Relationship of a dystrophin-associated glycoprotein to junctional acetylcholine receptor clusters in rat skeletal muscle

The relationship of a member of the transmembrane dystrophin-associated glycoprotein (DAG) complex to acetylcholine receptors (AChRs) was investigated using immunofluorescence techniques at rat neuromuscular junctions (NMJs) viewed en face. These results were compared with those from a similar previous study of dystrophin and an autosomal homologue (utrophin or dystrophin-related protein, DRP) (Bewick et al. Neuro Report 1992; 3: 857-860). The region of highest 43 K DAG (43DAG) labelling projected beyond the AChRs by approximately 0.3 microns, as does that for dystrophin. By contrast DRP labelling precisely co-localizes with the AChRs. These results suggest that at the NMJ, the region of high 43DAG concentration encompasses the area of highest intensity labelling for both DRP and dystrophin.     

Discrimination between periportal and pericentral necrosis of rat liver by determination of glutamine synthetase and other enzyme activities in serum

Periportal or pericentral necrosis of rat liver was produced by injection of allyl-alcohol or bromobenzene, respectively. Activities of predominantly periportal and perivenous enzymes were determined in serum during maximal necrosis. Aspartate aminotransferase, which is more or less homogeneously distributed in the liver acinus, exhibited similar activities in serum after periportal and pericentral injury. Serum activities of the mainly periportal enzymes alanine aminotransferase and fructose 1,6-bisphosphatase were 1.5- to 2-fold higher after periportal as compared to pericentral necrosis. Serum activity of the mainly pericentral glutamate dehydrogenase was 3-fold higher after pericentral than after periportal damage. However, due to individual variations necrosis could not be definitively localized in any case by measurement of these enzyme activities. Better discrimination between periportal and pericentral necrosis was achieved by the serum activity of the exclusively pericentral enzyme glutamine synthetase, which was 8-fold higher after pericentral as compared to periportal necrosis. Conclusive discrimination was obtained by the activity ratio fructose 1,6-bisphosphatase/glutamine synthetase in serum.     

The cytoplasmic loops between domains II and III and domains III and IV in the skeletal muscle dihydropyridine receptor bind to a contiguous site in the skeletal muscle ryanodine receptor

Excitation-contraction coupling in skeletal muscle is a result of the interaction between the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor or RyR1) and the skeletal muscle L-type Ca2+ channel (dihydropyridine receptor or DHPR). Interactions between RyR1 and DHPR are critical for the depolarization-induced activation of Ca2+ release from the sarcoplasmic reticulum, enhancement of DHPR Ca2+ channel activity, and repolarization-induced inactivation of RyR1. The DHPR III-IV loop was fused to glutathione S-transferase (GST) or His-peptide and used as a protein affinity column for 35S-labeled, in vitro translated fragments from the N-terminal three-fourths of RyR1. RyR1 residues Leu922-Asp1112 bound specifically to the DHPR III-IV loop column, but the corresponding fragment from the cardiac ryanodine receptor (RyR2) did not. Construction of chimeras between RyR1 and RyR2 showed that amino acids Lys954-Asp1112 retained full binding activity, whereas Leu922-Phe1075 had no binding activity. The RyR1 sequence Arg1076-Asp1112, previously shown to interact with the DHPR II-III loop (Leong, P., and MacLennan, D., H. (1998) J. Biol. Chem. 273, 7791-7794), bound to DHPR III-IV loop columns, but with only half the efficiency of binding of the longer RyR1 sequence, Lys954-Asp1112. These data suggest that the site of DHPR III-IV loop interaction contains elements from both the Lys954-Phe1075 and Arg1076-Asp1112 fragments. The presence of 4 +/- 0.4 microM GST-DHPR II-III or 5 +/- 0.1 microM His-peptide-DHPR III-IV was required for half-maximal co-purification of 35S-labeled RyR1 Leu922-Asp1112 on glutathione-Sepharose or Ni2+-nitrilotriacetic acid. Dose-dependent inhibition of 35S-labeled RyR1 Leu922-Asp1112 binding to GST-DHPR II-III and GST-DHPR III-IV by His10-DHPR II-III and His-peptide-DHPR III-IV was observed. These studies indicate that the DHPR II-III and III-IV loops bind to contiguous and possibly overlapping sites on RyR1 between Lys 954 and Asp1112.     

Degradative activity of granzyme A on skeletal muscle proteins in vitro: a possible molecular mechanism for muscle fiber damage in polymyositis

The importance of cytotoxic T lymphocytes (CTLs) in the autoimmune inflammatory myopathies, especially polymyositis (PM), has been emphasized. We have studied the degradative activity of granzyme A, a cytotoxic molecule with trypsin-like specificity in CTL granules, on several muscle proteins in vitro. Our study reveals that granzyme A hydrolyzes dystrophin, myosin, and nebulin, but not laminin, alpha-actinin, vinculin, and connectin in vitro. Among these proteins, nebulin is more susceptible to proteolysis, followed by dystrophin, myosin heavy chain, and myosin light chains, in that order. This result implies an important role of granzyme A in CTL-mediated muscle fiber damage in PM.     

A dual mechanism for impairment of GABAA receptor activity by NMDA receptor activation in rat cerebellum granule cells

It has been estimated that over three million workers in the USA are potentially exposed to silica or other mineral dusts. Results of epidemiological studies evaluating whether silica or glass fibers increase lung cancer risk to the exposed workers are inconclusive. Detection of DNA damage in cells exposed to genotoxic agents is being used to assess the carcinogenic potential of environmental agents. The alkaline (pH > 13) single cell gel/comet (SCG) assay was used to determine and compare DNA damage in cultured Chinese hamster lung fibroblasts (V79 cells) and human embryonic lung fibroblasts (Hel 299 cells) exposed to crystalline silica (Min-U-Sil 5), amorphous silica (Spherisorb), carbon black, and glass fibers (AAA-10). V79 or Hel 299 cells were exposed to these mineral dusts for 3 h at various concentrations. Min-U-Sil 5 and AAA-10, at almost all concentrations tested, caused a significant increase in DNA migration measured as tail length in both V79 and Hel 299 exposed cells. However, the increase was much higher in V79 then in Hel 299 cells for Min-U-Sil 5. Tail length was also increased relative to controls after amorphous silica treatment, but not to the same extent as that induced by crystalline silica. Exposure to carbon black did not induce DNA migration at any of the concentrations tested. These results indicate that silica and glass fibers, but not carbon black, can induce DNA damage in mammalian cells, and that crystalline silica has a higher DNA-damaging activity than amorphous silica. For glass fibers, induction of DNA damage in both V79 and Hel 299 cells was observed even at a concentration 10 times lower than silica and the response was similar in both cell lines. These results suggest that the SCG/comet assay is useful for the detection of DNA damage caused by occupationally related dusts/particles.     

The dependence on the extramitochondrial ATP/ADP-ratio of the oxidative phosphorylation in mitochondria isolated by a new procedure from rat skeletal muscle

A simple method for preparation of rat skeletal muscle mitochondria is presented using gentle mechanical homogenization in a syringe and nagarse treatment (EC 3.4.4.16). This method enables the preparation of skeletal muscle mitochondria, whose outer membrane is intact to 95%. Furthermore, with mitochondria prepared by this method the regulation of respiration and phosphorylation by the extramitochondrial ATP/ADP-ratio can be demonstrated. In accordance to rat liver and heart mitochondria and to mitochondria of rabbit reticulocytes, the regulation by the extramitochondrial ATP/ADP-ratio lies in the range from 5 (corresponding to 98% of the maximum respiration) to 100 (corresponding to state 4). At extramitochondrial ATP/ADP-ratios from 0.01 to 1 the respiration rate is nearly constant (maximum rate of respiration).     

Coordinated induction of MRP/GS-X pump and gamma-glutamylcysteine synthetase by heavy metals in human leukemia cells

We recently reported that GS-X pump activity, as assessed by ATP-dependent transport of the glutathione-platinum complex and leukotriene C4, and intracellular glutathione (GSH) levels were remarkably enhanced in cis-diamminedichloroplatinum(II) (cisplatin)-resistant human leukemia HL-60 cells (Ishikawa, T., Wright, C. D., and Ishizuka, H. (1994) J. Biol. Chem. 269, 29085-29093). Now, using Northern hybridization and RNase protection assay, we provide evidence that the multidrug resistance-associated protein (MRP) gene, which encodes a human GS-X pump, is expressed at higher levels in cisplatin-resistant (HL-60/R-CP) cells than in sensitive cells, whereas amplification of the MRP gene is not detected by Southern hybridization. Culturing HL-60/R-CP cells in cisplatin-free medium resulted in reduced MRP mRNA levels, but these levels could be induced to rise within 30 h by cisplatin and heavy metals such as arsenite, cadmium, and zinc. The increased levels of MRP mRNA were closely related with enhanced activities of ATP-dependent transport of leukotriene C4 (LTC4) in plasma membrane vesicles. The glutathione-platinum (GS-Pt) complex, but not cisplatin, inhibited ATP-dependent LTC4 transport, suggesting that the MRP/GS-X pump transports both LTC4 and the GS-Pt complex. Expression of gamma-glutamylcysteine synthetase in the cisplatin-resistant cells was also co-induced within 24 h in response to cisplatin exposure, resulting in a significant increase in cellular GSH level. The resistant cells exposed to cisplatin were cross-resistant to melphalan, chlorambucil, arsenite, and cadmium. These observations suggest that elevated expression of the MRP/GS-X pump and increased GSH biosynthesis together may be important factors in the cellular metabolism and disposition of cisplatin, alkylating agents, and heavy metals.     

Symmetry and pH dependency of the lactate/proton carrier in skeletal muscle studied with rat sarcolemmal giant vesicles

To study the pathogenesis of hyperlipoidemia and atheromatosis and the metabolism of lipoprotein, we have developed a colorimetric method for simultaneously determining the activities of post-heparinplasma lipoprotein lipase (LPL) and hepatic lipase (HL). The intralipid was kept for LPL and HL at 37 degrees C, pH8.3 for 30 min, with 100 microliters post-heparin plasma. The LPL and HL in the post-heparin plasma could hydrolyse the triglyceride in intralipid into glycerine and free fatty acid (FFA). Determining the amount of FFA by copper-reagent method, we could measure the activities of LPL and HL. The kinetics of LPL and HL in post-heparin plasma was observed. K(m) values for LPL and HL were 0.9 mumol/L and 2.4 mumol/L respectively. The C. V. for LPL and HL were 4.5% (n = 4), 2.9% (n = 6) and 6.4% (n = 6), 4.8% (n = 6) respectively.     

Posttranslational modifications of cardiac and skeletal muscle proteins by reactive oxygen species after burn injury in the rat

Hepatocytes derived either from rats fed a diet enriched in n-3 fatty acids or from rats fed a low-fat diet and cultured with an n-3 fatty acid (eicosapentaenoic acid, EPA) in vitro were used to distinguish between the dietary effects and the direct effects of n-3 fatty acids on hepatocellular apolipoprotein (apo) B metabolism and secretion. ApoB-48 and apoB-100 synthesis, degradation, and secretion as large (d<1.006) and small (d>1.006) particles were determined after a pulse label with [35S]methionine. These effects were compared with changes in triacylglycerol (TAG) synthesis and secretion and with changes in de novo fatty acid synthesis (using 3H2O incorporation) under identical conditions. When n-3 fatty acid was given via the dietary route, apoB-48 very low density lipoprotein (VLDL) secretion was inhibited, but there was no effect on the secretion of apoB-100 VLDL. There was no effect on the secretion of either apoB-48 or apoB-100 as small, dense particles (d>1.006). Cellular TAG synthesis was significantly inhibited under these conditions, and fatty acid synthesis de novo was inhibited by 80%. By contrast, after direct addition of EPA to hepatocytes from normal rats, the secretion of both apoB-48 and apoB-100 VLDL was suppressed. The secretion of apoB-48, but not of apoB-100, as dense particles was also inhibited. However, there was little or no effect on TAG synthesis nor on fatty acid synthesis de novo. In addition, whereas dietary administration of n-3 fatty acid gave rise to decreased net synthesis and degradation of apoB-48, direct administration in vitro resulted in increased degradation with no effect on net synthesis. We conclude that the effects of n-3 fatty acids on hepatic lipid and apoB metabolism differ according to whether they are administered in vivo, via the dietary route, or in vitro, via direct addition to hepatocyte cultures.     

In the rat, tumor necrosis factor alpha administration results in an increase in both UCP2 and UCP3 mRNAs in skeletal muscle: a possible mechanism for cytokine-induced thermogenesis?

Aedes thibaulti Dyar and Knab occurs from southern Louisiana, USA, to Ontario, Canada, but has an exceptionally patchy distribution over much of its range. Typical breeding habitat for this univoltine species includes cavities at the bases of trees growing in low-lying swampy areas or dark recesses within the root balls of upturned trees. Larvae have never been collected in the northeastern portion of the range, but adult records suggest that breeding populations are present. The lack of low-lying swampland in northern areas where adults have been collected suggests that the breeding habitat for this species may not be as specific as previously believed. On April 20, 1997, we collected Ae. thibaulti larvae from the flooded cavity of a red maple tree (Acer rubrum) growing next to a temporary snow pool in northern New Jersey. Larvae persisted in this habitat until mid-May when the cavity dried completely. The collection suggests that Ae. thibaulti is a cavity breeder in the northern portion of its range, but is able to utilize cavity habitats associated with temporary pools in dry forested areas.     

Inactivation of aconitase and oxoglutarate dehydrogenase in skeletal muscle in vitro by superoxide anions and/or nitric oxide

Strips of rat soleus muscle were incubated in media containing a superoxide generating system and/or the nitric oxide donor sodium nitroprusside (SNP) before the maximal catalytic activities of aconitase, citrate synthase, and oxoglutarate dehydrogenase were measured. The maximal activities of aconitase and oxoglutarate dehydrogenase were both decreased by 25-30% by superoxide anions; however, only the maximal activity of aconitase was decreased, by approximately 50%, by incubation of muscles with SNP. Furthermore, when both superoxide and NO were present in the medium, aconitase activity was decreased by 70%. The maximal activity of citrate synthase was not affected by any of the treatments. This is the first time that superoxide anions or NO has been shown to inactivate aconitase and oxoglutarate dehydrogenase in skeletal muscle. It is suggested that these effects may be responsible for some alterations in skeletal muscle metabolism, and these possibilities are discussed.     

Inhibition of cyclic AMP-dependent kinase by expression of a protein kinase inhibitor/enhanced green fluorescent fusion protein attenuates angiotensin II-induced type 1 AT1 receptor mRNA down-regulation in vascular smooth muscle cells

Expression of the angiotensin II type 1 receptor (AT1-R) mRNA in vascular smooth muscle cells (VSMC) is down-regulated by a variety of agonists, including growth factors, agonists of Galphaq protein-coupled receptors, and activators of adenylyl cyclase. To determine whether cAMP-dependent protein kinases (PKA) participates in AT1-R mRNA down-regulation controlled by multiple classes of receptors, a PKA inhibitor peptide (PKIalpha) was developed and expressed in rat VSMC as a fusion with the enhanced green fluorescent protein (eGFP). PKA activity elicited both by forskolin and angiotensin II is suppressed in cells expressing this fusion protein (PKIalpha-eGFP), but platelet-derived growth factor-BB does not stimulate PKA activity in this preparation. PKIalpha-eGFP expression fully inhibits the forskolin-stimulated down-regulation of AT1-R mRNA levels and blocks 50% of the effect elicited by angiotensin II. This indicates that PKA plays a substantial role in angiotensin II-stimulated AT1-R mRNA down-regulation. However, inhibition of PKA has no effect on AT1-R mRNA down-regulation caused by platelet-derived growth factor-BB. These findings show how agonists such as angiotensin II that are not normally considered as activators of PKA can use PKA-dependent processes to modulate gene expression. These findings also provide definitive evidence that PKA-dependent pathways are involved in modulation of AT1-R mRNA levels in VSMC.     

Change in the hexokinase activity of the rat myocardial and skeletal muscle mitochondria and cytoplasm in fasting and on a carbohydrate diet

Our experience using the lateral simplified thoracotomy incision for most chest work other than operations requiring median sternotomy is reported. The incision provides adequate exposure, yet preserves major muscle masses and decreases the postoperative morbidity. Return of normal ipsilateral arm function appears to be hastened in the postoperative period. In addition, the incision is easier to open and close than the standard incision.     

Activated alpha 2-macroglobulin promotes mitogenesis in rat vascular smooth muscle cells by a mechanism that is independent of growth-factor-carrier activity

Vascular smooth muscle cell (vSMC) proliferation is important in atherosclerosis. We previously demonstrated that methylamine-activated alpha 2-macroglobulin (alpha 2M) and transforming growth factor beta 1 (TGF-beta 1) cause a synergistic proliferative response in quiescent rat aortic vSMCs [Stouffer, G. A., La-Marre, J., Gonias, S. L. & Owens, G. K. (1993) J. Biol. Chem. 268, 18,340-18,344]. The first goal of this study was to determine whether the synergy is due to the ability of alpha 2M-methylamine (alpha 2M-MeNH2) to bind TGF-beta 1 and target the growth factor to vSMCs that express the alpha 2M receptor. Receptor-recognized alpha 2M derivatives without TGF-beta 1-binding activity, including ternary alpha 2M-trypsin, an 18-kDa proteolytic fragment of the alpha 2M subunit, and the corresponding recombinant receptor-binding fragment (rRBF) increased vSMC [3H]thymidine incorporation and cell number in a manner similar to alpha 2M-MeNH2. In combination with TGF-beta 1, each alpha 2M derivative caused a synergistic vSMC proliferative response. vSMCs responded comparably when treated with alpha 2M-MeNH2 and TGF-beta 1 simultaneously or in sequence. Furthermore, alpha 2M-MeNH2-TGF-beta 1 complexes increased [3H]thymidine incorporation no more than alpha 2M-MeNH2 alone. These results indicate that TGF-beta 1 binding to alpha 2M is not responsible for the synergistic mitogenic activity. Additional studies were undertaken to determine whether activated alpha 2M independently induces a signal-transduction response in vSMCs. alpha 2M-MeNH2 and rRBF caused a rapid, transient increase in vSMC inositol 1,4,5-trisphosphate. This response was pertussis-toxin insensitive. Receptor-associated protein (RAP; 170 nmol/L) inhibited 91-95% of the specific binding of 125I-alpha 2M-MeNH2 and 125I-rRBF to vSMC; however, RAP did not affect the inositol 1,4,5-trisphosphate response or the mitogenic response. These studies suggest that vSMCs express a receptor, other than low-density-lipoprotein-receptor-related protein, that transduces a signal in response to activated alpha 2M. This receptor may mediate the mitogenic activity of alpha 2M in vSMC culture.     

Increased oxidative stress in renal proximal tubules of the spontaneously hypertensive rat: a mechanism for defective dopamine D1A receptor/G-protein coupling

AIM: Defective dopamine D1A dopamine receptor/G-protein coupling has been demonstrated in renal proximal tubules of the spontaneously hypertensive rat (SHR). In the present study, we aimed to analyze the underlying mechanisms through which such defects are introduced into the D1A receptor protein of SHR. MATERIALS AND METHODS: The oxidative state of SHR proximal tubules was analyzed by measuring lipid peroxidation. D1A receptor/G-protein coupling was measured following the induction of oxidative stress in normotensive Wistar-Kyoto (WKY) rats. RESULTS: For the first time, an increased state of oxidative stress was demonstrated in SHR proximal tubules compared with those of normotensive controls, WKY and Sprague-Dawley rats. Lipid peroxidation levels in SHR were significantly higher by 66 and 79%, relative to WKY or Sprague-Dawley rats, respectively. Hydrogen peroxide treatment of proximal tubules from SHR, WKY and Sprague-Dawley rats induced an additional increase in lipid peroxidation in a dose-dependent manner, although the percentage induction was lower in SHR than in WKY and Sprague-Dawley rats. This induction of lipid peroxidation in WKY rats resulted in a loss of D1A/G-protein coupling, with no decrease in receptor protein. Treatment of WKY rat proximal tubules with an antioxidant, ascorbic acid, or a reducing agent, dithiothreitol, induced D1A receptor/G-protein coupling. CONCLUSIONS: These data indicate that D1A receptor/G-protein coupling is modulated by changes in redox states. Therefore, the D1A receptor/G-protein coupling in SHR may have been damaged by reactive oxygen species released as a result of the elevated oxidative stress seen in the proximal tubules.     

Effect of benzyl succinate on insulin receptor function and insulin action in skeletal muscle: further evidence for a lack of spare high-affinity insulin receptors

Benzyl succinate inhibited insulin binding and tyrosine receptor kinase in a concentration-dependent manner in the partially purified insulin receptor preparation from rat skeletal muscle. Benzyl succinate lowered the apparent number of high-affinity insulin binding sites. We have made use of the inhibitory effect of benzyl succinate to investigate the possible presence of spare high-affinity insulin receptors in muscle. Benzyl succinate inhibited the effect of a supramaximal concentration of insulin on 3-O-methylglucose uptake, 2-(methylamino)isobutyric acid uptake and lactate production by the incubated muscle. Furthermore, the inhibitory effect of benzyl succinate on insulin binding in vitro closely correlated with its inhibitory effect on insulin action in vivo. These findings suggest the absence of spare high-affinity insulin receptors in skeletal muscle. In contrast to data obtained in skeletal muscle, benzyl succinate did not affect the maximally insulin-stimulated glucose transport, although it caused a marked decrease in insulin sensitivity in isolated rat adipocytes, for which the existence of spare insulin receptors is well documented.     

Myoblast fusion is not a prerequisite for the appearance of calcium current, calcium release, and contraction in rat skeletal muscle cells developing in culture

During in vitro development of rat skeletal muscle cells, contraction and calcium currents progressively appear after fusion of myoblasts. To investigate whether muscle-specific functions are expressed in the absence of myoblast fusion, rat neonatal muscle cells were cultured in a differentiation medium under conditions that are well known to inhibit fusion: prolonged culture in a low-calcium medium or treatment with cytochalasin B. We have demonstrated that the fusion-arrested cells expressed differentiative properties in L-type calcium current, transient release of calcium ions from internal stores in response to caffeine and depolarizing agents, and contraction elicited by depolarization. Properties and potential-dependence of L-type calcium currents were similar to that in control fused cells, but T-type calcium currents were not observed, while both types coexist in myotubes. Properties of calcium transients and voltage dependence of contraction suggested that the excitation-contraction mechanisms were well established. However, comparing to well-developed myotubes at the same time of culture, the characteristics of calcium transients and contraction of fusion-arrested cells were closer to those of younger myotubes, which can be interpreted in terms of a delay in maturation of excitation-contraction coupling and contractile machinery. All these observations demonstrate that myoblast fusion is not necessary for triggering the establishment of calcium transport and release and contractile functions of rat muscle cells developing in culture. The appearance of muscle-specific functions is consistent with previous results demonstrating that the fusion-arrested cells express muscle-specific proteins and structures.     

Superior activity of a thromboxane receptor antagonist as compared with aspirin in rat models of arterial and venous thrombosis

We determined the effects of aspirin and a novel thromboxane A2/prostaglandin endoperoxide (TP)-receptor antagonist, BMS-180291, on thrombosis and bleeding times in skin and mesenteric arteries. In anesthetized rats, occlusive thrombosis was induced in the carotid artery by topical application of ferrous chloride and in the vena cava by blood flow stasis combined with either infusion of thromboplastin or hypotonic saline. Aspirin (1, 10, and 50 mg/kg) did not reduce arterial or venous thrombus weight significantly. BMS 180,291 (150 micrograms/kg/min) decreased arterial thrombus weight and hypotonic saline-induced caval thrombus weight by 58 and 57%, respectively. BMS-180291 lacked antithrombotic activity at a lower dose (50 micrograms/kg/min) and failed to inhibit thromboplastin-induced caval thrombosis. BMS-180291 (150 micrograms/kg/min) significantly reduced arterial thrombus weight by 40% when plasma epinephrine concentration was increased to 5 ng/ml. BMS-180291 and aspirin produced increases of only < or = 30% in bleeding times. These results demonstrate that BMS-180291 has antithrombotic activity in experimental aspirin-resistant arterial and venous thrombosis. Both aspirin and BMS-180291 have only modest effects on small artery hemostasis in rats.