ischaemic preconditioning of the human heart. - Leicester Research ...

espectively. Diazoxide lost its protective effect at doses
-500 jiM and pinacidil at doses ýI mM. At the above
optimal doses, the greatest degree of protection was
afforded when the KATP openers was applied for 10 min
before ischernia (pretreatment) followed by wash-out.
Glibenclamide abolished the protective effects of pre-
conditioning at doses ý! 10 RM. A dose response analysis
for 5-HD was performed between concentrations of 0 and
10 mM. The minimal effective concentration for 5-HD
which abolished protection afforded by preconditioning
was I mM. The dose-response analysis for HMR 1883
revealed that concentrations between the range 0 to 100
ýLM had no effect on the protection afforded by precondi-
tioning. Again pretreatment with these drugs for 10 min
before ischernia was the most effective protocol.
Fig. 2 shows that ischemia alone resulted in a significant
increase in CK leakage and that preconditioning complete-
ly reversed the effect of ischernia so that CK leakage was
similar to that seen in the aerobic control group. It also
shows that glibenclamide (10 RM), which
S. Ghosh et al. / Cardiovascular Research 45 (2000) 934-940
blocks KITP
channels both in the sarcolemma and the mitochondrial
inner membrane, partially blocked the beneficial effect of
preconditioning on CK leakage. 5-HD is a KATP channel
blocker which appears to show selectivity for mitoKATP
channels over sarcolemmal KATP*
Thus 5-HD has been shown to block KATP channels in
isolated rnitochondria [181 but did not affect sarcolemmal
KATP currents activated by cromakalim [191. In isolated
r_
0
cn
Co
Je
(0
9
8
7
2
1
0
Aerobic
Control
Ischemia
Alone
937
rabbit cardiac myocytes, Sato et al. [20] have recently
shown that 5-HD inhibited oxidation in response to the
opener pinacidil, but did not block the sarcolemmal K,,,
current activated by the same opener, consistent with 5-HD
showing selectivity for blocking of mitoKITP over sar-
colernmal KATP, Fig. 2 also shows that 5-HD (I mM)
abolished the protective effect of PC on CK leakage in
human myocardium. We also used the novel sul-
phonylthiourea HMR 1883, which is thought to have the
reciprocal selectivity to 5-HD, preferentially blocking the
sarcolemmal KATP channel [21,22]. HMR 1883 did not
block the protective effect of preconditioning.
The results in Fig. 2 also show the effects of pretreat-
ment with KATP channel openers in the absence of an
ischemic preconditioning stimulus. Both the non-selective
KATP channel opener, pinacidil and the selective opener of
mitoKATP channels, diazoxide were protective; with
diazoxide reducing CK leakage to levels not significantly
different from those obtained with PC itself and pinacidil
exhibiting a less potent effect than PC. In this connection,
it is worth noting that Garlid et al. [13] found that
diazoxide was around 2000-fold more potent at opening
mitoKATP than cardiac sarcolemmal KATP channels.
Fig. 3 shows the results of MTT reduction. In essence, it
reveals a mirror image of the results with CK leakage in
the aerobic control, ischernia alone and preconditioning
groups. Thus, ischernia alone caused a five-fold decrease in
MTT reduction values to those seen in the aerobic control
Alone Glibenclamide 5-HD HMR 1883 Pinacidil Diazoxide
I Preconditioning
Pig. 2. Leakage of creatinine kinase (CK) into the media during the 120-min reperfusion period (last 120 min in the aerobic control period). Data are
expressed as mean±S. E. M. of six experiments. * P