European Jou~al oj Pharmaeoio~, 191 (1990) 11-18

Elsevier EJP 51584

Reduction of ischemic damage in isolated rat hearts by the opener, I GaryJ. Grover, Steven Dzwonczyk and Paul G. Sleph Department of Pharmocologv, The Squibb Institutefor Medical Research, Route 206 and Provinceline Rod Princeton, NJ 08543-4OOQU.S.A.

P.O. Box 4000,

Received 8 March 1990. revised MS received 13 July 1990, accepted 28 August 1990

Potassium channel activators have been shown to protect ischemic myocardium. We studied the ability of the novel potassium channel activator, RP 52891, to also reduce ischemic damage in isolated globaily ischemic rat hearts. RP 52891 (l-100 FM) was given before the hearts were subjected to 25 min of &hernia and 30 min of r~rfusion. Before

ischemia, RP 52891reduced contractile function only at the highest concentration (100 PM). Significant reductions in ischernic damage were observed at 3 FM and higher concentrations. RP 52891 improved reperfusion contractile function and reduced lactate dehydrogenase release. Contracture was significantly reduced by RP 52891 during reperfusion. The protective effects of RP 52891 were completely reversed by glyburide and sodium S-hydroxydecanoate, both blockers of ATP-sensitive potassium channels. Thus, RP 52891 has direct cardioprotective efficacy, which may be related to activation of ATP-sensitive potassium channels. RP 52891; Glyburide; 5-Hydroxydecanoate,

sodium; Myocardial ischemia; K+ channel activators

Potassium channel activators are novel pharmacologic agents which may be efficacious in the treatment of hypertension and several other diseases. (Kardel et al., 1981; Cook et al., 1988). Their utility stems from their ability to hyperpolarize smooth muscle and thus reduce calcium influx through voltage-operated channels (Hamilton et al., 1986; Weir and Weston, 1986). Potassium channel activators also open potassium channels in myocytes, resulting in action potential duration (APD) shortening (Cain and Metzler, 1985; S~guinetti et al., 1988). It has been shown

Correspondence to: G.J. Grover, Department of Pharmacology, The Squibb Institute for Medical Research, Route 206 and Provinceline Road, P.O. Box 4000, Princeton, NJ 085434000. U.S.A. ~14.2999/90~~03.50

that ~rom~alim and pinacidil can open myocardial ATP-sensitive potassium channels, which are normally inhibited by physiological levels of ATP (Noma and Shibasaki, 1988; Escande et al., 1989). It has been suggested that these channels may open during periods of low ATP concentration such as seen during ischemia or hypoxia and may account for some of the potassium efflux seen during ischemia. This reasoning has led some investigators to speculate that blockers of myocardial ATP-sensitive potassium channels may result in myocardial protection as well as ~ti~h~~c activity and that activators may be contraindicated during ischemia {Fossett et al., 1988). We tested this concept using pinacidil and cromakalim and found them to protect ischemic/ reperfused myocardial tissue (Grover et al., 1989a). This was done with a buffer-perfused rat heart model of global ischemia and reperfusion. We also found that the ATP-sensitive potassium channel

b 1990 Ekevier Science Publishers B.V. (Biomedical Division)

de. did not affect ischemia when completely reversed the protective alone effects of pi~acidil and cromakalim. Another tassimn channel activator, RP 49356, which is s~~t~~~ different from cromakalim has recently been reported to open cardiac ATP-sensitive ~ot~i~ channels (Escande et al., 1989). Tbe purpose of the present study was to determine the anti-ischemic profile of its active enantiomer, RP 52891, in isolated perfused rat hearts. We also ned if the protective effects of this comcan be reversed by the ATP-sensitive potassium channel blockers, 8lyburide and sodium 5-hy~oxyd~~~te (HD). er. gly

Male Sprague-Dawley rats (450-550 g) were used in all in vitro experiments. The rats were anesthetized using 100 mg/kg sodium pentobarbid (i.p.), They were intubated then treated with iv. heparin (IO00 U/kg). While the rats were being mechanically ventilated, their hearts were perfused iu situ via retrograde cannulation of the aorta. The hearts were then excised and quickly moved to a Langendorff apparatus where they were perfused with Krebs-Henseleit bicarbonate buffer (mM: 112 NaCl, 25 NaHCO,, 5 KCl, 1.2 MgSQ, 1 KH,POd, 1.25 CaCi,, 11.5 dextrose and 2 pyruvate bubbled with 95% C&5% COz) at a constant perfusion presstire (75 mm Hg). A water-filled latex balloon attached to a metal cannula was then inserted into the left ventricle and connected to a Statham pressure transducer for measurement of left ventricular pressure. The hearts were allowed to equilibrate for 15 min then end diastolic pressure (EDP) was adjusted to 5 mm Hg and this balloon volume was maintained

for the duration of the experiment. Pre-ischemia or pre-drug function, heart rate and coronary flow (extraco~orea1 electromagnetic flow probe, Carolina Medical Electronics, King, NC) were then measured. Cardiac function was determined using LVDP which was calculated from the difference between left ventricular peak systolic pressure and EDP (Watts and Maiorano, 1987). Cardiac temperature was kept constant throughout the experiment by submerging the hearts in 37*C buffer which was allowed to accumulate in a stoppered, heated chamber. For the concentration-response determination, the hearts were treated with 1, 3, 7, 10, 30 or 100 PM RP 52891 (n = 4 for each concentration) or vehicle (0.04% DMSO, n = 8) added to the perfusate. The drug treatment was begun 10 min before the start of ischemia. Global &hernia was initiated by completely shutting off the perfusate flow and maint~ning this for 25 min. Reperfusion was then begun without drug and the hearts were allowed to recover for 30 min. Reperfusion cardiac function and flow were then measured. At this time the reperfusion effluent was sampled for cumulative lactate dehydrogenase (LDH) release as previously described (Hamm and Opie, 1983). LDH concentrations were determined using a kit supplied by Boerhinger Mannheim based on the technique of Wroblewski and La Due (1955). LDH release was expressed as U/g pre-ischemia heart weight for the 30~tin collection period. LDH release is a sensitive index of loss of cell viability (Van der Laarse et al., 1979). To test whether RP 52891 reduces the severity of ischemia via potassium channel activation, we determined if glyburide and HD can reverse its anti-ischemic effects. To accomplish this, we treated hearts with 1 PM glyburide alone (n = 7) or in combination with 100 FM RP 52891 (n = 4). q.s Me

0 Cl

----r-YNa OH giyburide

0

sodium Shydroxydecanoate

Fig. 1. The chemical structures of glyburide, sodium 5.hydroxyde~~~te

RP 52891

and RP 52981.

13

We also treated hearts with 100 FM HD (n = 4) alone or in combination with 30 (n = 4) or 100 pM (n = 4) RP 52891. Differences between groups were assessed using an analysis of variance. Multiple comparisons were done using the Dunnett procedure. All values are expressed as the mean + S.E. The chemical structures of the compounds used are shown in fig. 1.

3. Results The effects of RP 52891 on cardiac function and coronary flow are shown in table 1. Neither drug treatment nor ischemia had any effect on heart rate whenever it was measured. Before TABLE 1 The effects of RP 52891 (RP) on cardiac function and coronary flow before and after global &hernia. AU vahtes are means& S.E. Pre-ischemia Pre-drug

30 min postreperfusion

10 min post-drug

LVDP (mm Hg)

Vehicle (n = 8) luMRP(n=4) 3pMRP(n=4) lOpMRP(n=4) 30pMRP(n=4) 100 /LM RP (n = 4)

136 139 144 143 143

+ + * f +

4 6 5 6 4

146 + 6

130 124 133 130 127

rt + + f +

4 4 5 6 4

26 37 53 70 64

80 rt: 6 a*b

+4a + 8’ + 9 nxb + 7 Gb f 6 “_.b

74 + 4 a*b

HR (beats/min)

Vehicle (n = 8) 251 + 6 lpMRP(n=4) 289 f12 3pMRPln-44) 273 cl4 lOgMRP(n=4)278 +I1 30pM RP(n =4) 258 * 5 100 pM RP (n =4) 255 f16 CoronaryflowfmI/min per g) 18.7& 0.4 Vehicle (n = 8) 1 CIM RP(n =4) 22.0f 2.2 3 pM RP (n = 4) 22.72 2.1 10 PM RP(n = 4) 22.4k 1.6 30 pM RP(n =4) 16.4+ 1.2 100 PM RP (n = 4) 20.3k 1.6

245 270 256 272 253

+ 6 414 t15 +12 + 6

231 265 255 265 252

245 +ll

ltl0 +1X -+23 &ll f 9

260 +13

16.31t: 24.Ok 25.‘7* 26.85 21.2*

0.9 3.1 b 2.9 b 2.6 ’ 1.9 asb

ll.l+ 13.3& 15.2k 16.4+ 12.2+

0.8” 1.3 a 3.1 a 1.5 a.b 1.5

26.9f

3.7 b

14.0f

2.9

a Sig~ficantiy different from its respective pre-drug value (P -Z0.05). b Significantly different from its respective vehicletreated group value (P -z 0.05).

ischemia, RP 52891 only si~ficantly reduced LVDP at 100 PM. LVDP during reperfusion, was sig~fic~tly lower than the pre-ischemic values m vehicle-treated hearts. RP 52891 improved reperfusion LVDP in a concentration-dependent manner up to the 10 FM concentration in which no further improvements were observed. RP 52891 also increased coronary flow before ischemia and improved reflow after the ischemic episode. The reperfusion EDP was markedly elevated in vehicle-treated hearts, indicating contracture (fig 2). RP 52891 si~~c~tly reduced reperfusion EDP at 10 FM and higher concentrations. Significant LDH release was observed in ve~cl~treat~ hearts (fig. 2) and RP 52891 reduced the release of this enzyme in a ~ncentration-dependent manner. Additional experiments were designed to determine the importance of the coronary flow increases observed for RP 52891 for mediation of its cardioprotective effects. When pre-ischemic flow was held constant at 16.5 4: 0.7 ml/min per g and reperfusion flow was held at 12.0 f 0.8 ml/n-tin per g (flows similar to vehicle, see tables) 30 gM RP 52891 significantly improved LVDP (77 + 6 mm Hg) and reduced LDH release (11 + 2 U/g} during reperfusion. These values were not significantly different from those for hearts treated with 30 PM RP 52891 under constant pressure conditions. We tested whether the ATP-sensitive potassium channel blocker, HD, could reverse the protective effects of RP 52891 to see if RP 52891 was protecting the ischemic myocardium by opening potassium channels. We found that 300 PM HD alone had no effect on cardiac function and coronary flow before ischemia (tab!e 2). HD significantly reduced reperfusion contractile function, indicating some pro-ischemic activity (fig. 3 and table 2). HD alone had no effect on LDH release. When given in combination with 30 CM RP 52891, HD completely reversed the protective effects of the potassium channel opener on reperfusion function and LDH release. When given with 100 FM RP 52891, HD also significantly reduced its cardioprotective efficacy, though the protective effect of RP 52891 on reperfusion function was only partially reversed. The pre-ischemic cardiodepressant effect of 100 FM RP 52891 was

TABLE 2 The effects of RP 52891 (RP), and/or sodium 5-hydroxydecanoate (HD) on cardiac function and coranary flow before and after global &hernia. All values are mean& S.E. 30 mm postreperfusion

Pre-ischemia Pre-drug LVDP (mm Hg) Vehicle (n = 8) 100 prM HD (n=4) 30 gM RP (n=4) 1OOpM RP (n=Q) 30 /.tM RPI100 pM HD (n=4) lOO/.tM RP-t100 ).iM HD (n-4) HR fbenrs/mm) Vehicle (n = S) 100 pM HD (n = 4) 30 PM RP

v

30pM

Fig. 2. The effect of RP 52891 on cumulative repetfusion LDH r&ease and end diastolic pressure (EDP). These parameters were measured 30 min after the initiation of reperfusion. Asterisks denote differences fnxn ~e~cIe-tr~t~ hearts (V, P =z0.05).

not reversed by HD. We also tested the effects of glyburide, another blocker of the ATP-sensitive potassium channel blocker. At a concentration of 1 FM, glyburide alone did not affect pre-ischernic cardiac function or coronary flow. Glyburide significantly reduced reperfusion heart rate and

10 rnin post-drug

136

f4

130

* 4

128

+5

128

+ 6

143

*4

127

f

4

64

146

+6

80

f

6 a*b

74 f

4 z&b

141

i3

129

*

3

39 f

8”

130

98

f

3 a.b

50

+loa

251

245

+ 6

231

*IO

271

263

f

138 f61

253

+. 6

252

f

245

+I1

260

f13

275

f

228

+20

268

fll

252

+ 3

258 (n = 4) 100 FM RP (n = 4) 255 30gM RP+ 100 pM HD (n=4) 282 +6 10 pM RP+ 100 /rM ND (n =4) 274 f10 Caronuryflow {ml/min per g) Vehicle (n = 8) 18.7f 0.8 100 j&l HD (n = 4) 15.2& 2.2 30 PM RP cri = 4) w.4* 1.2 1rH)pM RP (n-4) 20.3f 1.6 30 pM RP+ 100 pM HD (n=4) 15.0a 1.5 100 gM RP+ 100 pM HD (n=4) 17.9f 2.8

7

6

26

f

4

6 4 3 Osb I

6 =&

9

16.3*

0.9

17.9f

1.9

21.2+

1.9 aB

12.2f

1.5

26.9f

3.7 ’

14.w

2.9

18.5-t

1.5

7.2-f

0.5 ab

21.44

3.9

10.5 f

2.9 =

ll.lf 7.7 f

O.Ba 1.9 a

a significantly different from its respective pm-drug value (P +z0.85). b Significanrty different from its respective vehicfem treated group value (P or 0.05).

15 LDH (U/g) 30

20

I *

-

10

,

0 V

1OOFM HD

30$4

RP

lOOphARP

30pM RP+HD lOOphARP+HD

Fig. 3. The effect of RP 52891 (RP) or sodium 5-hydroxydecanoate (HD, 100 pM) on cumulative reperfusion LDH release. This was measured 30 min after the initiation of reperfusion. Asterisks denote differences from vehicle-treated hearts (V, P c 0.05).

LVDP when given alone (table 3) although it had no effect on LDH release (fig. 4). When given in combination with 100 PM RP 52891, glyburide reversed its pm-ischemic cardiodepressant effects. Glyburide also reversed the protective effects of RP 52891 on ischemic/reperfused hearts.

4. Discussion It has been shown that several structurally diverse ~mpo~ds can open potassinm channels in smooth and cardiac muscle (Imanishi et al., 1983; Cook et al., 1988; Smallwood and Steinberg, 1988). Cromakalim, pinacidil and RP 49356 have been shown to open ATP-sensitive potassium channels in myocytes. Previous studies from our laboratory have shown that both cromakalim and pinacidil can reduce ischemicfreperfusion injury in isolated perfused rat hearts (Grover et al., 1989a). We showed that the protective effects of these compounds were completely reversed by glyburide, which is thought to block cardiac ATP-sensitive potassium channels (Fossett et al., 1988). It was also noted that glyburide alone had no effect on the severity of ischemic damage, indicating a paucity of open chamrels in this ischemia model. We

also showed that both cromakalim and pinacidil can reduce infarct size in a canine model of ischemia and reperfusion (Grover et al., 1989b). At the present time little is known about the cardioprotective effects of other potassium channel activators, but it would be useful to learn as much as possible about all of the currently available compounds. A new compound, RP 49356, has recently been reported to open ATP-sensitive potassium channels in guinea-pig myocytes and thus it may also protect ischemic cardiac tissue (Escande et al., 1989). RP 49356 has been shown to selectively open ATP-sensitive potassium channels in cardiac myocytes. It was found to activate a time-independent outward potassium current and this effect was fully antagonized by glyburide, a selective blocker of ATP-sensitive potassium channels (Escande et al., 1989). In inside-out patches, RP 49356 was found to open a high conductance potassium channel which could be blocked by ATP (Escande et al., 1989). since this compound is structurally different from cromakalim and pinacidil, we thought it would be useful to characterize it in our model of global ischemia and reperfusion in isolated rat hearts. In our experiments we used the active enantiomer of RP 49356, RP 52891.

as RP 52891 had little effect on pm-ischemic cardiac contractile function except at the highest concentration tested (100 FM). This was identical to the concentration at which cardiodepression was observed for cromakalim and pinacidil (Grover et al., 1989a). After ischemia, cardiac

LDH (U/g) 40 -

35 -

I

30 -

25 -

l-

TABLE 3 The effects of RP 52891 (RP) and/or glyburide on cardiac function and coronary flow before and after global ischemia. All vahles are means + SE. 30 mitt postreperfusion

Pre-ischemia 10 mm

prr-drug (mm

t

15 -

10 -

5-

post-drug LVDP

20-

Hg)

Vehicle (n = 8)

1 pM glyburide (n = 7) 100 pM RP (n=J) 1 pM glyburide +IOOyM RP (a=4)

136 f

4

150 + 5 146 + 6

129 i

7

o+

130 * 4

26

139 f

4

11 * 3 n*b

6 a.b

74

80

i

f4”

i

V

4 a.b

118

+ 5

245

+ 6

231

234

+ 6

157 540 a.b

245

+ll

260

*13

236

* 8

150

*5s a.b

16.3~

0.9

+lO

ll.lk

0.8 =

15.5& 1.8

13.5*

1.7a

26.9*

3.7

14.0f

2.9 a

21.7& 4.0

9.0*

2.6 a

EDP (mm Hg)

Vehicle (n = 8) 1 gM glyburide (n = 7) 100 pM RP (n = 4) 1 pM gfyburide +lOOgM RP (n = 4)

rl 1OOpM RP

RP+G

Fig. 4. The effect of RP 52891 (RP) or glyburide (G) on cumulative reperfusion LDH release. These parameters were measured 30 mitt after the initiation of reperfusion. Asterisks denote differences from vehicle-treated hearts (V. P < 0.05).

13 +6’

HR (bean/min)

251 + 6 Vehicle (n = 8) 1 u M glyburide (n = 7) 257 +10 100 ph4RP (n=4) 255 +16 1 PM glyburide +lOOpMRP (n = 4) 264 + 9 Coronq flou* (ml/min per g) Vehicle (n = 8) 18.7k 0.8 1 gM glyburide (n = 7) 20.1 f 2.0 100 pM RP (n = 4) 20.3& 1.6 1 PM glyburide +lOOpMRP (n=4) 17.5 f 2.9

1 @lG

5

5

65

f

5

5

88

+4b

5

5

35

+4b

5

5

58

f20

4

a Significrtly different from its respective pre-drug value (P < 0.05). Significantly different from all of the respective vehicle-treated group values (P < 0.05).

contractile function was markedly reduced in vehicle-treated animals and RP 52891 significantly improved reperfusion function, starting at the 3 PM concentration. Cromakalim was shown to improve reperfusion function at similar concentrations (Grover et al., 1989b). RP 52891 also significantly reduced reperfusion EDP, an indicator of contracture which may result from rigor bond formation (Tilton et al., 1985; Vogel et al., 1986; Wexler et al., 1986). RP 52891 also increased the time to the start of contracture (not shown), also used as an index of severity of ischemia (Wexler et al., 1986). Contracture occurs during ischemia and the inhibition of contracture formation indicates an effect of the drug during ischemia per se rather than a reperfusion effect. Cromakalim does not reduce the severity of ischemia when given only during reperfusion, even when reperfusion flow is increased (Grover et al., 1989b). LDH release was also significantly reduced by RP 52891. LDH release has been used as an index of cell or membrane integrity and has been shown to be closely correlated with histological tissue damage. While RP 52891 seemed to protect ischemic/ reperfused rat hearts, we also determined if this

17

was via activation of potassium channels. We thus tested if its cardioprotective effects could be reversed by potassium channel blockers. We chose two structurally dissimilar compounds reported to block ATP-sensitive potassium channels in myocardial tissue, sodium 5hydroxydecanoate and glyburide (Niho et al., 1987; Fossett et al., 1988). Alone, both blockers slightly increased the severity of ischemia, which is different from previously published results from our laboratory, showing that neither compound had any effect on ischemic damage (Grover et al., 1989a). Both blockers reversed the protective effects of RP 52891, although this effect was less pronounced at higher concentrations of RP 52891. We had found that both glyburide and HD reversed the protective effects of cromakalim and pinacidil (Grover et al., 1989b). It was interesting that glyburide reversed the cardiodepressant effects of RP 52891 in nonischemic tissue, while HD had no effect. Unpublished data from our laboratory indicates similar results for cromakalim and may suggest some degree of ischemia selectivity for the blocking actions of HD. The ATP-sensitive channels may represent a protective mechanism which the heart can use at very low intracellular ATP levels. Some potassium channels may open during ischemia as glyburide slightly worsened ischemia, indicating that opening of ATP-sensitive channels may represent a protective mechanism. The opening of this channel may inhibit ischemia-induced depolarization and thus reduce calcium entry through voltagegated calcium entry. Data from our laboratory indicated that cromakalim can prevent ischemiainduced depolarization in cardiac cells, although more work needs to be done to prove that this was its protective mechanism of action (Grover et al., 1989a). It is also possible that shortening of the action potential duration itself may also reduce calcium influx. If reduction of calcium influx is the protective mechanism of the potassium channel activators, their use would appear to be advantageous when compared to that of calcium antagonists because of their low propensity for cardiodepressant effects. The degree of protection observed for the potassium channel openers was similar to that seen for calcium channel blockers,

although the calcium blockers are typically > lofold more potent (Grover and Sleph, 1989). While pinacidil, cromakalim and RP 52891 do not seem to be negative inotropes at cardioprotective concentrations, they are still potent vasodilators and can reduce action potential duration. The vasodilator activity may result in a compromised subendocardial perfusion pressure or coronary steal (Sakamoto et al., 1989) and the APD reduction may result in an enhanced propensity for arrhythmias or fibrillation. Unfortunately the results in this respect are inconsistent with Kerr et al. (1985) finding pinacidil to reduce reperfusion arrhythmias and other groups finding pinacidil and cromakalim to be profibrillatory (Siegl et al., 1989; Uprichard et al., 1989). We have found cromakalim to be profibrillatory in isolated perfused rat hearts and anti-fibrillatory in reperfused dog hearts (Grover et al., 1989b). Thus, it might be useful to determine if potassium channel activators can be developed that would have cardiac or ischemic selectivity. It might also be useful to determine the importance of action potential shortening and to find if this feature can be minimized in second-generation potassium channel activators.

Acknowledgement We are grateful to Dr. 1. Cavero of Rhone-Poulenc Sante for his generous gift of RP 52891.

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via high affinity receptors that are linked to ATP-dependent R + channels. J. Biol. Chem. 263. 7933. Grover. G.J., JR. McCullough. D.E. Hemy, M.L. Conder and P.G. Sleph. 19S9a. Anti-ischemic effects of the potassium channel activators pinscidil and cromakalim and the reversal of these effects with the potassium channel blocker glyburide, J. Pharmacol. Exp. Ther. 251.98. Grover. G.J.. PG. Sleph. S. Dzwonczyk and C.S. Parham, 1989b. Anti-ischemic and antifibrillatory effects of the K+ channel activators cromakdim and pinacidil in dog and isolated rat hearts, Circulation 80 (Suppl. II). 499. Grow-er. G.J. and PG. Sleph. 1989. Dissociation of cardiodepression from cardioprotection with calcium antagonists: diltiazem protects ischemic rat myocadium with a lower functional cost as compared with verapamil or nifedipinr. J. Cardiovasc. Pharmacol. 14. 331. Hamilton. T.C.. S.W. Weir and A.H. Weston, 1986, Comparison of the effects of BRL 34915 and verapamil on electrical and mechanical activity in rat portal vein, Br. J. Pharmacol. S8. 103. Hanun. C.W. and L.H. Gpie. 1983. Protective effects of verapamil. nifedipine and diltiaxem in the coronary-ligated, isolated working rat heart. Circ. Res. 52 (Suppl. 1). 129. buanishi. S.. M. Arita. T. Kiyosue and M. Aomine, 1983, Effects of SG-75 (nicorandil) on electrical activity of canine cardiac purkirje: Possible increase in potassium conductance. J. Pharmacol. Exp. Tber. 225, 198. Kardel. T., T. Hilden. J.E. Carlsone and J. Trap-Jensen, 1981, N “-Cyano-N-4-pyridyl-N ‘-1.2.2-trimethyl propylguanidine a new vasodilator agent: Acute effect on blood pressure and pharmacokinetics in hypertensive patients, J. Cardiovasc. Pharmacol. 3. 1002. Kerr. M.J. and R.G. Shanks. 1985, Suppression of ventricular arrhythmias after coronary artery ligation by pinacidil, a vasodilator drug, J. Cardiovasc. Pharmacol. 7, 875. Niho. T.. T. Notsu, H. Ishikawa. H. Funato, M. Yatnazaki, I. Takahashi. 1. Tanaka, M. Kayamoto, T. Dabasaki, F. Yamas& T. Shinkawa. A. Uemura and M. Mizota, 1987, Study of mechanisms and effects‘ of sodium 5-hydroxydecanoate on experimental ischemic ventricular arrhythmias, Folia Pharmacol. Jap. 89, 155. Noma A. and T. Shibasaki. 1988. Intracelhtlar ATP and cardiac membrane currents, in Channels, Vol. 1, Chapter 5, ed. T. Narahashi (Plenum Press. New York) p. 183. Sakamoto. S., C. Liang, C.K. Stone and W.B. Hood, Jr., 1989. Effects of pinacidil on myocardial blood flow and infarct size after acute left anterior descending coronary artery

occlusion and reperfusion in awake dogs with and without a coexisting left circumflex coronary artery stenosis, J. Cardiovasc. Pharmacol. 14. 747. Sanguinetti. M.C., A.L. Scott, G.L. Zingaro and P.K.S. Siegl, 1988. BRL 34915 (cromakalim) activates ATP-sensitive K+-current in cardiac muscle, Proc. Natl. Acad. Sci. U.S.A. 85. 8360. Siegl. P., C. Wolleben, G. Zingaro and M. Sanguinetti, 1989. Effects of the ATP-sensitive potassium channel modulators, glyburide and BRL 34915 on ischemia-induced fibrillation in isolated rat hearts, FASEB J. 3, A847. Smallwood, J.K. and M.I. Steinberg, 1988. Cardiac electrophysiological effects of pinacidil and related pyridylcyanoguanidines: Relationship to antihypertensive activity, J. Cardiovasc. Pharmacol. 12, 102. Tilton. R.G., E.K. Williamson, P.A. Cole, K.B. Larson, C. Kilo and J.R. Williamson, 1985, Coronary vascular hemodynantic and permeability changes during reperfusion after no-reflow ischemia in isolated diltiazem treated rabbit hearts. J. Cardiovasc. Pharmacol. 7, 424. Van Der Laarse, A., L. Holaar and J.L.M. Van Der Volk, 1979, Release of alpha hydroxybutyrate from neonatal rat heart cell cultures exposed to anoxia and reoxygenation: Comparison with impairment of structure and function of damaged cardiac cells, Cardiovasc. Res. 13, 345. Vogel, W-M., A.W. Cerel and C.S. Apstein. 1986, Post-ischemic cardiac chamber stiffness and coronary vasomotion: The role of edema and effects of dextran, J. Mol. Cell Cardiol. 18, 1207. Uprichard, A.C.G.. L. Chi, E.M. Driscoll and B.R.. Lucchesi, 1989, Pinacidil is proarrhythmic in a conscious canine model of sudden death, J. Mol. Cell. Cardiol. 21 (Suppl. II). s. 13. Watts, J.A. and L. Maiorano, 1987, Effects of diltiazem upon globally ischemic rat hearts, European J. Pharmacol. 138, 335. Weir, S.W. and A.H. Weston, 1986, The effects of BRL 34915 and nicorandil on electrical and mechanical activity and on 56Rb efflux in rat blood vessels, Br. J. Pharmacol. 88, 121. Wexler, C.F., E.O. Weinberg, J.S. Ingwall and C.S. Apstein. 1986, Acute alterations in diastolic left ventricular chamber distensibility: Mechanistic differences between hypoxemia and ischemia in isolated perfused rabbit and rat hearts, Circ. Res. 59, 515. Wroblewski, F. and J.S. LaDue, 1955, Lactic dehydrogenase activity in blood, Proc. Sot. Exp. Biol. Med. 90, 210.