Excessive Intracellular Accumulation Research Articles

Elimination of a Hydroxyl Group in FTY720 Dramatically Improves the Phosphorylation Rate

The new immunosuppressant FTY720 (fingolimod), an analog of the endogenous lipid sphingosine, induces transient lymphopenia through the sequestration of lymphocytes in secondary lymphoid organs. Phosphorylation of FTY720 by sphingosine kinase 2 (SphK2) yields the active metabolite FTY720-phosphate (FTY-P), which induces lymphopenia through agonism of the sphingosine 1-phosphate receptor S1P(1) on endothelial cells and lymphocytes. Dephosphorylation of circulating FTY-P creates an equilibrium between FTY720 and its phosphate, and results with human patients indicate that phosphorylation of FTY720 could be rate limiting for efficacy. We report that the FTY720 derivative 2-amino-4-(4-heptyloxyphenyl)-2-methylbutanol [AAL(R)] is phosphorylated much more rapidly than FTY720 in cultured human cells and whole blood. The K(cat) for AAL(R) with recombinant SphK2 is 8-fold higher than for FTY720, whereas the K(m) for the two substrates is very similar, indicating that the increased rate of phosphorylation results from faster turnover by SphK2 rather than a higher binding affinity. Consequently, treating cells with AAL(R), but not FTY720, triggers an apoptotic pathway that is dependent on excessive intracellular accumulation of long-chain base phosphates. In agreement with the in vitro results, phosphorylation of AAL(R) is more complete than that of FTY720 in vivo (mice), and AAL(R) is a more potent inducer of lymphopenia. These differences may be magnified in humans, because phosphorylation of FTY720 is much less efficient in humans compared with rodents. Our results suggest that AAL(R) is a better tool than FTY720 for in vivo studies with S1P analogs and would probably be a more effective immunosuppressant than FTY720.

Reducing the effects of intracellular accumulation of thermolabile collagen II mutants by increasing their thermostability in cell culture conditions

Mutations in collagen II are associated with spondyloepiphyseal dysplasia, a group of heritable diseases whose common features include aberrations of skeletal growth. The mechanisms through which mutations in collagen II affect the cartilaginous tissues are complex and include both intracellular and extracellular processes. One of those mechanisms involves cellular stress caused by excessive accumulation of misfolded collagen II mutants. We investigated whether stabilizing the structure of thermolabile R789C and R992C collagen II mutants would improve their secretion from cells, thereby reducing cellular stress and apoptosis. Employing glycerol and trimethylamine N-oxide (TMAO), chemicals that increase the thermostability of collagen triple helices, we demonstrated that those compounds function as chaperones and stabilize the R789C and R992C mutants, accelerate their secretion, and improve cell survival. Our study provides a scientific basis for considering misfolded triple helices of collagen mutants a target for reducing the deleterious effects caused by their excessive intracellular accumulation.

Drug Transporters: Recent Advances and Therapeutic Applications

Drug Transporters: Recent Advances and Therapeutic Applications

Atherosclerosis and Autoimmunity

Atherosclerosis is a chronic pathological condition in which arteries undergo gradual thickness of the intima causing decreased elasticity, narrowing, and reduced blood supply to target organs. The blood vessels most commonly affected and of clinically relevance include the aorta, coronary, carotid, and cerebral arteries, collectively classified under cardiovascular disease. Several inherited and environmental risk factors have been identified as contributors to the initiation and progression of atherosclerosis. These risk factors are thought to disrupt a number of lipid regulatory and inflammatory mechanisms within the arterial wall. The appearance of lipid-laden foam cells in the arterial intima is a characteristic histological finding of early atherosclerosis. High plasma levels of low-density lipoprotein (LDL) in combination with arterial shear stress may contribute to vascular inflammation that promotes the oxidative modification of LDL. The causal relationship between oxidized LDL (oxLDL) and atherosclerosis is almost universally accepted. OxLDL transported into the subendothelial space is ingested by macrophages in the arterial intima via scavenger receptors, resulting in excessive intracellular accumulation of oxLDL and foam cell (plaque) formation. OxLDL is the principal plasma lipoprotein found in atherosclerotic lesions and it colocalizes with β2-glycoprotein I (β2GPI), the major antigenic target for antiphospholipid antibodies. Furthermore, there is wide consensus that anti-β2GPI antibodies play a central role in the development of the clinical complications that characterize the antiphospholipid syndrome (APS). It has been recently demonstrated that oxLDL forms complexes with β2GPI in atherosclerotic lesions and that such oxLDL/β2GPI complexes are present in the bloodstream of patients with

In Vitro Detection of Drug-Induced Phospholipidosis Using Gene Expression and Fluorescent Phospholipid Based Methodologies

Phospholipidosis (PLD) is characterized by the excessive intracellular accumulation of phospholipids. It is well established that a large number of cationic amphiphilic drugs have the potential to induce PLD. In the present study, we describe two facile in vitro methods to determine the PLD-inducing potential of a molecule. The first approach is based on a recent study by (Sawada et al., 2005, Toxicol. Sci. 83, 282-292) in which 17 genes were identified as potential biomarkers of PLD in HepG2 cells. To confirm the utility of this gene panel, we treated HepG2 cells with PLD-positive and -negative compounds and then analyzed gene expression using real-time PCR. Our initial analysis, which used a single dose of each drug, correctly identified five of eight positive compounds and four of four negative compounds. We then increased the doses of the three false negatives (amiodarone, tamoxifen, and loratadine) and found that the changes in gene expression became large enough to correctly identify them as PLD-inducing drugs. Our results suggest that a range of concentrations should be used to increase the accuracy of prediction in this assay. Our second approach utilized a fluorescently labeled phospholipid (LipidTox) which was added to the media of growing HepG2 cells along with compounds positive and negative for PLD. Phospholipid accumulation was determined using confocal microscopy and, more quantitatively, using a 96-well plate assay and a fluorescent plate reader. Using an expanded set of compounds, we show that this assay correctly identified 100% of PLD-positive and -negative compounds. Dose-dependent increases in intracellular fluorescent phospholipid accumulation were observed. We found that this assay was less time consuming, more sensitive, and higher throughput than gene expression analysis. To our knowledge, this study represents the first validation of the use of LipidTox in identifying drugs that can induce PLD.

Na + /Ca 2+ Exchanger Maintains Ionic Homeostasis in the Peri-Infarct Area

A prominent feature of cerebral ischemia is the excessive intracellular accumulation of both Na(+) and Ca(2+) ions, which results in subsequent cell death. The plasma membrane Na(+)/Ca(2+) exchanger (NCX), regulates the distribution of these ions acting either in the forward mode or in its reverse mode and it can play a critical role in brain ischemia. However, it is unclear whether the activity of NCX leads to detrimental or beneficial effects. Extracellular field potentials and whole-cell patch clamp recordings were obtained from rat corticostriatal brain-slice preparations in the peri-infarct area 24 hours after the permanent middle cerebral artery occlusion. Ischemia was induced in rats by permanent middle cerebral artery occlusion. Bepridil, an inhibitor of NCX, reduced in a concentration-dependent manner (IC(50)=68 micromol/L) the field potential amplitude recorded from the peri-infarct area of corticostriatal slices. Conversely, no change was observed in sham-operated animals. The effect of bepridil was mimicked by 5-(N-4-chlorobenzyl)-2',4'-dimethylbenzamil (CB-DMB) (IC(50)=6 micromol/L), a more selective inhibitor of NCX. In whole-cell patch clamp experiments, bepridil and CB-DMB caused an inward current in spiny neurons recorded from the peri-infarct area but not in the same cells recorded from controls. Interestingly, cholinergic interneurons recorded from the striatal peri-infarct area did not develop an inward current after the application of NCX inhibitors, suggesting that the electrophysiological alterations induced by NCX inhibition are cell-type specific. Bepridil and CB-DMB also induced a suppression of excitatory synaptic currents in most of spiny neurons recorded from the peri-infarct area. This effect was not coupled to a significant change of paired-pulse facilitation suggesting a postsynaptic site of action. Our data indicate that NCX plays a critical role in the maintenance of ionic homeostasis in the peri-infarct area.

Stroke: molecular mechanisms and potential targets for treatment.

Significant advances have been made over the past few years concerning the cellular and molecular events underlying ischemic cell death. The brain succumbs to ischemic injury as a result of loss of metabolic stores, excessive intracellular calcium accumulation, oxidative stress, and potentiation of the inflammatory response. Neurons can also die via necrotic or apoptotic mechanisms, depending on the nature and severity of the insult. While it has been widely held that ischemia is notable for cessation of protein synthesis, brain regions with marginal reduction in blood supply are especially capable of expressing a variety of genes, the functions of many of which are only beginning to be understood. Gene expression is also upregulated upon reperfusion and reoxygenation. As a result, a number of signaling pathways have been identified and are now known to contribute to ischemic progression or, in some cases, attempts at self preservation. This review will focus on the roles of stress genes, apoptosis-related genes, and inflammation. Knowledge of such molecular events has fueled interest in developing specific molecular targets with the hope of someday affecting outcome in clinical stroke.

Giant muscle fibres in pigs with different Ryr1 genotype.

This study examined the frequency, morphological and immunohistochemical characteristics of the giant fibres in the longissimus muscle of local Krsko polje pigs with different Ryr1 genotypes. Giant fibres were round-shaped and had significantly increased cross-sectional area compared with normal muscle fibres. Only fast-twitch glycolytic fibres were affected, usually showing enhanced succinate dehydrogenase activity. On the ultrastructural level, the dilation of the sarcoplasmic reticulum, swelling of mitochondria and destruction of myofilaments was observed. The incidence of giant fibres was the highest in Ryr1 dimutant pigs (Ryr1 nn), which also exhibited lower muscle pH1 than heterozygous (Ryr1 Nn) or pigs with the wild Ryr1 gene (Ryr1 NN). However, the giant fibres were also present in pigs free of Ryr1 gene mutation. Our results suggest that the giant fibre syndrome depends mostly upon the rate and intensity of early post-mortem glycolysis, which results in acidity of muscle tissue. We suppose that the giant fibre formation is a result of excessive intracellular lactate accumulation in some fast-twitch glycolytic fibres. This process could also explain the ultrastructural alterations and the consequent changes in the oxidative enzymes and myofibrillar ATPase staining pattern observed in our and some previous studies.

Evidence that neurones accumulating amyloid can undergo lysis to form amyloid plaques in Alzheimer's disease.

Amyloid has recently been shown to accumulate intracellularly in the brains of patients with Alzheimer's disease (AD), yet amyloid plaques are generally thought to arise from gradual extracellular amyloid deposition. We have investigated the possibility of a link between these two apparently conflicting observations. Immunohistochemistry and digital image analysis was used to examine the detailed localization of beta-amyloid(42) (A beta 42), a major component of amyloid plaques, in the entorhinal cortex and hippocampus of AD brains. A beta 42 first selectively accumulates in the perikaryon of pyramidal cells as discrete, granules that appear to be cathepsin D-positive, suggesting that they may represent lysosomes or lysosome-derived structures. AD brain regions abundantly populated with pyramidal neurones exhibiting excessive A beta 42 accumulations also contained evidence of neuronal lysis. Lysis of these A beta 42-burdened neurones apparently resulted in a local, radial dispersion of their cytoplasmic contents, including A beta 42 and lysosomal enzymes, into the surrounding extracellular space. A nuclear remnant was found at the dense core of many amyloid plaques, strengthening the idea that each amyloid plaque represents the end product of a single neuronal cell lysis. The inverse relationship between the amyloid plaque density and pyramidal cell density in the AD brain regions also supports this possibility, as does the close correlation between plaque size and the size of local pyramidal cells. Our findings suggest that excessive intracellular accumulation of A beta 42-positive material in pyramidal cells can result in cell lysis, and that cell lysis is an important source of amyloid plaques and neuronal loss in AD brains.

Intracellular calcium accumulation during the evolution of hypoxic–ischemic brain damage in the immature rat

An excessive intracellular accumulation of calcium (Ca 2+) in neurons and glia has been proposed to represent a major ‘final common pathway’ for cell death arising from hypoxia–ischemia. To clarify the role of altered calcium flux into the perinatal brain undergoing hypoxic–ischemic damage, 7-day postnatal rats underwent unilateral common carotid artery ligation followed by systemic hypoxia with 8% oxygen. This insult is known to produce brain damage in the form of selective neuronal death or infarction largely limited to the cerebral hemisphere ipsilateral to the arterial occlusion. Either prior to or following hypoxia–ischemia, the rat pups received a s.c. injection of 45CaCl 2, and specimens of blood, cerebrospinal fluid (CSF), and brain were obtained for isotopic measurements and the calculation of the extent of brain intracellular radioactivity. During hypoxia–ischemia, there was a modest increase in intracellular Ca 2+ radioactivity (+28–47%) in both cerebral hemispheres only after 2 h of hypoxia–ischemia. During recovery from 2 h of hypoxia–ischemia, intracellular Ca 2+ accumulated progressively only in the ipsilateral cerebral hemisphere for up to 24 h, during which interval intracellular Ca 2+ decreased in the contralateral hemisphere. No such progressive accumulation was noted during recovery in animals previously exposed to only 1 h of hypoxia–ischemia. The results suggest that a disruption of intracellular Ca 2+ homeostasis is a major contributing factor in the evolution of perinatal hypoxic–ischemic brain damage. Ca 2+ accumulation is a relatively modest and late event during the hypoxic–ischemic phase, and a progressive overload occurs during the recovery phase only if infarction occurs. The question remains as to whether or not the intracellular Ca 2+ overload occurring during recovery is a contributor to or a consequence of the ultimate brain damage.

Availability of oncogene activated production system for mass production of light chain of human antibody in CHO cells.

We previously established a ras-oncogene amplified Chinesehamster ovary (CHO) cell line, named ras clone I, as anuniversal host cell line for oncogene activated production(OAP) system to mass-produce recombinant protein by activationof the cytomegalovirus immediate early (CMV) promoter with ras protein. The lambda light chain(C5lambda) of human monoclonal antibody HB4C5 is expected tobe potentially useful for lung cancer targeting. We generated aC5lambda hyper-producing cell line by transfecting ras cloneI with the C5lambda gene expression plasmid regulated by theCMV promoter, of which productivity was 5.3 times greater thanthe hyper productive CHO cell line generated by using conventional CHO cells. Introduction of the adenovirus E1A geneinto the hyper-producing cell line derived from ras clone I resulted in further 9.5 times enhancement of the productivity,suggesting the synergistic effect of E1A and ras oncogenes on the recombinant protein production driven by the CMV promoter. In addition, intracellular accumulation of C5lambda andupregulation of BiP was found in hyper-producing cell lineswhich were introduced E1A and ras oncogene. This resultsuggests that excessive intracellular accumulation ofC5lambda protein, which might be caused by that the amount of produced C5lambda in ER is beyond the ability of CHO cells to secrete, might signal the BiP promoter. Our data imply that ras clone I is available as a general host cell for establishing the recombinant protein hyper-producing CHOcells by the OAP system, and suggest that further mass production of recombinant proteins in the OAP system can be possible by clarifying the accurate role of upregulated BiP protein.

Inhibition of different pathways influencing Na + homeostasis protects organotypic hippocampal slice cultures from hypoxic/hypoglycemic injury

A prominent feature of cerebral ischemia is the excessive intracellular accumulation of both Na + and Ca 2+, which results in subsequent cell death. A large number of studies have focused on pathways involved in the increase of the intracellular Ca 2+ concentration [Ca 2+] i, whereas the elevation of intracellular Na + has received less attention. In the present study we investigated the effects of inhibitors of different Na + channels and of the Na +/Ca 2+ exchanger, which couples the Na + to the Ca 2+ gradient, on ischemic damage in organotypic hippocampal slice cultures. The synaptically evoked population spike in the CA1 region was taken as a functional measure of neuronal integrity. Neuronal cell death was assessed by propidium iodide staining. The Na + channel blocker tetrodotoxin, and the NMDA receptor blocker MK 801, but not the AMPA/kainate receptor blocker NBQX prevented ischemic cell death. The novel Na +/Ca 2+ exchange inhibitor 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), which preferentially acts on the reverse mode of the exchanger, leading to Ca 2+ accumulation, also reduced neuronal damage. At higher concentrations, KB-R7943 also inhibits Ca 2+ extrusion by the forward mode of the exchanger and exaggerates neuronal cell death. Neuroprotection by KB-R7943 may be due to reducing the [Ca 2+] i increase caused by the exchanger.

Alternative RNA Splicing Generates a Glycosylphosphatidylinositol-anchored Form of Ceruloplasmin in Mammalian Brain

Ceruloplasmin is a copper-containing ferroxidase that is essential for normal iron homeostasis. Whereas ceruloplasmin in plasma is produced and secreted by hepatocytes, in the brain a glycosylphosphatidylinositol (GPI)-anchored form of ceruloplasmin is expressed on the surface of astrocytes. By using a cDNA cloning approach, we have now determined that the GPI-anchored form of ceruloplasmin is generated by alternative RNA splicing. The splicing occurs downstream of exon 18 and replaces the C-terminal 5 amino acids of the secreted form with an alternative 30 amino acids that signal GPI anchor addition. RNase protection analysis demonstrates that the GPI-anchored form is the major form in the brain, whereas the secreted form predominates in the liver. Individuals with aceruloplasminemia, a hereditary deficiency of ceruloplasmin, have severe iron deposition in a number of organs, including the brain where it results in neurodegeneration. Therefore, this novel GPI-anchored form of ceruloplasmin is likely to play an important role in iron metabolism in the central nervous system.

Reduced sarcolemmal dystrophin distribution and upregulation of utrophin in the cardiac and skeletal muscles of CHF-146 dystrophic hamsters.

Abnormalities in the dystrophic gene product, dystrophin, have been implicated in initiating the primary membrane defect and excessive intracellular calcium accumulation (EICA), which play fundamental pathogenic roles in hereditary muscular dystrophy (HMD). Two other cytoskeletal proteins, spectrin and utrophin, bear remarkable structural and functional homologies to dystrophin. CHF-146 strain dystrophic hamsters (DH), like patients with Duchenne muscular dystrophy (DMD), die prematurely from cardiopulmonary insufficiency, focal myonecrosis, and progressive degeneration of the cardiac and skeletal muscles with EICA. Although DH present a suitable model for HMD, there are controversies concerning their dystrophin and utrophin status. Using immunocytochemistry and Western blotting, we studied dystrophin, spectrin and utrophin anomalies in the cardiac and skeletal muscles of 6-mo-old male DH. Age- and sex-matched CHF-148 strain albino normal hamsters (NH) served as controls. Sarcolemmal dystrophin staining was much weaker and interruptive in the DH. The densitometric analysis of the immunoblots revealed that dystrophin is reduced in DH by 83% in cardiac muscle (p < 0.0001), and by 50% in skeletal muscle (p < 0.0001). We conclude that sarcolemmal dystrophin distribution is markedly reduced and discontinuous in the cardiac and skeletal muscles of DH, with simultaneous upregulation of utrophin and a varied degree of spectrin labelling. This observation suggests that reduced sarcolemmal dystrophin is associated with membrane hyperpermeability, which leads to progressive muscle degeneration via EICA and segmental necrosis in DH. As in DMD, utrophin appears to play an important compensatory role in hamster dystrophinopathy.

Is calcium accumulation post-injury an indicator of cell damage?

It is generally agreed that excessive intracellular calcium accumulation is the main culprit for nerve cell damage following brain injury. Many autoradiographic studies of the post-injury brain have demonstrated an accumulation of 45Ca2+ in regions exhibiting neuronal damage. We have recently observed, after cortical contusion trauma [10], that there was a discrepancy between the extent of cell damage and the extent of 45Ca2+ in autoradiograms; rather the distribution of 45Ca2+ followed that of serum proteins. In addition 45Ca2+ was also observed in white matter, which had no signs of damage. We tested the hypothesis that 45Ca2+ accumulation was coupled to the presence of protein by directly injecting albumin into the brain cortex. There was a highly significant correlation between the content of 45Ca2+ and of albumin as measured by ELISA. A similar pattern was found after a cortical freeze-lesion in the contralateral hemisphere. However, in the ipsilateral hemisphere where cell damage was observed, the relation broke down and calcium accumulated in excess. We conclude that calcium accumulation in the brain is not only the result of cell damage but also the presence of calcium-binding proteins, e.g. albumin.

Halothane suppresses the increase in intracellular calcium concentration of isolated rat myocytes during hydrogen peroxide perfusion.

Ischemia-reperfusion injury is probably caused by the generation of oxygen free radicals. The final common pathway to cell injury may be mediated by intracellular calcium overloading induced by oxygen free radicals. Volatile anesthetics have been shown to improve myocardial function following reperfusion. To determine whether or not oxygen radicals are involved in the mechanism by which volatile anesthetics improve myocardial function following reperfusion, we investigated the effects of hydrogen peroxide (H2O2) on the intracellular calcium concentration ([Ca2+]i) in isolated rat ventricular cells. First, the effects of volatile anesthetics, halothane, isoflurane, or sevoflurane, on [Ca2+]i were studied in the absence of H2O2. Next, myocytes were perfused with volatile anesthetics in the presence of H2O2. [Ca2+]i was measured using fura-2, a Ca2+-sensitive fluorescent dye. None of the volatile anesthetics changed [Ca2+]i in the absence of H2O2. In the presence of H2O2, [Ca2+]i gradually increased during H2O2 perfusion. Halothane delayed the onset of the increase in [Ca2+]i induced by H2O2, whereas sevoflurane and isoflurane accelerated the onset. Furthermore, sevoflurane caused more pronounced accumulation of intracellular calcium than did halothane and isoflurane. Therefore, the reduction of excessive intracellular calcium accumulation caused by halothane may have beneficial effects on myocardial function following reperfusion.

Regulation of intracellular cholesterol metabolism is defective in lymphoblasts from Niemann-Pick type C and type D patients

Regulation of intracellular cholesterol metabolism has been studied in Epstein-Barr virus-transformed lymphoblasts from patients with Niemann-Pick type C (NPC) and the Nova Scotia type D (NPD) disease. Addition of LDL to normal lymphoblasts cultured in lipoprotein-deficient medium increased cholesterol esterification 10-fold (to a maximum of 1.0 nmol/h/mg protein at 15 h), while little stimulation was seen in NPC cells. The response by NPD lymphoblasts was intermediate, reaching approximately half of normal values by 12–24 h. Lymphoblasts from both NPC and NPD obligate heterozygotes exhibited 50% of normal LDL-stimulated cholesterol esterification at 6 h, when activity was s 1 ̌ 0% of normal values in patient cells. Fluorescence staining with filipin indicated excessive intracellular accumulation of LDL-derived cholesterol in both NPC and NPD lymphoblasts. Downregulation of LDL receptor mRNA levels by LDL, measured by S1 nuclease protection assay, was also impaired in NP lymphoblasts and fibroblasts (NPC > NPD), although a similar rate of receptor protein down-regulation by LDL ( t 1 2 = 10–15 h ) was observed in normal and NP lymphoblasts. In contrast, LDL down-regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA did not appear to be affected in NP cells: LDL produced a 3-fold (lymphoblasts) of > 10-fold (fibroblasts) decrease by 12 h in both normal and affected cells. Thus, NPC and NPD lymphoblasts exhibit distinct defects in cholesterol esterification and storage, similar to those observed in mutant fibroblasts. Other regulatory responses are also impaired in NPC lymphoblasts but appear to be less affected in NPD cells. Lymphoblasts should provide a valuable immortalized cell line model for study of defective regulation of cholesterol esterification and transfort in Niemann-Pick type II disease, and may also suitable for diagnosis and carrier detection.

Reversal of impaired oxidative phosphorylation and calcium overloading in the in vitro cardiac mitochondria of CHF-146 dystrophic hamsters with hereditary muscular dystrophy

Membrane-mediated excessive intracellular calcium accumulation (EICA), and diminished cellular energy charge are invariably present in the myocardium of CHF-146 strain dystrophic hamsters (DH) with hereditary muscular dystrophy (HMD) and hypertrophic cardiomyopathy (HC). Therefore, we investigated respiratory dysfunctions and Ca 2+ overloading in the isolated cardiac mitochondria from young and old DH, and whether these abnormalities can be reversed by controlling EICA in the in vitro mitochondria upon chelating excessive Ca 2+ from the isolation medium with EDTA. Age- and sex-matched CHF-148 strain albino normal hamsters (NH) served as the disease controls. As an index of membrane-mediated EICA and chronic cellular degeneration, Ca and Mg concentrations were quantitated in the ventricular myocardium and in the cardiac mitochondria harvested in two different isolation media. Mitochondria from young and old DH, isolated in the absence of 10 mM EDTA (B 0 medium), revealed poor coupling of oxidative phosphorylation, diminished stimulated oxygen consumption rate, and lower respiratory control and ADP O ratios, than those seen in NH. However, incorporation of 10 mM EDTA in the isolation medium (B medium) restored the mitochondrial functions and reduced massive Ca 2+-overloading in the dystrophic organelles. Ca concentration in the in vitro mitochondria from DH was significantly higher than in NH, irrespective of the composition of the isolation medium and age of the hamsters. Furthermore, the dystrophic organelles isolated in B medium had a much lower Ca concentration, and markedly improved oxidative phosphorylation as seen in the cardiac mitochondria from NH, compared to those prepared using B 0 medium. Tissue Ca levels in the hearts of both young and old DH were also significantly elevated compared to their normal counterparts, bearing a positive correlation with the intramitochondrial Ca 2+-overloading in DH. However, Mg levels in the myocardium and cardiac mitochondria were similar in NH and DH. These data suggest that mitochondrial dysfunctions and Ca 2+-overloading in HMD with HC may be reversible, if membrane-mediated EICA in the dystrophic myocardium is pharmacologically mitigated by a potent Ca 2+-channel blocker.

Combined Therapy with Dizocilpine and Levemopamil Does Not Reduce Ischemic Neuronal Injury in Rats

Excessive intracellular accumulation of calcium has been postulated to result in ischemic neuronal death. Reduction of intracellular calcium entry should therefore be expected to reduce ischemic neuronal injury. Two pathways through which extracellular calcium ions can enter neurons are voltage-sensitive and N-methyl-D-aspartate receptor-linked cation pores. Combined blockade of both these types of channels might be more effective in reducing intracellular calcium accumulation than the blockade of either channel alone. We therefore evaluated the cerebroprotective effects of dizocilpine, an N-methyl-D-aspartate receptor antagonist, and levemopamil, a phenylalkylamine calcium channel blocker, administered singly or in combination, in a model of forebrain ischemia in the rat. Four groups of rats (n = 8 each) were studied. In the first group, dizocilpine, 5 mg/kg, was administered before ischemia. In the second group, levemopamil, 5 mg/kg, was given both preischemia and 2 h postischemia. In the third group, both dizocilpine (5 mg/kg) and levemopamil (5 mg/kg) were given preischemia and levemopamil (5 mg/kg) was given postischemia. The control group received saline placebo. The rats were subjected to forebrain ischemia by bilateral carotid artery occlusion for 10 min with simultaneous hypotension to 35 mm Hg. Neuronal injury was evaluated 3 days after ischemia. Dizocilpine reduced postischemia neuronal injury in the ventral hippocampus (p = 0.045). Levemopamil and the combination of levemopamil and dizocilpine did not protect neurons from ischemic injury. The present study does not provide support for the strategy of combined therapy with dizocilpine (administered before ischemia) and levemopamil (administered before and after ischemia) to protect neurons from injury produced by severe incomplete forebrain ischemia.

Regulation of membrane-mediated chronic muscle degeneration in dystrophic hamsters by calcium-channel blockers: diltiazem, nifedipine and verapamil

Membrane-mediated excessive intracellular calcium accumulation (EICA) is a fundamental pathogenetic event associated with chronic muscle degeneration in patients with Duchenne muscular dystrophy (DMD), and in animals with hereditary muscular dystrophy (HMD). Because of potential Ca 2+-channel blocking properties, we investigated the relative efficacies of chronic diltiazem (DTZM) (50 mg/kg/d), nifedipine (NFDN) (6 mg/kg/d), and verapamil (VPML) (25 mg/kg/d) therapies in reducing EICA and improving dystrophic pathobiology beginning in 30-day-old male BIO-14.6 strain dystrophic hamsters (DH). Each agent, and sterile distilled water as vehicle control, was given in a single daily oral dose for 180 days to four groups each of DH and BIO-F1B strain normal hamsters (NH). Plasma [Ca] and [Mg]; plasma aldolase (ALD), creatine kinase (CK), and lactate dehydrogenase (LDH) activities; relative cardiac hypertrophy and relative soleus hypertrophy; tissue [Ca] and [Mg] of the heart and rectus femoris muscle, histology of rectus femoris, and overall mortality rate were quantitated. Muscle Mg was not modified in DH, or by any of these agents. NFDN produced significant edema in the soleus and myocardium. During the 6-month therapeutic trial, 45% DH and 18% NH died on VPML, 27% DH and 9% NH on NFDN, and 20% DH controls on distilled water, but none on DTZM; suggesting that DTZM treated DH lived longer than DH controls. Relative efficacy in regulating EICA in both the cardiac and skeletal muscles; plasma ALD, CK, and LDH; and improving associated dystrophic pathobiology was found to be DTZM > > > NFDN > VPML. DTZM appears to be the most effective and safest agent in mitigating EICA in cardiac and skeletal muscles, efflux of intracellular enzymes, histopathology of dystrophic muscle with sporadic necrosis, and chronic muscle degeneration in DH with HMD. DTZM therapy also halted the high morbidity and mortality associated with the dystrophic pathobiology inherent in DH.