 100 times lower than the concentration used in the present study. Because the intrinsic sympathomimetic activity of pindolol can be defined by the drug-induced increase in the activity of cardiac mechanical function (such as heart rate), which is inhibited by /~-adrenoceptor antagonists without intrinsic sympathomimetic activity (Abrahamsson, 1986; Aelling, 1983), we examined whether timolol reduced the pindolol-induced functional recovery during reperfusion. When the heart was treated with both pindolol and timolol, mechanical function recovered less than when the heart was treated with pindolol alone, indicating that timolol reduced the pindolol-induced functional recovery during reperfusion. It is suggested, therefore, that the intrinsic sympathomimetic activity of pindolol possibly contributes to its postischemic functional recovery. Recent findings have suggested that an improvement of glycolysis or a stimulation of glucose utilization contributes to the improvement of postischemic functional recovery (Lopaschuk et al., 1988, 1990; McVeigh and Lopaschuk, 1990; Lewandowski et al., 1991). Reperfusion of ischemic myocardium enhances glucose uptake and utilization (Myears et al., 1987). It is suggested, therefore, that pindolol may stimulate myocardial /3-adrenoceptors temporarily because of its intrinsic sympathomimetic activity, resulting in accelerated glycolysis and hence improvement in the myocardial energy state during reperfusion. The results clearly show that pindolol increased the levels of ATP, energy-charge potential and creatine phosphate in the heart during reperfusion. Accordingly, accelerated glycolysis may lead to an elevation of cytosolic ATP concentration, which can be used preferentially at cell membranes as an energy substrate for ionic pumps to maintain the cellular ion homeostasis (Bricknell et al., 1981). It is of interest to note that stimulation of glucose utilization may not be beneficial during ischemia but beneficial during reperfusion (McVeigh and Lopaschuk, 1990), and therefore the intrinsic sympathomimetic activity of pindolol may contribute to its cardioprotective effect during reperfusion but not during ischemia. Pindolol also causes vasodilation (Clark et al., 1982; Aellig, 1983; McDevitt, 1983), because it stimulates vascular /~2-adrenoceptors (Abrahamsson, 1986).

180 T h e r e f o r e p i n d o l o l m a y i n c r e a s e o x y g e n s u p p l y to t h e p o s t i s c h e m i c m y o c a r d i u m u p o n r e p e r f u s i o n by d i l a t i n g c o r o n a r y vessels. M a r s h a l l a n d P a r r a t t (1976) o b s e r v e d that /3-adrenoceptor antagonists with intrinsic sympat h o m i m e t i c activity s e e m to h a v e a b e n e f i c i a l a c t i o n when administred after coronary occlusion. Conseq u e n t l y , it is likely t h a t t h e i n t r i n s i c s y m p a t h o m i m e t i c activity o f p i n d o l o l , at l e a s t in p a r t , m a y b e r e s p o n s i b l e for t h e c a r d i o p r o t e c t i v e e f f e c t o f t h e d r u g , e s p e c i a l l y in the postischemic myocardium. I n c o n c l u s i o n , t h e r e s u l t s s h o w t h a t t h e r e is a d i f f e r e n c e in t h e c a r d i o p r o t e c t i v e e f f e c t o f p i n d o l o l a n d timilol; pindolol prevented both ischemic damage and reperfusion damage and accelerated the recovery of cardiac mechanical function and tissue metabolites d u r i n g r e p e r f u s i o n , b u t t i m o l o l did n o t p r e v e n t isc h e m i c d a m a g e a n d did n o t a c c e l e r a t e f u n c t i o n a l rec o v e r y . T h e r e s u l t s also i n d i c a t e t h a t t h e b e n e f i c i a l effect of pindolol on recovery of tissue metabolites s u c h as A T P , e n e r g y - c h a r g e p o t e n t i a l a n d c r e a t i n e p h o s p h a t e d u r i n g r e p e r f u s i o n is m o r e p r o m i n e n t w h e n t h e d r u g is a p p l i e d to t h e h e a r t d u r i n g t h e e n t i r e reperfusion period. This beneficial effect of pindolol d u r i n g p o s t i s c h e m i c r e p e r f u s i o n m a y b e r e l a t e d , at l e a s t in p a r t , to its i n t r i n s i c s y m p a t h o m i m e t i c activity.

Acknowledgements We are grateful to Mr. Tadahiko Yokoyama for his technical assistance, to Mrs. Junko Nakata for her secretarial work and to Miss Aoi Rikiyama for her excellent assistance in making the illustrations. We also thank Dr. Akiyoshi Hara for his valuable advise and Sandoz Yakuhin Co. Ltd., Tokyo, Japan, for their gift of pindolol.

References Abrahamsson, T., 1986, The /31- and /32-adrenoceptor stimulatory effects of alprenol, oxprenolol and pindolol: a study in the isolated right atrium and uterus of the rat, Br. J. Pharmacol. 87, 657. Aellig, W.H., 1983, Clinical pharmacology of fl-adrenoceptor blocking drugs possessing partial agonist activity, with a special reference to pindolol, J. Cardiovasc. Pharmacol. 5, S16. Bricknell, O.L., P.S. Daries and L.H. Opie, 1981, A relationship between adenosine triphosphate, glycolysis and ischemic contracture in the isolated rat heart, J. Mol. Cell. Cardiol. 13, 941. Clark, B.J., K. Menninger and A. Bertholet, 1982, Pindolol - The pharmacology of a partial agonist, Br. J. Clin. Pharmacol. 13, 1498. Frishman, W., J. Kostis, J. Strom, M. Hossler, U. Elkayam, 8. Goldner, R, Silverman, R. Davis, J. Weinstein and E. Sonnenblick, 1979, Clinical pharmacology of the new beta-adrenergic blocking drugs: Part 6: a comparison of pindolol and propranolol in treatment of patients with angina pectoris: the role of intrinsic sympathomimetic activity, Am. Heart J. 98, 526. Grover, G.J., J.B. Kostis, H.R. Weiss, J.K. Li, T. Kovacs and J. Kedem, 1988, Effect of beta-adrenoceptor stimulation or blockade on regional myocardial function and regional O 2 consump-

tion during myocardial ischemia, Arch. Int. Pharmacodyn. Ther. 291, 68. Gutmann, I. and A.W. Wahlefeld, 1974, L-Lactate. Determination with lactate dehydrogenase and NAD, in: Methods of Enzymatic Analysis, ed. H.U. Bergmeyer (Academic Press, New York) p. 1464. Hara, Y., K. Nakamura, Y. Nasa, K. Ichihara and Y. Abiko, 1990, Changes in myocardial nonesterified fatty acids during ischemia and reperfusion in isolated, perfused, working rat hearts, Heart Vessels 6, 21. Ichihara, K. and Y. Abiko, 1983, Effects of diltiazem and propranolol on irreversibility of ischemic cardiac function and metabolism in the isolated perfused rat heart, J. Cardiovasc. Pharmacol. 5, 745. Kaumann, A.J. and J.R. Blinks, 1980, Stimulant and depressant effects of/3-adrenoceptor blocking agents on isolated heart muscle, Naunyn-Schmiedeb. Arch. Pharmacol. 311, 205. Lamprecht, W., P. Stein, F. Heinz and H. Weisser, 1974, Creatine phosphate. Determination with creatine kinase, hexokinase, and glucose-6-phosphate dehydrogenase, in: Methods of Enzymatic Analysis, ed. H.U. Bergmeyer (Academic Press, New York) p. 1777. Lamprecht, W. and I. Trautschold, 1974, Adenosine-5-triphosphate. Determination with hexokinase and glucose-6-phosphate dehydrogenase, in: Methods of Enzymatic Analysis, ed. H.U. Bergmeyer (Academic Press, New York) p. 2101. Lange, R., M.S. Nieminin and R.A. Kloner, 1984, Failure of pindolol and metoprolol to reduce the size of non-perfused infarcts in dogs using area at risk techniques, Cardiovasc. Res. 18, 37. Lefer, A.M., J.R. Cohn and G.H. Osman, Jr., 1977, Protective action of timolol in acute myocardial ischemia, European J. Pharmacol. 41,379. Lewandowski, E.D., D.L. Johnston and R. Roberts, 1991, Effects of inosine on glycolysis and contracture during myocardial ischemia, Circ. Res. 68, 578. Lopaschuk, G.D., S.R. Wall, P.M. Olley and N.J. Davies, 1988, Etomoxir, a carnitine palmitoyltransferase I inhibitor, protects hearts from fatty acid-induced ischemic injury independent of changes in long chain acylcarnitine, Circ. Res. 63, 1036. Lopaschuk, G.D., M.A. Spafford, N.J. Davies and S.R. Wall, 1990, Glucose and palmitate oxidation in isolated working rat hearts reperfused after a period transient global ischemia, Circ. Res. 66, 546. Marie, P.Y., F. Zannad, M. Parisot and R.J. Royer, 1989, Role of ancillalry properties of /3-adrenoceptor antagonists in protecting the heart from anoxia, Eur. J. Pharmacol. 163, 337. Marshall, R.J. and J.R. Parratt, 1976, Comparative effects of propranolol and practolol in the early stages of experimental canine myocardial infarction, Br. J. Pharmacol. 57, 295. McDevitt, D.G., 1983, /3-Adrenoceptor blocking drugs and partial agonist activity, Drugs 25, 331. McVeigh, J.J. and G.D. Lopaschuk, 1990, Dichloroacetate stimulation of glucose oxidation improves recovery of ischemic rat heart, Am. J. Physiol. 259, H1079. Myears, D.W., R.E. Sobel and S.R. Bergmann, 1987, Substrate use in ischemic and reprefused canine myocardium: quantitative considerations, Am. J. Physiol. 253, H107. Nasa, Y., K. Ichihara and Y. Abiko, 1990a, Myocardial non-esterifled fatty acids during normoxia and ischemia in the Langendorff and working rat hearts, Jap. J. Pharmacol. 53, 129. Nasa, Y., K. Ichihara and Y. Abiko, 1990b, Both d-cis- and I-cis-diltiazem have anti-ischemic action in perfused, working rat heart, J. Pharmacol. Exp. Ther. 255, 680. Rasmussen, M.M., K.A. Reimer, R.A. Kloner and R.B. Jennings, 1977, Infarct size reduction by propranolol before and after coronary ligation in dogs, Circulation 56, 794. Reimer, K.A., M.M. Rasmussen and R.B. Jennings, 1973, Reduction

181 by propranolol of myocardial necrosis following temporary coronary artery occlusion in dogs, Circ. Res. 33, 353. Takeo, S., H. Yamada, K. Tanonaka, M. Hayashi and N. Sunagawa, 1990, Possible involvement of membrane-stabilizing action in beneficial effect of beta adrenoceptor blocking agents on hypoxic and posthypoxic myocardium, J. Pharmacol. Exp. Ther. 254, 847. Tamargo, J. and E. Delpon, 1990, Optimization of /3-blockers' pharamcology, J. Cardiovasc. Pharmacol. 16 (Suppl. 5), S10.

Van Bilsen, M., G.J. Van der Vusse, P.H.M. Willemsen, W.A. Coumans, T.H.M. Roemen and R.S. Reneman, 1989, Lipid alterations in isolated, working rat hearts during ischemia and reperfusion: its relation to myocardial damage, Circ. Res. 64, 304. Wood, A.J.J. and N. Term, 1984, Pharmacologic differences between beta blockers, Am. Heart J. 108, 1070.