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)
Structural heterogeneity of the binding sites of HSA for phenyl-groups and medium-chain fatty acids. Demonstration of equilibrium between different binding conformations.
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A new facet of the very heterogeneous albumin molecule is described. Chromatography at pH 6-9 of human serum albumin (HSA) on a phenyl-sepharose column separates it into two nonconvertible conformations that are, in turn, in equilibrium with its binding and nonbinding forms. The hydrophobic interaction of HSA with phenyl-sepharose depends on ionic strength, pH, and time of contact with the immobilized ligand. Binding as a function of pH shows a minimum at pH 6.5, and the binding profile at pH 7-9 fits the titration of a weak monoprotic acid with a pKa of 7.3. There was no observable difference in the CD spectra or the masses of the two forms. The equilibrium between the albumin forms was examined under defined conditions and cannot be explained by a simple two-state model. Thus rechromatography of the nonbinding fraction derived from a sample in which 50% of the protein was originally retained resulted only in 10-20% bound protein. Correspondingly only 70-80% of the binding form was retained. A model explaining the observations can be derived if two species, I and II, exist in the solution, both being in an equilibrium with a binding and a nonbinding form, but in which I is not in equilibrium with II. The rate of conversion between the binding and nonbinding conformations was determined to be faster than 15 s at room temperature. (+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)
Paracrine immunotherapy with interleukin-2 and local chemotherapy is synergistic in the treatment of experimental brain tumors.
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Potent immune responses against malignant brain tumors can be elicited by paracrine intracranial (i.c.) immunotherapy with interleukin (IL)-2. Additionally, i.c. delivery of carmustine via biodegradable polymers has been shown to significantly prolong survival in both animal models and clinical trials. In this study, we show that the combination of paracrine immunotherapy, with nonreplicating genetically engineered tumor cells that produce IL-2, and local delivery of chemotherapy by biodegradable polymers prolongs survival in a synergistic manner in mice challenged intracranially with a lethal murine brain tumor. Animals receiving IL-2-transduced cells and polymers containing 10% 1,3-bis(2-chloroethyl)-1-nitrosourea had significantly improved survival compared with animals receiving IL-2-transduced cells or 10% 1,3-bis(2-chloroethyl)-1-nitrosourea alone. Median survival for the control group was 19 days. Survival in animals receiving IL-2-transduced cells and 1% carboplatin-containing polymers was also significantly improved compared with either therapy alone. Histopathological examination on day 14 of animals receiving combination treatment showed rare degenerating tumor cells. In addition to tissue necrosis surrounding the polymer, a marked inflammatory reaction was observed. In long-term survivors (all animals receiving combination treatment), no tumor was observed and the inflammatory reaction was completely resolved. The brains of animals receiving combination therapy showed both tissue necrosis due to local chemotherapy and strong inflammation due to paracrine immunotherapy. The demonstration of synergy between paracrine IL-2 and local i.c. delivery of antineoplastic drugs is novel and may provide a combined treatment strategy for use against both primary and metastatic i.c. tumors. (+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)
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)
The cyanogenic glucoside, prunasin (D-mandelonitrile-beta-D-glucoside), is a novel inhibitor of DNA polymerase beta.
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A DNA polymerase beta (pol. beta) inhibitor has been isolated independently from two organisms; a red perilla, Perilla frutescens, and a mugwort, Artemisia vulgaris. These molecules were determined by spectroscopic analyses to be the cyanogenic glucoside, D-mandelonitrile-beta-D-glucoside, prunasin. The compound inhibited the activity of rat pol. beta at 150 microM, but did not influence the activities of calf DNA polymerase alpha and plant DNA polymerases, human immunodefficiency virus type 1 reverse transcriptase, calf terminal deoxynucleotidyl transferase, or any prokaryotic DNA polymerases, or DNA and RNA metabolic enzymes examined. The compound dose-dependently inhibited pol. beta activity, the IC(50) value being 98 microM with poly dA/oligo dT(12-18) and dTTP as the DNA template and substrate, respectively. Inhibition of pol. beta by the compound was competitive with the substrate, dTTP. The inhibition was enhanced in the presence of fatty acid, and the IC(50) value decreased to approximately 40 microM. In the presence of C(10)-decanoic acid, the K(i) value for substrate dTTP decreased by 28-fold, suggesting that the fatty acid allowed easier access of the compound to the substrate-binding site. (+info)
Tumor radiosensitization by sustained intratumoral release of bromodeoxyuridine.
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We have previously reported that the use of the polymer bis(p-carboxyphenoxy)propane-sebacic acid (20:80) for intratumoral delivery of cis-platinum in a mouse tumor model (RIF-1) potentiated the effects of acute and fractionated radiation. This mode of drug delivery seems particularly applicable to the administration of radiosensitizing drugs because an optimum concentration of radiosensitizer can be maintained in the tumor over the prolonged period required for fractionated radiation treatment. We have now investigated, in the same tumor model, radiosensitization by the thymidine analogue bromodeoxyuridine (BrdUrd). BrdUrd (20%, w/w) was incorporated into bis(p-carboxyphenoxy)propane-sebacic acid (20:80) and polymer rods containing the drug implanted in the RIF-1 tumor. Preliminary in vitro studies of the rate of release of BrdUrd from the polymer showed an initial rapid loss over 24 h, followed by a slower release extending over the next 5 days. In experiments in which tumor cells, which had incorporated BrdUrd in vivo from implanted polymer, were excised and a single cell suspension irradiated in vitro radiosensitization indicative of BrdUrd incorporation was associated mainly with an increase in the alpha constant for the linear quadratic model of cell survival. Radiosensitization was seen for tumor cells harvested between 5 and 10 days after polymer implant, a finding that is consistent with results of experiments in which the percentage of cells that had incorporated BrdUrd were measured by flow cytometry at various times after polymer/BrdUrd implant. The proportion of tumor cells positive for BrdUrd was 40-50% between 3 and 8 days after polymer implant. When tumors were irradiated in situ and response measured in terms of tumor growth delay (TGD), radiosensitization was not seen for an acute dose of 16.5 Gy. In contrast, significant radiosensitization was seen for fractionated treatments when polymer/BrdUrd was implanted 3 days before the first radiation dose. For a dose of 5 x 6 Gy, TGD was increased from 22 days for radiation alone to 27 days for radiation plus polymer implant. For 10 x 6 Gy fractions, TGD increased from 45-77 days for those mice in whom the tumor eventually regrew, whereas for 25% of the mice in this group the tumor volume was reduced to a point where it was no longer detectable and there was no recurrence for at least 120 days after treatment. (+info)