Muscle Microsomes Research Articles

Porcine oxymyoglobin and lipid oxidation in vitro

The present study was carried out to investigate the effect of dietary α-tocopherol supplementation on porcine oxymyoglobin (OxyMb) and lipid oxidation (TBARS) in model lipid systems, and to determine the influence of 4-hydroxynonenal (4-HNE), a secondary product of lipid oxidation, on porcine OxyMb stability. Porcine metmyoglobin (MetMb) formation was greater in the presence of 4-HNE than the control ( P<0.05). Western blot analyses confirmed the covalent modification of myoglobin (Mb) histidine residues by 4-HNE. When combined with microsomes, both equine and porcine OxyMb oxidation increased with time of incubation, and was greater at 37 than at 25 °C ( P<0.05). Lower TBARS values were observed in microsomes prepared from vitamin E-supplemented than control pork livers ( P<0.05). α-Tocopherol concentration did not affect OxyMb oxidation in microsomes at 25 or 37 °C ( P>0.05). These results differ from those observed with beef muscle microsomes where both OxyMb and lipid oxidation were delayed with elevated α-tocopherol levels. These results suggest that the factors affecting Mb and lipid oxidation interactions may be species-dependent.

Role of endogenous regucalcin in transgenic rats: suppression of kidney cortex cytosolic protein phosphatase activity and enhancement of heart muscle microsomal Ca2+-ATPase activity.

Rats were generated by pronuclear injection of the transgene with a cDNA construct encoding rat regucalcin that is a regulatory protein of Ca2+ signaling. Transgenic (TG) founders were fertile, transmitted the transgene at the expected frequency, and bred to homozygote. Western analysis of the cytosol prepared from the tissue of TG female rats (5-week-old) showed a remarkable expression of regucalcin (3.3 kDa) protein in the liver, kidney cortex, heart, lung, stomach, brain, spleen, muscle, colon, and duodenum. Regucalcin expression of TG male rats was seen in the liver, kidney cortex, heart, and lung. In wild-type (wt) male and female rats, regucalcin was mainly present in the liver and kidney cortex. Regucalcin inhibited protein phosphatase activity in rat kidney cortex cytosol and activated Ca2+-ATPase activity in rat heart muscle microsomes. The suppressive effect of regucalcin on protein phosphatase activity was significantly enhanced in the cytosol of kidney cortex of TG male and female rats as compared with those of wt rats. Likewise, heart muscle microsomal Ca2+-ATPase activity was significantly enhanced in TG rats. The changes in their enzyme's activities in TG rats were completely abolished in the presence of anti-regucalcin monoclonal antibody (100 ng/ml) in the enzyme reaction mixture. Moreover, the body weight of TG female rats was significantly lowered as compared with that of wt rats. Serum inorganic phosphorus concentration was significantly increased in TG male and female rats, while serum calcium, glucose, triglyceride, free cholesterol, albumin, and urea nitrogen concentrations were not significantly altered in TG rats. Regucalcin TG rats should be a useful model to define a regulatory role of endogenous regucalcin in the tissues in vivo.

Influence of dietary vitamin E and selenium on muscle fatty acid composition in pigs

A total of 28 female pigs were fed a basal diet containing a low amount of α-tocopherol (10.3 mg/kg; control), and diets supplemented with 0.3 mg selenium/kg (group Se) or with 200 mg α-tocopherol/kg (group V) at the growing-finishing period. Increasing dietary level of vitamin E resulted in higher concentration of α-tocopherol in plasma and muscle immediately after slaughter and 4 h later ( P⩽0.05). The fatty acid composition of muscle microsomes and mitochondria was slightly affected by the diet. Corresponding to the minor changes of the membrane fatty acid composition the fluidity was unaffected by the diet. A positive relationship was observed between the resistance to in vitro stimulation of peroxidation and the vitamin E content of the muscle samples. Supplementation with selenium did not reduce the lipid oxidation after stimulation. In conclusion, even though the effect was minor, vitamin E improved the antioxidative status in pork.

Ryanodine Receptor Channel-Dependent Glutathione Transport in the Sarcoplasmic Reticulum of Skeletal Muscle

We found that glutathione transport across endo/sarcoplasmic reticulum membranes correlates with the abundance of ryanodine receptor type 1 (RyR1). The transport was the fastest in muscle terminal cisternae, fast in muscle microsomes and slow in liver, heart, and brain microsomes. Glutathione influx could be inhibited by RyR1 blockers and the inhibitory effect was counteracted by RyR1 agonists. The effect of blockers was specific to glutathione, as the transport of other small molecules was not hindered. Therefore, the glutathione transport activity seems to be associated with RyR1 in sarcoplasmic reticulum.

FKBP12 associates tightly with the skeletal muscle type 1 ryanodine receptor, but not with other intracellular calcium release channels

This study compared the relative levels of ryanodine receptor (RyR) isoforms, inositol 1,4,5-trisphosphate receptor (IP 3R) isoforms, and calcineurin, plus their association with FKBP12 in brain, skeletal and cardiac tissue. FKBP12 demonstrated a very tight, high affinity association with skeletal muscle microsomes, which was displaced by FK506. In contrast, FKBP12 was not tightly associated with brain or cardiac microsomes and did not require FK506 for removal from these organelles. Furthermore, of the proteins solubilised from skeletal muscle, cardiac muscle and brain microsomes, only skeletal muscle RyR1 bound to an FKBP12–glutathione- S-transferase fusion protein, in a high affinity FK506 displaceable manner. These results suggest that RyR1 has distinctive FKBP12 binding properties when compared to RyR2, RyR3, all IP 3R isoforms and calcineurin.

Peroxynitrite-induced oxidation of lipids: implications for muscle foods.

Peroxynitrite (ONOO(-)), formed from the nearly diffusion limited reaction between nitric oxide and superoxide, could be an important prooxidant in muscle foods. The objective of this study was to determine whether peroxynitrite caused oxidation of pyrogallol red, liposomes, muscle microsomes, and skeletal muscle homogenate. Oxidation of pyrogallol red, liposomes, and microsomes initiated by peroxynitrite continuously produced by 3-morpholinosydnonimine (SIN-1, 2 mM) was time-dependent and enhanced by CO(2) (1 mM). Reagent peroxynitrite (2 mM) caused concentration-dependent oxidation of pyrogallol red, liposomes, and muscle microsomes that was very rapid with no change after 5 min. Peroxynitrite-induced oxidation was suppressed by CO(2) and low pH. Skeletal muscle homogenate oxidized by reagent peroxynitrite (0.5 mM) exhibited gradual oxidation with time and was suppressed by CO(2), low pH, and metal chelators. These data suggest that peroxynitrite could be an important prooxidant in muscle foods.

Pyrrolization and antioxidant function of proteins following oxidative stress.

The consequences of oxidative stress on microsomal proteins were analyzed by studying their pyrrolization and the antioxidative activity of the modified proteins produced. The microsomal system consisted of freshly prepared trout muscle microsomes, which were oxidized in the presence of 5 microM Cu(2+), 1 mM Fe(3+)/5 mM ascorbate, or 1 mM Cu(2+)/10 mM H(2)O(2). Pyrroles on proteins were detected by forming Ehrlich adducts with p-(dimethylamino)benzaldehyde and by determination of epsilon-N-pyrrolylnorleucine (Pnl) by capillary electrophoresis. Their antioxidative activity was studied by testing two model pyrrolized proteins (dimeric and monomeric modified bovine serum albumin: DBSA and MBSA, respectively), which were produced in the reaction of BSA and 4,5(E)-epoxy-2(E)-heptenal. These proteins were assayed at a concentration of 10-40 microg/mL, which was selected because at this concentration both DBSA and MBSA had a concentration of Pnl similar to the Pnl concentration produced in oxidized microsomes. Both DBSA and MBSA significantly (p < 0.05) protected against lipid peroxidation, assessed by the formation of thiobarbituric acid reactive substances (TBARS), and protein damage, evaluated by amino acid analysis, for the three systems assayed, and this protection was always higher than that exhibited by BSA, which was used as control. The order of effectiveness was DBSA > MBSA > BSA and was parallel to the Pnl content in the assayed proteins. These results suggest that antioxidative activity of BSA may also be related to its ability to react with lipid oxidation products and to produce modified BSA with antioxidative activity. This mechanism may also be contributing to the antioxidative activity exhibited by many proteins.

Effects of three different Ca 2+ pump ATPase inhibitors on evoked contractions in rabbit aorta and activities of Ca 2+ pump ATPases in porcine aorta

Using vascular smooth muscle, we describe the actions of three pharmacological tools, cyclopiazonic acid (CPA), thapsigargin (TG) and 2,5-di-( tert-butyl)-1,4-benzohydroquinone (tBHQ), which are presumed to act as selective inhibitors of the sarco-endoplasmic reticulum Ca 2+-ATPases (SERCAs). In porcine aortic smooth muscle microsomes two Ca 2+-ATPase activities have been described, one vanadate-sensitive and one vanadate-resistant, representing the Ca 2+-ATPase activities of the plasma membrane and SERCAs, respectively. In agreement, CPA, TG and tBHQ, in the concentration range 0.1 μM to 0.1 mM, dose-dependently inhibit the Ca 2+-ATPase activity only in the vanadate-resistant microsomes. However, 0.1 mM tBHQ also significantly inhibited the Ca 2+-ATPase activity of vanadate-sensitive microsomes. In rabbit aortic rings, all three SERCA inhibitors produced a dose-dependant inhibition of contractions evoked by 20 mM caffeine or 1 μM phenylephrine (PE) in a Ca 2+-free physiological solution. However, in PE-contracted rings, tBHQ (≥30 μM) also significantly inhibited the ability of cromakalim to induce relaxation. In conclusion, the data suggest that CPA, TG and tBHQ can all act as selective SERCA inhibitors in both porcine and rabbit aortic smooth muscle. However, in contrast to CPA and TG, high concentrations of tBHQ can exhibit some nonspecific effects, which include inhibition of the plasma membrane Ca 2+-ATPase and possibly K + channels regulated by cromakalim.

Increase in Content of Glycogen Phosphorylase during Ischemia in Isolated Rabbit Heart.

The amount of a 97-kDa protein in the sarcoplasmic reticulum-enriched microsomal fraction from rabbit heart changed in a manner dependent on the oxygenated condition after global hypothermic ischemia and subsequent normothermic reperfusion. Isolated hearts were immersed in physiologic saline for 0h (control group) or 1, 2 and 3h at 4°C (ischemia group) and were subjected to 40-min reperfusion at 37°C after 1h of hypothermic ischemia (reperfused group). The amount of the 97-kDa protein in the microsomal fraction apparently increased after ischemia, but decreased after reperfusion. The N-terminal amino acid of the protein was blocked and two internal sequences were determined, demonstrating the 97-kDa protein to be glycogen phosphorylase. A polyclonal antibody against rabbit skeletal muscular glycogen phosphor-ylase (GP) stained only the 97-kDa protein in the microsomal fraction in Western blot analysis. Microsomal fractions isolated from rabbit brain, liver, spleen, and white skeletal muscle were also examined by Western blot analysis using anti-GP. Liver and skeletal muscle contained a 97-kDa protein that reacted with anti-GP in control preparations. Ischemia induced a modest increase in the amount of the 97-kDa protein skeletal muscle microsomes, whereas no change in the amount of the protein occurred in liver microsomes. The results indicate that alteration in the energy metabolism from ischemia to the subsequent reperfusion is associated with the amount of GP.

Increased butyrylcholinesterase levels in microsomal membranes of dystrophic Lama2dy mouse muscle.

The proportions and the glycosylation of butyrylcholinesterase (BuChE) forms in vesicles rich in sarcoplasmic reticulum from normal (NMV) and dystrophic (DMV) muscle were analyzed, using merosin-deficient dystrophic mice. BuChE activity in DMV was two- to threefold that in NMV. Globular amphiphilic G1A, G2A, and G4A and hydrophilic G4H BuChE forms were identified in NMV and DMV. The amount of G2A forms increased sevenfold in DMV, and the other forms increased about twofold. The higher BuChE level in DMV might reflect a maturational defect, with dystrophy preventing the down-regulation of BuChE with muscle development. About half of G1A, G2A, and G4H BuChE forms in NMV or DMV bound to Lens culinaris agglutinin (LCA), a higher fraction to wheat germ agglutinin (WGA), and little to Ricinus communis agglutinin (RCA). Most of the G4A forms in NMV or DMV bound to LCA or WGA; those from NMV failed to bind to RCA, whereas most of the variants in DMV bound to it, suggesting that the excess of tetramers in DMV is mainly RCA-reactive. The differential interaction of lectins with BuChE components from muscle microsomes, serum, and nerves confirmed that the microsomal BuChE was muscle-intrinsic. The results provide clues regarding the alterations that dystrophy produces in the biosynthesis of BuChE forms in muscle.

Alpha-tocopherol oxidation in beef and in bovine muscle microsomes.

The oxidation of alpha-tocopherol (TH) in beef was analyzed using a stable isotope dilution capillary gas chromatography-mass spectrometry assay. TH decreased while alpha-tocopherolquinone (TQ) and 2,3-epoxy-alpha-tocopherolquinone (TQE(2)) increased in ground longissimus lumborum (LL) and psoas major (PM) muscles during storage (P < 0.10). In LL steaks, the relative concentrations of TH decreased and TQ and TQE(2) increased in surface samples; changes were less dramatic in deep samples. Deuterated alpha-tocopherolhydroquinone (THQ) standard was not recovered and endogenous THQ was not detected in meat; THQ was measurable in microsomes isolated from PM and incubated in the presence of 2, 2'-azobis(2-amidopropane)HCl (ABAP) or myoglobin. ABAP-challenged microsomes yielded a tocopherol product profile which favored 5, 6-epoxy-alpha-tocopherolquinone (TQE(1)) and TQE(2), while the use of myoglobin as prooxidant resulted in a higher proportion of TQ and THQ. Results demonstrated that concentrations of TH decreased and TQ and TQE(2) increased in meat during storage and are consistent with the peroxy-radical scavenging function of tocopherol.

Synthesis of an unsaturated fatty acid analogue (18-(4′-azido-2′-hydroxybenzoylamino)-oleic acid) and its interaction with lysophosphatidylcholine: acyl-CoA-O-acyltransferase

Acylation/deacylation reactions represent a basic requirement of triglyceride as well as phospholipid metabolism, and maintenance of membrane lipid composition. In order to examine enzymes participating in these pathways, we synthesized 18-(4'-azido-2'-hydroxybenzoylamino)-oleic acid, an iodinable photoaffinity analogue of oleic acid as a new tool for analyzing enzymes, especially those binding unsaturated fatty acids or acyl-CoAs. For the synthesis of omega-amino-oleic acid, coupling two bifunctional Cg-components was used. The described synthesis scheme is also suited for the specific generation of other fatty acid analogues with distinct positions of the double bond. The functionality of 18-(4'-azido-2'-hydroxybenzoylamino)-oleic acid was investigated with the enzyme lysophosphatidylcholine:acyl-CoA-O-acyltransferase (LAT) [EC 2.3.1.23], an enzyme that shows high specificity towards (poly)unsaturated fatty acyl-CoAs. It could be shown that the photolabel, esterified with coenzyme A, acts in the dark as a reversible inhibitor of the enzyme activity, but photolysis of the label results in irreversible inactivation of LAT. This inactivation could be prevented by addition of the native substrate arachidonyl-CoA during photolysis. Several proteins could be specifically visualized using the iodinated analogue. The data indicate that this new photoaffinity label may have application to identify and characterize lipid biosynthetic enzymes using unsaturated fatty acids as well as acyl-CoA binding proteins and the active site of these proteins.

Temperature Dependency of 5′-Nucleotidase and Acid Phosphatase in Muscle Microsomes of Fish

Relationships between kinetics of IMP dephosphorylation by muscle microsomal enzymes and lipid composition of muscle microsomes were compared among fish of different habitat temperature, yellowtail, grunt, Spanish mackerel, rainbow trout and Pacific cod. The Km values of microsomal 5′-nucleotidase of fish except cod markedly increased at a temperature range below the break point on the Arrhenius plots of the enzyme activity. The break point temperatures in Arrhenius plots for microsomal 5′-nucleotidase were correlated with the content of saturated and polyunsaturated fatty acids in phospholipids of muscle microsomes in the fish examined. The results suggest that the occurrence of a break point in the Arrhenius plot of microsomal 5′-nucleotidase may be caused by the lipid phase transition of membranes that bind 5′-nucleotidase. Break point temperature may depend on microsomal lipid composition which is closely related to the habitat temperature of the fish.

Kinetic basis of quantal calcium release from intracellular calcium stores

The kinetics of Ca2+ release from canine cerebellum and rabbit skeletal muscle microsomes, mediated by the inositol 1,4,5-trisphosphate (IP 3) receptor (IRC) and the ryanodine receptor (RyRC), respectively, were analyzed by a model, which considers that Ca 2+ release channels undergo spontaneous inactivation. We found that: (i) both the initial rate of release (v o) and the rate of inactivation (v 1) were saturable functions of the activating ligand concentration (C L); and (ii) the ratio of v i/v o, termed the relative tendency for inactivation, decreased with increasing C L. Equilibrium [ 3H]-IP 3 binding studies, on the other hand, revealed the presence of one single class of non-co-operative IP 3 sites in cerebellum membranes (K deq = 47 nM and Hill coefficient = 1.1). Based on the above v i−v o relationship and the IP 3-binding data, we propose that quantal Ca 2+ release through IRCs might be a result of spontaneous channel inactivation, whose rate is controlled by the ratio of IP 3-occupied/free monomers in the tetrameric release channel units. Furthermore, because of the kinetic similarities between the IRC- and RyRC-mediated Ca 2+ release processes, as well as between quantal Ca 2+ release and channel adaptation, the same mechanism is also proposed to apply to the RyRC-mediated Ca 2+ release as well as to constitute the basis of release channel adaptation.

Differential interaction of the monoclonal antibody AE-1 with acetylcholinesterase oligomers and monomers from rabbit muscle microsomes, human brain and fetal bovine serum

The monoclonal antibody AE-1 raised against acetylcholinesterase (AChE) from human erythrocytes (HE) is shown to react with active asymmetric and tetrameric AChE components from rabbit muscle microsomes (RMM), and with tetrameric forms from human brain (HB) or fetal bovine serum (FBS). However, it failed to bind to AChE monomers from RMM or HB. The results of Western blot revealed that the determinant for AE-1 consisted of a conformational domain, not a primary sequence region, in the AChE subunit. The antibody recognized HE monomers and FBS dimers, but not FBS monomers. The formation of labile immunocomplexes between AE-1 and AChE subunits may explain the lack of interaction between the antibody and the monomers from non-erythrocyte sources.

Feed-Back Inhibition of Oxidative Stress by Oxidized Lipid/Amino Acid Reaction Products

Three oxidized lipid/amino acid reaction products (OLAARPs): 1-methyl-4-pentyl-1,4-dihydropyridine-3,5-dicarbaldehyde, 1-(5-amino-1-carboxypentyl)pyrrole, and N-(carbobenzyloxy)-1(3)-[1-(formylmethyl)hexyl]-l-histidine dihydrate, were prepared and tested for antioxidative activity in a microsomal system in order to investigate the effect that OLAARP formation may be playing in the oxidative stress process. The microsomal system consisted of freshly prepared trout muscle microsomes, which were oxidized in the presence of 5 microM Cu2+, 1 mM Fe3+/5 mM ascorbate, or 1 mM Cu2+/10 mM H2O2, and the compound to be tested as antioxidant added at 50 microM. At different periods of time, samples were tested for lipid peroxidation, assessed by the formation of thiobarbituric acid reactive substances (TBARS), and protein damage, which was evaluated by the formation of protein carbonyls and amino acid analysis. The three OLAARPs and butylated hydroxytoluene significantly (p < 0.05) protected against lipid peroxidation and protein damage for the three systems assayed. On the contrary, neither the amino acids used in the preparation of OLAARPs nor alpha-tocopherol, mannitol, aminoguanidine, or 4, 5-dihydroxy-1,3-benzenedisulfonic acid exhibited this constant protection. Because OLAARPs were produced at inhibitory levels during microsomal lipid peroxidation, these results suggest that OLAARP formation may be an antioxidative defense mechanism by which oxidative stress is feed-back-inhibited, delaying the damage caused by reactive oxygen species.

Effect of extensive feeding on α-tocopherol concentration and oxidative stability of muscle microsomes from Iberian pigs

AbstractThe effect of extensive feeding, confinement and diet supplementation with a-tocopheryl acetate (100 mg/kg) on the fatty acid composition and tocopherol concentration of microsome extracts and their susceptibility to oxidation was studied in Iberian pigs. The diet of pigs raised extensively was mostly composed of acorn and grass. The a-tocopherol contents of acorn and grass were 20 and 171 mg/kg dry matter, respectively. Microsomal fatty acid composition showed no differences among groups. Pigs feeding extensively had a higher concentration of a-tocopherol in muscle and microsomes than pigs given mixed diet with the basal level of a-tocopheryl acetate (P &lt; 0·05) but lower values than pigs given supplementary levels (100 mg/kg) (P &lt; 0·05). Microsomal fractions from pigs given mixed diet with a basal level of a-tocopheryl acetate were significantly more susceptible to iron-induced lipid oxidation than extract from pigs given diets containing a supplementary level (P &lt; 0·05). Microsomal extracts from pigs feeding extensively had the lowest oxidation rate (P &lt; 0·05), suggesting that other dietary constituents may play a role in the stabilization of microsomal lipids.

Membrane phosphorylation protects the cardiac sarcoplasmic reticulum Ca(2+)-ATPase against chlorinated oxidants in vitro.

The calcium (Ca) pump of cardiac sarcoplasmic reticulum (SR) membranes is vulnerable to oxidation and hence likely to be damaged by chlorinated compounds, specifically hypochlorite (NaOCl) and monochloramine (NH2Cl), the most potent oxidants produced upon neutrophil activation. This could occur during prolonged ischemia or myocardial infarction when tissue levels of catecholamines are high. Phospholamban (PLN), the phosphorylatable regulator of the Ca pump, plays a central role in the effects of beta-adrenergic agonists on the heart. The purpose of this study was to investigate a possible role of PLN in determining the pump's sensitivity to NaOCl and NH2Cl. Ca-uptake and Ca(2+)-ATPase activities in purified phosphorylated and control canine cardiac microsomes, incubated at increasing concentrations of NaOCl or NH2Cl, were related to the extent of PLN phosphorylation by protein kinase A, which was quantitated by PhosphorImager analysis. Our data indicate that microsomal phosphorylation protects the Ca pump fully against 10 microM NaOCl or NH2Cl, which inhibit Ca-uptake by 21-41% when assayed at 25 or 37 degrees C and saturating Ca2+ in unphosphorylated microsomes, and protects partially at higher oxidant concentrations. The protective effect of protein kinase A on Ca-uptake is proportional to the amount of phosphorylated PLN. No comparable protection against similar oxidative damage of the Ca pump is observed when light fast skeletal muscle microsomes, which lack PLN, are incubated under conditions favorable for phosphorylation nor when PLN's inhibition of the cardiac Ca pump is relieved by proteolytic cleavage of its cytoplasmic domain. Our findings contribute toward an understanding of possible endogenous protective mechanisms that may promote calcium homeostasis in myocardial cells in inflammatory states associated with neutrophil activation and may suggest an approach toward development of protective strategies against oxidative damage in the heart.

Extracellular interaction of the voltage-dependent Ca2+ channel alpha2delta and alpha1 subunits.

The role of the extracellular domain of the voltage-dependent Ca2+ channel alpha2delta subunit in assembly with the alpha1C subunit was investigated. Transiently transfected tsA201 cells processed the alpha2delta subunit properly as disulfide linkages and cleavage sites between the alpha2 and delta subunits were shown to be similar to native channel protein. Coimmunoprecipitation experiments demonstrated that in the absence of delta subunits, alpha2 subunits do not assemble with alpha1 subunits. Furthermore, the transmembrane and cytoplasmic sequences in delta can be exchanged with those of an unrelated protein without any effect on the association between the alpha2delta and alpha1 proteins. Extracellular domains of the alpha2delta subunit are also shown to be responsible for increasing the binding affinity of [3H]PN200-110 (isopropyl-4-(2,1, 3-benzoxadiazol-4-yl)-1,4-dihydro-2, 6-dimethyl-5-([3H]methoxycarbonyl)-pyridine-3-carboxylate) for the alpha1C subunit. Investigation of the corresponding interaction site on the alpha1 subunit revealed that although tryptic peptides containing repeat III of native alpha1S subunit remain in association with the alpha2delta subunit during wheat germ agglutinin chromatography, repeat III by itself is not sufficient for assembly with the alpha2delta subunit. Our results suggest that the alpha2delta subunit likely interacts with more than one extracellular loop of the alpha1 subunit.

An investigation into the role of rat skeletal muscle as a site for xenobiotic metabolism using microsomes and isolated cells

1. The role of skeletal muscle microsomes as a site of extrahepatic xenobiotic metabolism using n-hexane as a model substrate was investigated. The observed cytochrome P450-dependent metabolism was compared with that found with liver, and brain microsomal fractions. 2. Rat skeletal muscle microsomes metabolised n-hexane to 1-, 2- and 3-hexanol at rates 40-300 times lower than observed with rat liver microsomes. 3. Fast-twitch extensor digitorum longus muscle (EDL) microsomes had twice as much n-hexane hydroxylase activity as the slow-twitch soleus and furthermore the EDL microsomes produced 2-hexanol, a bioactivation product of n-hexane, as a major metabolite. 4. Metabolism of hexane to 1-, 2- and 3-hexanol and 2-hexanon was demonstrated in cultured rat myoblasts. 5. Ethoxyresorufin and pentoxyresorufin O-dealkylation were not detected in either muscle microsomes or myoblasts although immunocytochemical localisation studies were suggestive of the presence of cytochrome P-450. 6. In conclusion, rat skeletal muscle has a low level of xenobiotic metabolism activity. The relevance to neuromuscular toxicity of n-hexane is discussed.