NoteImproved method for high energy nucleotide analysis of canine cardiac muscle using reversed-phase high-performance liquid chromatography
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Cited by (51)
A comparative study of the most widely used solutions for cardiac graft preservation during hypothermia
2002, Journal of Heart and Lung TransplantationReports conflict on the benefits of preservative solutions. We investigated the efficacy of the most widely used cardioplegic solutions by comparing extracellular solutions such as Celsior solution, St. Thomas Hospital solutions 1 and 2 (STH-1, STH-2), the modified University of Wisconsin solution (UW-1), Lyon Preservation solution (LYPS) from our laboratory, and intracellular solutions such as standard University of Wisconsin solution (UW), Bretschneider solution (HTK), Stanford solution (STF), and Euro-Collins solution (EC).
Male rats (n = 110) were randomized into 11 groups: LYPS, Celsior, STH-1, STH-2, UW-1, UW, HTK, STF, EC, and normal saline solution groups, and a control group. All hearts, except controls, were preserved by cold storage (8 hours at 4°C) in the various solutions. We used an isolated non-working-heart model and biopsy specimens to assess heart preservation (n = 5/group).
Hearts stored in the EC and saline solutions had poor left ventricular developed pressure (LVDP) × heart rate (HR) (1,407.5 ± 154 and 1,390 ± 439 mm Hg/mn, respectively). In contrast, hearts stored in LYPS and Celsior had a LVDP × HR close to control hearts (31,349 ± 1,847, 27,620 ± 1,207, and 36,627 ± 1,322 mm Hg/mn, respectively), whereas hearts stored in STH-1, STH-2, UW-1, UW, HTK, and STF had intermediate functional response (14,278 ± 2,176, 12,402 ± 1,571, 11,428 ± 1,629, 11,603 ± 2,521, 7,045 ± 537, and 7,086 ± 1,206 mm Hg/mn, respectively). Hearts preserved with STH-2, UW, HTK, STF, EC, and saline solution showed significantly increased release of creatine kinase and lactate dehydrogenase than did control hearts or hearts preserved in Celsior, LYPS, STH-1, and UW-1. The energetic charge (EC = [(0.5 adenosine diphosphate + adenosine triphosphate) ÷ (adenosine triphosphate + adenosine diphosphate + adenosine monophosphate)]) in STH-2, UW, HTK, STF, EC, and saline groups was significantly lower (p < 0.05) than in the other groups.
Extracellular-type solutions provided better preservation than did intracellular-type solutions. However, UW and UW-1 (intracellular- and extracellular-type solutions) provided equivalent preservation of cardiac function. Preservation quality may be attributed to calcium, often added to extracellular solutions. Among extracellular solutions, Celsior and LYPS solution showed comparable efficacy on left ventricular function and seemed to offer better preservation than the other solutions tested in this study.
Evaluation of a new preservative solution for cardiac graft during hypothermia
2000, Journal of Heart and Lung TransplantationBackground: Reports conflict on the beneficial effects of several cardioplegic solutions such as University of Wisconsin and St. Thomas’ Hospital solutions. Therefore our objective was to assess the efficacy of several cardioplegic solutions for cardiac preservation by comparing University of Wisconsin modified solution (UW-1 and UW-1 + calcium = UW-2), St. Thomas’ Hospital solution N°1 (STH-1), Celsior solution, and a solution from our laboratory, Lyon preservation solution (LYPS).
We randomized male rats (n = 70) to 7 groups: LYPS, Celsior, STH-1, UW-1, UW-2, normal saline solution, and control. All hearts, except control hearts were preserved by cold storage (8 hours, 4°C) in the various solutions. We used isolated non–working-heart preparations (left ventricular function evaluation, n = 5/group) or biopsy specimens (energetic store evaluation, n = 5/group) to assess quality of heart preservation.
Hearts stored with the saline solution had a mean left ventricular developed pressure (LVDP) of 3.6 ± 1.3 mm Hg. In contrast, LYPS and Celsior hearts had mean LVDP close to that of the control hearts (97 ± 2.6, 92.1 ± 2.2, and 122 ± 1.9 mm Hg, respectively), whereas STH-1, UW-1, and UW-2 hearts presented an intermediate functional response (48 ± 4, 39.9 ± 4.1, and 69 ± 1.8 mm Hg, respectively). The STH-1 and saline hearts showed increased release of creatine kinase (541.9 ± 168 and 1,080.8 ± 126.2 UI/liter, respectively). The energetic charge (EC = [(0.5 ADP + ATP)/ATP + ADP + AMP]) in Celsior, UW-2, and saline was significantly lower (p < 0.001) than in the other groups.
The composition of the preservation solutions had a notable effect on myocardial viability. Our results indicated that LYPS and Celsior solutions had comparable efficacy for left ventricular function. However these solutions may offer better preservation than do UW-1, UW-2, or STH-1 solutions.
ATP depletion induces a loss of respiratory epithelium functional integrity and down-regulates CFTR (cystic fibrosis transmembrane conductance regulator) expression
1997, Journal of Biological ChemistryTo mimic the effect of ischemia on the integrity of airway epithelium and expression of cystic fibrosis transmembrane conductance regulator (CFTR), we induced an ATP depletion of the respiratory epithelium from upper airway cells (nasal tissue) and human bronchial epithelial 16HBE14o− cell line. Histological analysis showed that 2 h of ATP depletion led to a loss of the epithelium integrity at the interface between basal cells and columnar cells. The expression of connexin 43 (Cx43, subunit of the gap junctions) and desmoplakins 1 and 2 (DPs 1 and 2, major components of the desmosomes) proteins was inhibited. After 90 min of ATP depletion, a significant decrease of the transepithelial resistance (25%) was observed but was reversible. Similar results were obtained with the 16HBE14o− human bronchial epithelial cell line. ATP depletion led to actin filaments depolymerization. The expression of the mature CFTR (170 kDa) and fodrin proteins at the apical domain of the ciliated cells was down-regulated. The steady-state levels of CFTR, Cx43, DPs 1 and 2 mRNAs, semiquantified by RT-polymerase chain reaction kinetics, remained constant throughout ATP depletion in nasal tissue as in the homogeneous cell population of 16HBE14o− human bronchial epithelial cell line. This suggests that the down-regulation of these proteins might be posttranscriptional. The intercellular diffusion through gap junctions of Lucifer dye was completely inhibited after 90 min of ATP depletion but was reversible. The volume-dependent and the cAMP-dependent chloride secretion were inhibited in a nonreversible way. Taken together, these results suggest that an ATP depletion in human airway epithelium, mimicking ischemia, may induce a marked alteration in the junctional complexes and cytoskeleton structure concomitantly with a loss of apical CFTR expression and chloride secretion function.
Coincubation of rat renal proximal tubules with hepatic subcellular fractions potentiates the effect of para-aminophenol
1997, Fundamental and Applied ToxicologyTreatment of rats withpara-aminophenol (PAP) (300 mg/kg ip) produced decreases in renal nonprotein sulfhydryl (NPSH) content, oxygen consumption, and adenine nucleotide concentrations 2–4 hr following administration. In contrast, incubation of rat renal tubules with up to 1 mmPAP for 4 hr produced inconsistent changes in renal tubules. This discrepancy suggested that extrarenal metabolism of PAP may be involved in PAP bioactivation and nephrotoxicity. We designed the present studies to test the hypothesis that hepatic metabolism of PAP potentiates the effects of PAP on renal tubules. Incubation of renal tubules with 0.5 mmPAP and 10 mg protein from hepatic postmitochondrial supernatant (S9 fraction) in the absence of glutathione (GSH) for 4 hr did not alter renal oxygen consumption or adenine nucleotide metabolite concentrations. We observed no changes when we incubated tubules with 0.5 mmPAP and 1 mmGSH in the absence of hepatic S9 fraction. However, incubation of renal tubules with 0.5 mmPAP, 1 mmGSH, and 10 mg hepatic S9 protein for 4 hr significantly decreased renal oxygen consumption and adenosine triphosphate and total nucleotide concentrations. These data suggest that the effects of PAP in renal tubules may be potentiated by enzymatic metabolism of PAP, possibly involving oxidation and GSH conjugation. From experiments using hepatic microsomes or cytosol instead of S9 fraction, we found that changes were produced when we incubated tubules with PAP in the presence of hepatic microsomes, but not cytosol. These data suggest that hepatic microsomal metabolism of PAP may contribute to the production of changes in renal tubulesin vitro.PAP-induced changes in renal proximal tubules were prevented when we included a β-nicotinamide adenine dinucleotide phosphate (NADPH) generating system in the incubation medium. The protective effect of NADPH persisted when microsomes were inactivated by incubation with 1-aminobenzotriazole, a cytochrome P450 inhibitor. These data suggest that cytochrome P450-dependent oxidation is not involved in the production or prevention of PAP-induced changes in renal tubules. The enzyme(s) responsible for PAP bioactivation and the mechanism(s) by which NADPH protects renal tubules from PAP-induced decrements in oxygen consumption and adenine nucleotide concentrations are currently unclear.
Protein arylation precedes acetaminophen toxicity in a dynamic organ slice culture of mouse kidney
1996, Fundamental and Applied ToxicologyAcetaminophen (APAP) is an analgesic and antipyretic agent which may cause hepatotoxicity and nephrotoxicity with overdose in man and laboratory animals.In vivostudies suggest thatin situactivation of APAP contributes to the development of nephrotoxicity. Associated with target organ toxicity is selective arylation of proteins, with a 58-kDa acetaminophen binding protein (58-ABP) being the most prominent cytosolic target. In this study a mouse kidney slice model was developed to further evaluate the contribution ofin situactivation of APAP to the development of nephrotoxicity and to determine the selectivity of protein arylation. Precision cut kidney slices from male CD-1 mice were incubated with selected concentrations of APAP (0–25 mM) for 2 to 24 hr. APAP caused a dose- and time-dependent decrease in nonprotein sulfhydryls (NPSH), ATP content, and K+retention. Preceding toxicity was arylation of cytosolic proteins, the most prominent one being the 58-ABP. The association of 58-ABP arylation with APAP toxicity in this mouse kidney slice model is consistent with earlier,in vivoresults and demonstrates the importance ofin situactivation of APAP for the development of nephrotoxicity. Precision cut renal slices and dynamic organ culture are a good model for further mechanistic studies of APAP-induced renal toxicity..
Lack of correlation between para-aminophenol toxicity in vivo and in vitro in female Sprague-Dawley rats
1996, Fundamental and Applied ToxicologyThe present study was designed to test the hypothesis thatpara-aminophenol (PAP) nephrotoxicity is due to autooxidation. We compared renal functional responses following PAP administration to female Sprague–Dawley rats and following incubation of renal proximal tubules with PAP. The concentrations of PAP selected forin vitroincubations produced cytotoxicity (for example, a decrease in oxygen consumption or adenine nucleotide concentration) in rat renal epithelial cells or rabbit proximal tubule suspensions. In rats, PAP (300 mg/kg ip) caused proximal tubular necrosis within 24 hr. Changes in renal function 24 hr following PAP administration included increased kidney weight and blood urea nitrogen concentration and decreased renal glutathione (GSH) content and adenine nucleotide concentrations. PAP did not cause hepatic damage. Within 2–4 hr following PAP administration, renal GSH content and adenine nucleotide concentrations were significantly decreased. In renal cortical slices prepared from PAP-treated rats, oxygen consumption and accumulation of organic ions (para-aminohippurate and tetraethylammonium) were significantly decreased compared with renal cortical slices prepared from control rats. In liver, GSH content was significantly decreased from 1 to 4 hr following PAP administration. In contrast to the effects of PAPin vivo,renal proximal tubules showed little evidence of injury when incubated with 0.1 or 0.5 mMPAP for up to 4 hr in the presence or absence of amino acids in the incubation medium. When tubules were incubated with 1 mMPAP for 4 hr in the presence of amino acids, GSH content, AMP concentration, and TEA uptake were significantly decreased. When amino acids were removed from the incubation medium, 1 mMPAP caused decreases in oxygen consumption and ATP concentration after 4 hr of incubation. Functional changes observed during incubation with PAPin vitrowere not consistent with functional changes observedin vivo.The discrepancy between PAP toxicityin vivoandin vitrosuggests that autooxidation is unlikely to be responsible for PAP nephrotoxicity and that nephrotoxicityin vivois primarily mediated by extrarenal bioactivation. Further, depletion of hepatic GSH content prior to changes in renal function suggests that PAP or a PAP metabolite may conjugate with hepatic GSH. These observations suggest that PAP nephrotoxicity may be mediated by PAP–GSH conjugates rather than autooxidation of PAP in the kidney.