Preconditioning in immature rabbit hearts: role of KATP channels.
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BACKGROUND: The protective effects of ischemic preconditioning have been shown to occur in adult hearts of all species studied. We determined whether immature hearts normoxic or chronically hypoxic from birth could be preconditioned, the time window or memory of the cardioprotective effect, and the involvement of the KATP channel. METHODS AND RESULTS: Isolated immature rabbit hearts (7 to 10 days old) were subjected to 0, 1, or 3 cycles of preconditioning consisting of 5 minutes of global ischemia plus 10 minutes of reperfusion. This was followed by 30 minutes of global ischemia and 35 minutes of reperfusion. Normoxic hearts (FIO2=0.21) subjected to 1 cycle of preconditioning recovered 70+/-7% of left ventricular developed pressure compared with 43+/-8% recovery in nonpreconditioned controls. Three cycles of preconditioning did not result in additional recovery (63+/-8%). Hearts from rabbits raised from birth in hypoxic conditions (FIO2=0.12) and subjected to 1 and 3 preconditioning cycles did not show increased recovery (68+/-8% and 65+/-5%) compared with nonpreconditioned hypoxic controls (63+/-9%), although the recovery was greater in chronically hypoxic hearts than in age-matched normoxic controls. Increasing the recovery period after the preconditioning stimulus from 10 to 30 minutes resulted in a loss of cardioprotection. Pretreatment of normoxic hearts for 30 minutes with the KATP channel blocker 5-hydroxydecanoate (300 micromol/L) completely abolished preconditioning (70+/-7% to 35+/-9%) but had no effect on nonpreconditioned hearts (40+/-8%). CONCLUSIONS: Immature hearts normoxic from birth can be preconditioned, whereas immature hearts hypoxic from birth cannot. Preconditioning in normoxic immature hearts is associated with activation of the KATP channel. (+info)
Demonstration of a new mammalian isoleucine catabolic pathway yielding an Rseries of metabolites.
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1. Normal human urine contains small amounts (less than 4 mg/g of creatinine) of 2-ethylhydracrylic acid, formed, we believe, by a previously undisclosed endogenous catabolic pathway for the oxidation of a newly described series of R metabolites of isoleucine. 2. Urinary excretion of 2-ethylhydracrylic acid is variably increased in defects of isoleucine oxidation at distal steps in the catabolic pathway (3-oxoacyl-CoA thiolase deficiency and methylmalonyl-CoA mutase deficiency) and is diminished when proximal steps of the oxidative pathway are blocked as in branched-chain oxo acid decarboxylase deficiency ('maple-syrup-urine' disease). 3. Precursors of R-pathway metabolites [R(-)-2-methylbutyrate and 2-ethylacrylate ] lead to increased 2-ethylhydracrylate excretion in the mammal(rat, rabbit and dog); the corresponding S metabolites [S(+)-2-methylbutyric acid and tiglic acid ], when given in equimolar amounts, have little effect on its excretion, suggesting that little or no interconversion between S and R metabolites occurs in vivo. 4. Studies with 2H-labelled precursors indicate that conversion of R 2-methylbutyrate into 2-ethylhydracrylic acid occurs by a direct pathway (apparently via 2-ethylacrylic acid). 5. The further oxidation of 2-ethylhydracrylic acid to ethylmalonic acid was demonstrated, and may be analogous to S-metabolite oxidation via methyl malonate. 6. Valine metabolites do not interact with the R=isoleucine pathway under the conditions of these experiments in vivo. (+info)
Ischemic preconditioning depends on interaction between mitochondrial KATP channels and actin cytoskeleton.
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Both mitochondrial ATP-sensitive K+ (KATP) channels and the actin cytoskeleton have been proposed to be end-effectors in ischemic preconditioning (PC). For evaluation of the participation of these proposed end effectors, rabbits underwent 30 min of regional ischemia and 3 h of reperfusion. PC by 5-min ischemia + 10-min reperfusion reduced infarct size by 60%. Diazoxide, a mitochondrial KATP-channel opener, administered before ischemia was protective. Protection was lost when diazoxide was given after onset of ischemia. Anisomycin, a p38/JNK activator, reduced infarct size, but protection from both diazoxide and anisomycin was abolished by 5-hydroxydecanoate (5-HD), an inhibitor of mitochondrial KATP channels. Isolated adult rabbit cardiomyocytes were subjected to simulated ischemia by centrifuging the cells into an oxygen-free pellet for 3 h. PC was induced by prior pelleting for 10 min followed by resuspension for 15 min. Osmotic fragility was assessed by adding cells to hypotonic (85 mosmol) Trypan blue. PC delayed the progressive increase in fragility seen in non-PC cells. Incubation with diazoxide or pinacidil was as protective as PC. Anisomycin reduced osmotic fragility, and this was reversed by 5-HD. Interestingly, protection by PC, diazoxide, and pinacidil could be abolished by disruption of the cytoskeleton by cytochalasin D. These data support a role for both mitochondrial KATP channels and cytoskeletal actin in protection by PC. (+info)
Re-evaluation of the primary structure of Ralstonia eutropha phasin and implications for polyhydroxyalkanoic acid granule binding.
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Sequence analysis of several cDNAs encoding the phasin protein of Ralstonia eutropha indicated that the carboxyl terminus of the resulting derived protein sequence is different from that reported previously. This was confirmed by: (1) sequencing of the genomic DNA; (2) SDS-PAGE and peptide analysis of wild-type and recombinant phasin; and (3) mass spectrometry of wild-type phasin protein. The results have implications for the model proposed for the binding of this protein to polyhydroxyalkanoic acid granules in the bacterium. (+info)
Pharmacological and histochemical distinctions between molecularly defined sarcolemmal KATP channels and native cardiac mitochondrial KATP channels.
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A variety of direct and indirect techniques have revealed the existence of ATP-sensitive potassium (KATP) channels in the inner membranes of mitochondria. The molecular identity of these mitochondrial KATP (mitoKATP) channels remains unclear. We used a pharmacological approach to distinguish mitoKATP channels from classical, molecularly defined cardiac sarcolemmal KATP (surfaceKATP) channels encoded by the sulfonylurea receptor SUR2A and the pore-forming subunit Kir6.2. SUR2A and Kir6.2 were expressed in human embryonic kidney (HEK)293 cells, and their activities were measured by patch-clamp recordings of membrane current. SurfaceKATP channels are activated potently by 100 microM pinacidil but only weakly by 100 microM diazoxide; in addition, they are blocked by 10 microM glibenclamide, but are insensitive to 500 microM 5-hydroxydecanoate. This pharmacology, which was confirmed with patch-clamp recordings in intact rabbit ventricular myocytes, contrasts with that of mitoKATP channels as indexed by flavoprotein oxidation. MitoKATP channels in myocytes are activated equally by 100 microM diazoxide and 100 microM pinacidil. In contrast to its lack of effect on surfaceKATP channels, 5-hydroxydecanoate is an effective blocker of mitoKATP channels. Glibenclamide's effects on mitoKATP channels are difficult to assess, because it independently activates flavoprotein fluorescence, consistent with a previously described primary uncoupling effect. Confocal imaging of the subcellular distribution of expressed fluorescent Kir6.2 in HEK cells and in myocytes revealed no targeting of mitochondrial membranes. The differences in drug sensitivity and subcellular localization indicate that mitoKATP channels are distinct from surface KATP channels at a molecular level. (+info)
The role of arginine 120 of human prostaglandin endoperoxide H synthase-2 in the interaction with fatty acid substrates and inhibitors.
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Arg-120 is located near the mouth of the hydrophobic channel that forms the cyclooxygenase active site of prostaglandin endoperoxide H synthases (PGHSs)-1 and -2. Replacement of Arg-120 of ovine PGHS-1 with a glutamine increases the apparent Km of PGHS-1 for arachidonate by 1,000-fold (Bhattacharyya, D. K., Lecomte, M., Rieke, C. J., Garavito, R. M., and Smith, W. L. (1996) J. Biol. Chem. 271, 2179-2184). This and other evidence indicate that the guanido group of Arg-120 forms an ionic bond with the carboxylate group of arachidonate and that this interaction is an important contributor to the overall strength of arachidonate binding to PGHS-1. In contrast, we report here that R120Q human PGHS-2 (hPGHS-2) and native hPGHS-2 have very similar kinetic properties, but R120L hPGHS-2 catalyzes the oxygenation of arachidonate inefficiently. Our data indicate that the guanido group of Arg-120 of hPGHS-2 interacts with arachidonate through a hydrogen bond rather than an ionic bond and that this interaction is much less important for arachidonate binding to PGHS-2 than to PGHS-1. The Km values of PGHS-1 and -2 for arachidonate are the same, and all but one of the core residues of the active sites of the two isozymes are identical. Thus, the results of our studies of Arg-120 of PGHS-1 and -2 imply that interactions involved in the binding of arachidonate to PGHS-1 and -2 are quite different and that residues within the hydrophobic cyclooxygenase channel must contribute more significantly to arachidonate binding to PGHS-2 than to PGHS-1. As observed previously with R120Q PGHS-1, flurbiprofen was an ineffective inhibitor of R120Q hPGHS-2. PGHS-2-specific inhibitors including NS398, DuP-697, and SC58125 had IC50 values for the R120Q mutant that were up to 1,000-fold less than those observed for native hPGHS-2; thus, the positively charged guanido group of Arg-120 interferes with the binding of these compounds. NS398 did not cause time-dependent inhibition of R120Q hPGHS-2, whereas DuP-697 and SC58125 were time-dependent inhibitors. Thus, Arg-120 is important for the time-dependent inhibition of hPGHS-2 by NS398 but not by DuP-697 or SC58125. (+info)
Direct preconditioning of cardiac myocytes via opioid receptors and KATP channels.
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Previous studies demonstrated that opioid receptor activation mimics the cardioprotective effect of ischemic preconditioning via KATP channels in the intact heart. However, it is unknown whether this beneficial effect is exerted at the level of the cardiac myocyte or coronary vasculature or is mediated via the sarcolemmal or the mitochondrial KATP channel. Thus, the purpose of the present study was to investigate whether opioid receptor stimulation could mimic the cardioprotective effect of preconditioning in a cardiac myocyte model of simulated ischemia. Cardiac ventricular myocytes cultured from chick embryos 14 days in ovo were used as an in vitro model for ischemic preconditioning. A 5-minute exposure of the myocytes to the opioid receptor agonist morphine protected the myocytes during a subsequent 90-minute period of simulated ischemia, which was manifested as a pronounced reduction in the percentage of cardiac cells killed and the amount of creatine kinase released during ischemia. The preconditioning-like effect of morphine was concentration-dependent, reached a maximal effect at 1 micromol/L, and was reversed by naloxone (0.1 to 10 micromol/L). When KATP channel antagonists, such as glibenclamide, or the mitochondrial selective inhibitor 5-hydroxydecanoic acid were present during preexposure to morphine, they abolished the protective effect of morphine. Thus, cardiac myocytes express functional opioid receptors, and their activation mimics the cardioprotective effect of ischemic preconditioning. These results provide direct evidence that the preconditioning-like effect of morphine in the intact heart can be exerted at the level of cardiac myocytes and is most likely the result of mitochondrial KATP channel activation. (+info)
Role of an ATP-sensitive potassium channel opener, YM934, in mitochondrial energy production in ischemic/reperfused heart.
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We examined a possible mechanism of action of an ATP-sensitive potassium (K(ATP)) channel opener, YM934, for the improvement of energy metabolism in hearts subjected to 35-min ischemia and 60-min reperfusion. The treatment with 30 nM YM934 for the final 15 min of preischemia enhanced postischemic recovery of left ventricular developed pressure, attenuated the postischemic rise in left ventricular end-diastolic pressure, and suppressed the release of creatine kinase and ATP metabolites during reperfusion. The treatment also restored myocardial ATP and creatine phosphate contents and attenuated the decrease in mitochondrial oxygen consumption rate during reperfusion. The higher mitochondrial function was also seen in YM934-treated hearts at the end of ischemia. In another set of experiments, myocardial skinned bundles were incubated for 30 min under hypoxic conditions in the presence and absence of YM934, and then mitochondrial oxygen consumption rate was determined. Hypoxia decreased the mitochondrial oxygen consumption rate of skinned bundles to approximately 40% of the prehypoxic value. In contrast, the treatment of skinned bundles with 30 nM YM934 preserved the mitochondrial oxygen consumption rate during hypoxia. The effect of YM934 on the hypoxic skinned bundles was abolished by combined treatment with either the K(ATP) channel blocker glyburide or the mitochondrial K(ATP) channel blocker 5-hydroxydecanoate in a concentration-dependent manner. The results suggest that YM934 is capable of attenuating ischemia/reperfusion injury of isolated perfused hearts due to preservation of mitochondrial function during ischemia, probably through opening of mitochondrial K(ATP) channels. (+info)