Comparison of protection by propranolol,bepridil, verapamil,and captopril on depleting ATP,ADP, and AMP in heart,brain, and liver by anoxia plus isoprenaline

Mice were injected with 5 mg/kg isoprenaline 45 min prior to being subject to 12 min global anoxia and ATP, ADP, and AMP were assayed in the heart, brain, and liver by reverse phase HPLC with UV detection at 254 nm. In myocardium the depletion of ATP by global anoxia was 72.5% of the total and only 7.5% due to the addition of isoprenaline. In the brain and liver, globally anoxic depletion of ATP was much less; a large fraction of ATP depletion was caused by isoprenaline. The depletion of ATP, ADP, and AMP in the heart by anoxia plus isoprenaline was partially protected by propranolol, and the protection by bepridil, verapamil, and captopril was weak. In the cerebrum bepridil, propranolol, and verapamil were potent protectors of ATP and total high energy bonds. Bepridil was the most effective, and its protection against cerebral ischemia was reproduced by limiting the infarcted zone and relieving the abnormal behavior after occlusion of the middle cerebral artery in rats. In liver all four drugs exerted a mild protection. In summary, isoprenaline is more toxic to the brain and liver in depleting ATP in the presence of global ischemia and the protection by bepridil against cerebral ischemia is attributed to its dual blocking effect on sodium and calcium channels. Drug Dev. Res. 39:125–130. © 1997 Wiley-Liss, Inc.     

ATP metabolism in an ethanol induced fatty liver.

A marked decrease in the hepatic concentration of ATP lowers the phosphate potential of the liver of rats fed ethanol for prolonged periods of time. Either a decreased synthesis or an increased demand could account for this change in ATP. The experiments presented here were designed to investigate both possibilities. Changes in the utilization of ATP were assessed by measuring the activity of the [Na⁺ + K⁺]-activated ATPase system, and changes in the synthesis of ATP by the activity of the adenine nucleotide translocase system. Since the level of long-chain CoA derivatives of fatty acids regulate the translocation of ADP into mitochondria, their total cellular content and mitochondrial level were also determined. The experiments were conducted on male Sprague-Dawley rats (275–300 g) maintained on a liquid diet having 36 per cent of the caloric intake as ethanol. After 2 weeks, the experimental animals had a fatty liver. This change was not associated with any significant alteration in the ATP content of the liver or in the activity of the adenine nucleotide translocase system. After 4 weeks, the excess of neutral lipid in the liver still persisted, but was associated with a marked increase in the level of long-chain CoA derivatives of fatty acids. The level of ATP in the liver was only 50 per cent of normal, and the rate of translocation of ADP into the mitochondria was decreased. The activity of the adenine nucleotide translocase system could be restored to normal values if the long-chain CoA derivatives of fatty acids were removed from the surface of the mitochondrial membrane by treatment of the preparation with defatted albumin. At this time there was only a slight (15%) enhancement of the [Na⁺ + K⁺]-activated ATPase system. With the removal of ethanol from the diet the ATP level returned to normal rather quickly. Changes in the level of hepatic ATP correlated well with the activity of the adenine nucleotide translocase system but did not parallel changes in the activity of the Na⁺ pump system. These findings indicate that the dominant feature leading to the decrease in the ($\text{ATP}\text{ADP}\text{× P}{}_{\mathrm{i}}$) ratio is decreased synthesis of ATP.     

A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates

1. Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of diseases with excess bone resorption. On the basis of their molecular mechanism of action, bisphosphonates can be divided into two pharmacological classes; nitrogen-containing (N-BPs) and non-nitrogen-containing bisphosphonates (non-N-BP). Both classes induce apoptosis but they evoke it differently; N-BPs by inhibiting the intracellular mevalonate pathway and protein isoprenylation, and non-N-BPs via cytotoxic ATP analog-type metabolites. N-BPs are not metabolized to ATP analogs, but we report here that these bisphosphonates can induce formation of a novel ATP analog (ApppI) as a consequence of the inhibition of the mevalonate pathway in cells. We also investigated whether ApppI is involved in the apoptosis induced by N-BPs. 2. Mass spectrometry and NMR were used to identify ApppI in N-BP treated osteoclasts, macrophages and glioma cells. The potency of different bisphosphonates to promote ApppI production was tested in J774 macrophages. The effects of ApppI on ADP/ATP translocase in isolated mitochondria and its capability to induce apoptosis in osteoclasts were also studied. 3. ApppI production correlated well with the capacity of N-BPs to inhibit mevalonate pathway. ApppI inhibited the mitochondrial ADP/ATP translocase and caused apoptosis in osteoclasts. 4. In conclusion, these findings provide the basis for a new mechanism of action for N-BPs. Some of these very potent bisphosphonates, such as zoledronic acid, represent a third class of bisphosphonates that can act both via the inhibition of the mevalonate pathway and by the blockade of mitochondrial ADP/ATP translocase, which is known to be involved in the induction of apoptosis.     

Effect of Freezing Rate and Dendritic Ice Formation on Concentration Profiles of Proteins Frozen in Cylindrical Vessels

The process of freezing protein solutions can perturb the conformation of the protein and potentially lead to aggregate formation during long-term storage in the frozen state. Radial macroscopic freeze concentration and temperature profiles for bovine serum albumin (BSA) solutions in small cylindrical stainless steel vessels were determined for various freezing rates. The measured concentrations of both BSA and immunoglobulin G2, as well as trehalose in sampled ice sections, increased by up to twofold to threefold toward the bottom and radial center for slow freezing rates produced in stagnant air freezers. The concentration and temperature profiles result in density gradients that transport solutes by convective flow. For faster external cooling by either forced convection of air or a liquid coolant, the increased freezing rate raised the ice front velocity resulting in enhanced dendritic ice growth. The ice trapped the solutes more effectively before they were removed from the ice front by diffusion and convection, resulting in more uniform solute concentration profiles. The dynamic temperature profiles from multiple radial thermocouples were consistent with the independently measured freeze concentration profiles. The ability to control the protein concentration profile in the frozen state offers the potential to improve stability of protein in long-term frozen storage.     

Sex and hormonal influences on platelet sensitivity and coagulation in the rat.

Platelet sensitivity to adenosine di-phosphate (ADP), thrombin, collagen, arachidonic acid and prostaglandin I2 (PGI2) and the activity of the coagulation system as measured by the activated partial thromboplastin time, prothrombin time, Russell's viper venom time and plasma fibrinogen have been examined in male and female rats, female rats during the oestrous cycle and female rats treated with oestrogen and a progestogen. Male rat platelets were less sensitive to thrombin and more sensitive to inhibition by PGI2 than those from females and fibrinogen levels in male rat plasma were approximately twice those seen in females. During the oestrous cycle, platelets were more sensitive to ADP and less sensitive to thrombin at dioestrus. Following 6 weeks treatment with 17 beta-oestradiol or ethynyl oestradiol, both platelet aggregation and release of granular ATP induced by collagen were significantly reduced. Platelet sensitivity to other agents, ADP, arachidonic acid, thrombin and PGI2 was, however, unchanged following oestrogen treatment. Activation of factor X by Russell's viper venom was accelerated in rats treated with ethynyl oestradiol, although this enhancement was not reflected in the overall clotting times.     

Changes in adenylate energy charge and total adenine nucleotide concentration in mouse testis induced by cadmium administration

Subcutaneous administration of CdCl2 (3 mg/kg body weight) caused a decrease in adenylate energy charge, (ATP + 0.5ADP)/(ATP + ADP + AMP), and a depletion of the adenine nucleotide pool in the testis of mice. This preceded a decrease in LDH-X activity in the testicular extracts. These results biochemically support the hypothesis that the testicular damage induced by cadmium is secondary to anoxia caused by vascular lesion.     

Ice nucleation temperature influences recovery of activity of a model protein after freeze drying

The objective of this study was to determine whether a relationship exists between ice nucleation temperature and recovery of activity of a model protein, lactate dehydrogenase, after freeze drying. Aqueous buffer systems containing 50 µg/mL of protein were frozen in vials with externally mounted thermocouples on the shelf of a freeze dryer, then freeze dried. Various methods were used to establish a wide range of ice nucleation temperatures. An inverse relationship was found between the extent of supercooling during freezing and recovery of activity in the reconstituted solution. The data are consistent with a mechanism of inactivation resulting from adsorption of protein at the ice/freeze–concentrate interface during the freezing process. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3495–3498, 2009     

Further insight into mechanism of action of clodronate: inhibition of mitochondrial ADP/ATP translocase by a nonhydrolyzable, adenine-containing metabolite

Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of diseases with excess bone resorption. Recent studies have shown that bisphosphonates can be divided into two groups with distinct molecular mechanisms of action depending on the nature of the R(2) side chain. Alendronate, like other nitrogen-containing bisphosphonates, inhibits bone resorption and causes apoptosis of osteoclasts and other cells in vitro by preventing post-translational modification of GTP-binding proteins with isoprenoid lipids. Clodronate, a bisphosphonate that lacks a nitrogen, does not inhibit protein isoprenylation but can be metabolized intracellularly to a beta-gamma-methylene (AppCp-type) analog of ATP, which is cytotoxic to macrophages in vitro. The detailed molecular basis for the cytotoxic effects of adenosine-5'-[beta,gamma-dichloromethylene]triphosphate (AppCCl(2)p) has not been determined yet. We addressed this question by studying the effects of alendronate, clodronate, and the clodronate metabolite AppCCl(2)p on isolated mitochondria, mitochondrial fractions, and mitochondrial membrane potential in isolated human osteoclasts. We found that AppCCl(2)p inhibits mitochondrial oxygen consumption by a mechanism that involves competitive inhibition of the ADP/ATP translocase. Alendronate or the native form of clodronate did not have any immediate effect on mitochondria. However, longer treatment with liposome-encapsulated clodronate caused collapse of the mitochondrial membrane potential, although prominent apoptosis was a late event. Hence, inhibition of the ADP/ATP translocase by the metabolite AppCCl(2)p is a likely route by which clodronate causes osteoclast apoptosis and inhibits bone resorption.     

Adenine nucleotide analogues, including gamma-phosphate-substituted analogues, are metabolised extracellularly in innervated frog sartorius muscle.

The metabolism of adenine nucleotides and of their analogues by ecto-enzymes in the innervated frog sartorius muscle was investigated with HPLC. The breakdown of beta, gamma-methylene-ATP was also evaluated by studying the ability of the adenosine uptake inhibitor, dipyridamole, and of the adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), to modify the effect of beta, gamma-methylene-ATP on nerve-evoked twitches. ATP-gamma-S at low (10 microM) but not at high (> or = 100 microM) concentrations was quickly metabolised into a compound with a higher negative charge. L-ATP, homo-ATP and 2-methylthio-ATP were metabolised into compounds with a lower negative charge. Beta-gamma-Imido-ATP and gamma-anilino-ATP were only metabolised slightly. As determined by HPLC, beta, gamma-methylene-ATP was not metabolised. In contrast, this ATP analogue inhibited nerve-evoked twitch responses, an effect which was potentiated by dipyridamole and antagonised by DPCPX. Alpha, beta-Methylene-ATP was dephosphorylated into alpha, beta-methylene-ADP, which was virtually resistant to metabolism in the absence of ATP. In the presence of ATP, alpha, beta-methylene-ADP was transiently phosphorylated into alpha, beta-methylene-ATP. Formation of ATP from ADP was observed even in the absence of an exogenous phosphate donor, and was prevented by the adenylate kinase inhibitor, P1P5-di-(adenosine-5')pentaphosphate (AP5A). AP5A caused only partial inhibition of AMP formation from ADP. The results suggest that some ATP analogues with substitutions in the gamma-phosphate, such as ATP-gamma-S and beta, gamma-methylene-ATP, are metabolised in the innervated frog sartorius muscle. The ADP analogue, alpha, beta-methylene-ADP, might be a substrate for an ecto-nucleoside diphosphate kinase. ADP, besides being dephosphorylated, is also a substrate for an ecto-adenylate kinase in innervated frog sartorius muscle.     

Effects of clofibric acid on mRNA expression profiles in primary cultures of rat,mouse and human hepatocytes

The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1alpha (HNF1alpha) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis.     

小牛血去蛋白提取物调节急性缺氧模型小鼠肝能量代谢及其作用靶点的研究

目的：考察小牛血去蛋白提取物（DECB）在急性缺氧条件下对小鼠耐缺氧的作用,并初步探索其调节肝脏中能量代谢的可能机制。方法：构建急性缺氧小鼠模型,考察空白组（CON）、模型组（MOD）,模型给药组（MOD+DECB）小鼠耐缺氧时间,并检测小鼠肝脏、血清中糖、乳酸、ATP、酮体、三酰甘油（TG）及总胆固醇（TC）的含量,采用mRNA芯片筛选DECB处理后小鼠肝脏中差异表达基因,并进行聚类分析及Real time PCR验证。结果：MOD+DECB组小鼠耐缺氧时间明显高于MOD组,差异具有显著性（P<0.05）;MOD+DECB组血清中糖、乳酸含量显著低于MOD组,酮体含量显著高于MOD组,MOD+DECB组肝脏中糖、ATP和酮体的含量显著高于MOD组,乳酸含量显著低于MOD组,差异具有显著性（P<0.01）。肝脏和血清中TG及TC含量各组之间无显著差异。mRNA表达谱芯片分析共筛选出1 186个差异表达基因,其中上调表达基因495个,占基因总数的41.7%,下调表达基因691个,占基因总数的58.3%,其中Hmgcs2、Cpt1a、Angptl4、Cyp8b1、Ehhadh基因参与小鼠机体能量代谢密切相关,Real time PCR验证结果与芯片分析结果一致。结论：DECB可通过调控Hmgcs2、Cpt1a、Angptl4、Cyp8b1、Ehhadh基因的表达,调节肝脏中能量的代谢,从而提高小鼠耐缺氧的能力。     

Retinoid X receptor alpha regulates glutathione homeostasis and xenobiotic detoxification processes in mouse liver

Retinoid X receptor alpha (RXRalpha) plays a pivotal role in regulating liver metabolism. RXRalpha-mediated gene expression involved in amino acid metabolism was examined using the NIA Mouse 15K cDNA microarray containing 15,000 different expressed sequence tags. Seven amino acid metabolic genes, three of which encode enzymes involved in phase II detoxification process, were identified as RXRalpha target genes in mouse liver. Glutamate-cysteine ligase catalytic subunit (GCLC), glutathione S-transferasemu, and glutathione peroxidase 1 were down-regulated in the liver of hepatocyte RXRalpha-deficient mice. The down-regulation of GCLC in RXRalpha-deficient mice led to 40% and 45% reductions in the rate of glutathione (GSH) synthesis and level of hepatic GSH, respectively. Primary hepatocytes from RXRalpha-deficient mice were more sensitive to t-butylhydroperoxide-induced oxidative stress. However, GSH diminished RXRalpha-deficient mice were resistant to acetaminophen (APAP)-induced hepatotoxicity. Analysis of phase I detoxification genes revealed that CYP1A2 and CYP3A11 were up-regulated in wild-type mice but down-regulated in RXRalpha-deficient mice after APAP administration. Taken together, the data indicate that RXRalpha centrally regulates both phase I and phase II drug metabolism and detoxification. Regulation of hepatic GSH levels by RXRalpha is essential to protect hepatocytes from oxidative stress, whereas up-regulation of phase I drug metabolism genes by RXRalpha may render the liver more sensitive to APAP-induced toxicity.     

Extracellular metabolism of adenine nucleotides and adenosine in the innervated skeletal muscle of the frog.

The effects of coformycin, alpha,beta-methylene ADP, dipyridamole in the absence and presence of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), nitrobenzylthioinosine (NBTI), mioflazine and ouabain on the metabolic pathways of exogenously applied ATP and its metabolites in the frog innervated sartorius muscle were investigated. ATP catabolism yielded ADP, AMP, IMP, adenosine and inosine; the ecto-ATPase in situ was shown to be Ca(2+)- or Mg(2+)-activated with a Kmapp for ATP of 767 +/- 48 microM. AMP catabolism yielded IMP, adenosine and inosine; inosine was formed from either exogenous IMP or exogenous adenosine. Catabolism of AMP into IMP was blocked by coformycin, which enhanced adenosine and inosine formation from AMP. alpha,beta-Methylene ADP blocked adenosine formation from AMP and inosine formation from IMP; formation of IMP from AMP was enhanced by alpha,beta-methylene ADP. Complete blockade of AMP degradation was achieved with the simultaneous use of coformycin and alpha,beta-methylene ADP. Dipyridamole attenuated but did not completely block extracellular adenosine removal and inosine appearance in the bath. EHNA, applied in the presence of dipyridamole, did not cause any further attenuation of extracellular adenosine removal. Mioflazine, NBTI and ouabain did not affect adenosine disappearance from the bath. The results suggest that, in the frog innervated sartorius muscle, ATP can be sequentially catabolized into AMP which is then catabolized either into IMP or into adenosine. This extracellular degradation of AMP into IMP might then constitute a shunt-like mechanism to control the levels of adenosine formed from adenine nucleotides.     

Distribution of P1- and P2- purinoceptors in the guinea-pig and frog heart

1 The effects of adenyl compounds were examined on the guinea-pig and frog heart in terms of the P₁/P₂-purinoceptor hypothesis.

2 The effects of two slowly degradable adenosine 5′-triphosphate (ATP) analogues; β,γ-methylene adenosine 5′-triphosphate (APPCP) and α,β-methylene adenosine 5′-triphosphate (APCPP) were also examined.

3 Adenosine, adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP), ATP and APPCP produced inhibitory effects in guinea-pig atria. These inhibitory effects were antagonized competitively by theophylline and potentiated by dipyridamole. APCPP did not produce a similar inhibitory response.

4 Guinea-pig ventricles were insensitive to adenyl compounds.

5 ATP and ADP produced initial excitatory effects in frog atria which were followed by inhibitory effects. Adenosine and AMP produced inhibitory effects alone whereas APCPP produced excitatory effects only. The inhibitory effects were antagonized competitively by theophylline and potentiated by dipyridamole.

6 ATP, ADP, APPCP and APCPP evoked excitatory responses in frog ventricles. These responses were not affected by theophylline or dipyridamole. Adenosine and AMP were inactive on frog ventricles.

7 It is concluded that only P₁-receptors are present in guinea-pig atria; that both P₁- and P₂-receptors are present in frog atria; and that only P₂-receptors are present in frog ventricles. No evidence was found for the presence of either P₁- or P₂-purinoceptors in guinea-pig ventricles.

     

Effects of the lipophilic biscation, bis-pyridinium oxime BP12, on bioenergetics and induction of permeability transition in isolated mitochondria

In order to further investigate the mechanism of action of bridged lipophilic bis-pyridinium oximes previously observed to interfere with mitochondrial metabolism and to induce growth arrest and apoptosis in HeLa cells (Nocentini et al., Biochem Pharmacol 53: 1543-1552, 1997), we studied the effects of a bis-pyridinium oxime with a polymethylene chain N = 12 (BP12) on isolated rat liver mitochondria. Respiration in the absence of ADP with succinate plus rotenone as substrate was not affected after treatment with various concentrations of BP12 up to 10 microM, while the ADP-stimulated respiration was slowed down, with a parallel decrease in ATP synthesis. No effects of BP12 were detected on membrane potential, ATPase activity, and inorganic phosphate transport, but the adenine nucleotide translocase was inactivated and a permeability transition of the inner membrane was induced in the presence of calcium. These data suggest that mitochondrial impairment of ATP synthesis and the formation of the permeability transition pore may be responsible for apoptotic cell death already observed in cells treated with BP12.     

3-isobutyl-1-methylxanthine (IBMX) sensitizes cardiac myocytes to anoxia

Cardiac myocytes incubated with 3-isobutyl-1-methylxanthine (IBMX), a nonspecific cyclic nucleotide phosphodiesterase inhibitor, formed rigor complexes under anoxic conditions more readily than cells incubated with other phosphodiesterase inhibitors. Cardiac myocytes were incubated for 1 hr with either (a) no additions, (b) 150 microM zaprinast, or (c) 1 mM IBMX, and then were rendered anoxic for periods up to 60 min. Cells were >80% viable throughout the anoxic period; viability was unaffected by either drug. Rod count decreased more rapidly after the onset of anoxia in the IBMX-treated cells than in control or zaprinast-treated cells (11% rods vs. roughly 47% rods after 30 min of anoxia). IBMX-treated cell groups also formed more "contracted" myocytes (box-like rods) than their untreated or zaprinast-treated counterparts (50% contracted vs. roughly 27% contracted after 30 min of anoxia). While nucleotide degradation patterns were similar in all experimental groups, the ratio of ATP to ADP was lower in IBMX-treated cells than in control or zaprinast-treated cells. The L-type calcium channel was apparently not involved in this phenomenon; while cyclic AMP was elevated in the IBMX-incubated cells, verapamil did not protect IBMX-incubated cells from premature damage by anoxia. Incubation with 8-cyclopentyl-1,3-dipropylxanthine (CDPX), an A1 receptor antagonist, at concentrations up to 1 microM in place of 1mM IBMX did not reproduce the IBMX effect. We concluded that IBMX sensitizes cardiac myocytes to anoxia through a mechanism related to its effect on ATP/ADP, and unrelated to an elevation of intracellular calcium or preconditioning phenomena.     

Evidence of partial unfolding of proteins at the ice/freeze-concentrate interface by infrared microscopy

The goal of this research was to use infrared spectroscopy in combination with a freeze drying stage to gain a better understanding of the mechanism of loss of protein integrity due to the stresses associated with freezing. Infrared spectra were collected in triplicate for the interstitial space between ice crystals and through ice crystals in a partially frozen system. Spectra were collected for lactate dehydrogenase (LDH) and human immune globulin (IgG) both in the presence and absence of an added surfactant (polysorbate 80). Spectra collected in the interstitial space, distant from the surface of ice crystals, were very similar to spectra collected from the initial solution regardless of the presence of a surfactant. Spectra collected through ice crystals, without added surfactant, were significantly different than spectra collected from the initial solution. An increase in bands characteristic of intermolecular β-sheet structures (main component of aggregates) were present in these spectra. The presence of surfactant in both protein formulations resulted in a decrease in intermolecular β-sheet signals in spectra of the proteins on the ice crystal surface. Additionally, much of the native state structure of LDH initially lost on the surface of ice crystals returned when surfactant was added to the formulation prior to freezing. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3239–3246, 2009