Reduced resting potentials in dystrophic (mdx) muscle fibers are secondary to NF-κB-dependent negative modulation of ouabain sensitive Na+-K+ pump activity.

To examine potential mechanisms for the reduced resting membrane potentials (RPs) of mature dystrophic (mdx) muscle fibers, the Na(+)-K(+) pump inhibitor ouabain was added to freshly isolated nondystrophic and mdx fibers. Ouabain produced a 71% smaller depolarization in mdx fibers than in nondystrophic fibers, increased the [Na(+)](i) in nondystrophic fibers by 40%, but had no significant effect on the [Na(+)](i) of mdx fibers, which was approximately double that observed in untreated nondystrophic fibers. Western blots indicated no difference in total and phosphorylated Na(+)-K(+) ATPase catalytic α1 subunit between nondystrophic and mdx muscle. Examination of the effects of the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) indicated that direct application of the drug slowly hyperpolarized mdx fibers (7 mV in 90 min) but had no effect on nondystrophic fibers. Pretreatment with ouabain abolished this hyperpolarization, and pretreatment with PDTC restored ouabain-induced depolarization and reduced [Na(+)](i). Administration of an NF-κB inhibitor that utilizes a different mechanism for reducing nuclear NF-κB activation, ursodeoxycholic acid (UDCA), also hyperpolarized mdx fibers. These results suggest that in situ Na(+)-K(+) pump activity is depressed in mature dystrophic fibers by NF-κB dependent modulators, and that this reduced pump activity contributes to the weakness characteristic of dystrophic muscle.

The influence of passive stretch and NF-κB inhibitors on the morphology of dystrophic muscle fibers.

The triangularis sterni (TS) is an expiratory muscle that is passively stretched during inspiration. The magnitude of passive stretch depends upon the location of individual fibers within the TS muscle, with fibers located more caudally being stretched ∼ 5% to 10% more than fibers in the cephalad region. In the mdx mouse model for muscular dystrophy, the TS exhibits severe pathological alterations that are ameliorated by treatment with inhibitors of the NF-κB pathway. The purpose of this study was to assess the influence of passive stretch in vivo on fiber morphology in nondystrophic and mdx TS muscles, and the morphological benefits of treating mdx mice with two distinct NF-κB inhibitors, pyrrolidine dithiocarbamate (PDTC), and ursodeoxycholic acid (UDCA). Transmission electron microscopy revealed Z-line streaming, hypercontraction, and disassociation of the plasma membrane from the basal lamina in mdx fibers. In both nondystrophic and mdx TS muscles, fiber density was larger in more caudal regions. In comparison with nondystrophic TS, fibers in the mdx TS exhibited substantial reductions in diameter throughout all regions. In vivo treatment with either PDTC or UDCA tended to increase fiber diameter in the middle and decrease fiber diameter in the caudal TS, while reducing centronucleation in the middle region. These results suggest that passive stretch induces hypercontraction and plasma membrane abnormalities in dystrophic muscle, and that differences in the magnitude of passive stretch may influence fiber morphology and the actions of NF-κB inhibitors on dystrophic morphology.

Excessive collagen accumulation in dystrophic (mdx) respiratory musculature is independent of enhanced activation of the NF-kappaB pathway.

Skeletal muscle fibrosis is present in the diaphragm of the mdx mouse, a model for Duchenne dystrophy. In both the mouse and human, dystrophic muscle exhibits pronounced increases in NF-kappa B signaling. Various inhibitors of this pathway, such as pyrrolidine dithiocarbamate (PDTC) and ursodeoxycholic acid (UDCA), have been shown to have beneficial effects on dystrophic (mdx) muscle. The present study characterizes the development of fibrosis in the mdx musculature, and determines the fibrolytic efficacy of PDTC and UDCA. The results indicate that collagen accumulation and the expression of fibrogenic (TGF-beta1) and fibrolytic (MMP-9) mediators are dependent on muscle origin in both nondystrophic and mdx mice. Excessive collagen accumulation is observed in the mdx respiratory musculature prior to substantial muscle degeneration and cellular infiltration, and is associated with dystrophic increases in the expression of TGF-beta1 with no corresponding increases in MMP-9 expression. Treatment with PDTC or UDCA did not influence collagen deposition or TGF-beta1 expression in the mdx respiratory musculature. These results indicate that dystrophic increases in collagen are the result of NF-kappaB-independent signaling abnormalities, and that efforts to reduce excessive collagen accumulation will require treatments to more specifically reduce TGF-beta1 signaling or enhance the expression and/or activity of matrix metalloproteases.

Chlortetracycline and tylosin runoff from soils treated with antimicrobial containing manure.

This study assessed the runoff potential of tylosin and chlortetracycline (CTC) from soils treated with manure from swine fed rations containing the highest labeled rate of each chemical. Slurry manures from the swine contained either CTC at 108 microg/g or tylosin at 0.3 microg/g. These manures were surface applied to clay loam, silty clay loam, and silt loam soils at a rate of 0.22 Mg/ha. In one trial, tylosin was applied directly to the soil surface to examine runoff potential of water and chemical when manure was not present. Water was applied using a sprinkler infiltrometer 24-hr after manure application with runoff collected incrementally every 5 min for about 45 min. A biofilm crust formed on all manure-treated surfaces and infiltration was impeded with > 70% of the applied water collected as runoff. The total amount of CTC collected ranged from 0.9 to 3.5% of the amount applied whereas tylosin ranged from 8.4 to 12%. These data indicate that if surface-applied manure contains antimicrobials, runoff could lead to offsite contamination.

Manure placement depth impacts on crop yields and N retained in soil.

The objective of this study was to determine the impact of manure placement depth on crop yield and N retention in soil. Experimental treatments were deep manure injection (45 cm), shallow manure injection (15 cm), and conventional fertilizer-based management with at least three replications per site. Water infiltration, and changes in soil N and P amounts were measured for up to 30 months and crop yield monitored for three seasons following initial treatment. Deep and shallow manure injections differed in soil inorganic N distributions. For example, in the manure slot the spring following application, NO(3)-N in the surface 60 cm was higher (p < .01) when injected 15 cm (21.4 micro g/g) into the soil than 45 cm (11.7 micro g/g), whereas NH(4)-N had opposite results with shallow injection having less (p = 0.045) NH(4)-N (102 micro g/g) than deep (133 micro g/g) injection. In the fall one year after the manure was applied, NO(3)-N and NH(4)-N were lower (p = 0.001) in the shallow injection than the deep injection. The net impact of manure placement on total N was that deep injection had 31, 59, and 44 more kg N ha(- 1) than the shallow injection treatment 12, 18, and 30 months after application, respectively. Deep manure injection did not impact soybean (Glycine max L.) yield, however corn (Zea mays L.) yield increased if N was limiting. The higher corn yield in the deep injected treatment was attributed to increased N use efficiency. Higher inorganic N amounts in the deep injection treatment were attributed to reduced N losses through ammonia volatilization, leaching, or denitrification. Results suggest that deep manure placement in glacial till soil may be considered a technique to increase energy, N use efficiency, and maintain surface and ground water quality. However, this technique may not work in glacial outwash soils due to the inability to inject into a rocky subsurface.

Changes in the motility of B16F10 melanoma cells induced by alterations in resting calcium influx.

Alterations in the extracellular Ca(2+) or K(+) concentration had significant influences on the motility of B16F10 melanoma cells measured in the absence of exogenous integrins using a conventional Boyden chamber assay. At normal K(+) concentrations, motility increased slightly when the concentration of Ca(2+) was increased 10-fold. At normal Ca(2+) concentrations, motility increased by 290% when the extracellular K(+) concentration was reduced 10-fold (from control of 5.4 mM to 0.54 mM), and increased to 250% of control levels when the K(+) concentration was increased between 30 and 54 mM, but was relatively uninfluenced at K(+) concentrations between 5 and 30 mM. Simultaneous application of low concentrations (20 microM) of GdCl(3) completely prevented the effects of low and high K(+) on motility. Exposure to Gd(3+) or Tb(3+) also produced a flattening of the cells and enhanced cell attachment. Although the steady state intracellular Ca(2+) concentration was not significantly influenced by the K(+) concentration, the resting permeability to divalent cations, determined from Mn(2+) quench rates in fura-loaded cells, was significantly increased by a reduction in the K(+) concentration. These results indicate that resting Ca(2+) influx is critical to the movement of B16F10 melanoma cells, and demonstrate that lanthanides, which block resting Ca(2+) influx pathways, are potent antimotility agents.

Adult dystrophic (mdx) endplates exhibit reduced quantal size and enhanced quantal variation.

Examination of miniature endplate potential (MEPP) distributions indicated that the average quantal size is significantly reduced by approximately 44% in young adult mdx mice (5-7 weeks). The average quantum declined further to approximately 37% of non-dystrophic levels in mature (6- to 24-month) mdx mice. Young adult non-dystrophic and mdx endplates and mature non-dystrophic endplates exhibited a linear relationship between the mean and variance of uniquantal MEPP amplitude distributions. Mature mdx endplates, however, exhibited a distinctly nonlinear relationship characterized by large increases in variance at larger mean MEPP amplitudes. These results indicate a reduced average density of functional acetylcholine (ACh) receptors (AChRs) at mdx endplates that, in mature preparations, is associated with greater temporal or spatial variability in the density of functional AChRs apposed to individual release sites.

Involvement of mitochondrial ferredoxin and Cox15p in hydroxylation of heme O.

Cox15p is essential for the biogenesis of cytochrome oxidase [Glerum et al., J. Biol. Chem. 272 (1997) 19088-19094]. We show here that cox15 mutants are blocked in heme A but not heme O biosynthesis. In Schizosaccharomyces pombe COX15 is fused to YAH1, the yeast gene for mitochondrial ferredoxin (adrenodoxin). A fusion of Cox15p and Yah1p in Saccharomyces cerevisiae rescued both cox15 and yah1 null mutants. This suggests that Yah1p functions in concert with Cox15p. We propose that Cox15p functions together with Yah1p and its putative reductase (Arh1p) in the hydroxylation of heme O.

Spontaneous changes in acetylcholine receptor and calcium leakage activity in cell-attached patches from cultured dystrophic myotubes.

Calcium leakage activity (CLA) was recorded in association with acetylcholine receptor (AChR) activity in cell-attached patches from cultured nondystrophic and dystrophic (mdx) myotubes. Cell-attached recordings from dystrophic myotubes exhibited spontaneous transitions in the activity pattern that were characterized by an instability of AChR function and a decrease in the frequency of AChR events. Recordings from nondystrophic myotubes could be maintained for similar time periods without observing any consistent changes in the distribution of CLA and AChR events, thus indicating that the conditions of the experiment were not conducive to developing AChR instability or desensitization in nondystrophic myotubes. In dystrophic myotubes, the decrease in AChR event frequency was associated with an increase in small-conductance events which had the characteristics of CLA, and the subsequent acquisition of an inside-out patch appeared to restore the AChR activity. Examination of baseline current-voltage relationships indicated that dystrophic and nondystrophic patches exhibited the same general pattern of seal maturation with temporal increases in the total-patch circuit resistance. These results are discussed in relation to the AChR aggregation hypothesis, which proposes that the absence of dystrophin leads to abnormal AChR-cytoskeletal interactions and CLA that can be reversed by removing the influence of intracellular signal transduction enzymes that aggregate and stabilize AChR clusters.

The dystrophinopathies: an alternative to the structural hypothesis.

Abnormal expression of the cytoskeletal protein dystrophin has deleterious consequences for skeletal muscle, cardiac muscle, and the central nervous system. A complete failure to express the protein produces Duchenne muscular dystrophy (DMD), in which there is extensive and progressive skeletal muscle necrosis, the development of a life-threatening dilated cardiomyopathy, and mild mental retardation. Dystrophin binds the F-actin cytoskeleton and is normally expressed in a complex of transmembrane proteins (the "dystrophin protein complex") that interact with external components of the basal lamina. One pathogenic model for DMD (the "structural hypothesis") suggests that this complex forms a structural bridge between the external basal lamina and the internal cytoskeleton and that the absence of dystrophin produces a defect in membrane structural support that renders skeletal muscle susceptible to plasmalemmal ruptures (or "tears") during the course of contractile activity. This review attempts to critically evaluate the structural hypothesis for DMD and presents an opposing model (the "channel aggregation model") that highlights the role of dystrophin in organizing the membrane cytoskeleton and the role of the cytoskeleton in aggregating ion channels and neurotransmitter receptors. Since ion channel aggregation is a process that is common across organ systems, the idea that channel function can be altered when aggregated ion channels interact with a dystrophic cytoskeleton has immediate implications for the expression of the dystrophinopathies in skeletal muscle, cardiac muscle, and the central nervous system.

The relationship between the resistance of a membrane patch and predicted changes in total patch circuit resistance secondary to spontaneous or induced alterations in patch geometry.

The objective of this study was to determine the relationship between the magnitude of the membrane resistance in the free area of a cell-attached patch-clamp recording and the change in total patch circuit resistance that would be produced by the introduction of a series resistance, such as would be observed upon acquiring a patch-clamped membrane vesicle. The results describe a method for determining the magnitude of the membrane resistance in the free area of a membrane patch, and demonstrate that: (a) at a given value of shunt resistance, areas of membrane with higher resistivity produce smaller proportional increases in total patch circuit resistance upon acquiring a membrane vesicle; and (b) a presumption of spherical vesicle formation provides a lower limit estimate of the membrane resistance. The described procedures and relationships are useful in developing new techniques for examining channel activity in membrane patches where individual events are below the present limits of detection, for examining changes in membrane resistivity and/or shunt resistance in patches undergoing cytoskeletal re-organization, and for assessing the potential influence of series resistance changes on single channel parameters in longer term cell-attached patch-clamp recordings.

Acetylcholine receptor and calcium leakage activity in nondystrophic and dystrophic myotubes (MDX)

To determine whether the lack of dystrophin alters the occurrence of calcium leakage activity (CLA) and acetylcholine receptor (AChR) activity, the frequency of each event class was determined from several cell attached patches on nondystrophic and dystrophic (mdx) myotubes. The frequency of CLA observed in the presence of ACh was significantly (P < 0.05) elevated in mdx myotubes, an effect which was partly due to a significant (P < 0.05) increase in the proportion of cell attached patches that exhibited 100% CLA with no AChR activity. Areas of mdx and nondystrophic membrane that exhibited reduced or absent AChR activity had significantly (P < 0.01) and substantially elevated calcium leakage event frequencies. This inverse and discontinuous relationship between CLA and AChR activity provides further evidence that some CLA in dystrophic muscle is produced by clusters of AChRs that form unusual physical associations with the dystrophic cytoskeleton during the processes associated with receptor localization and stabilization.

Single channel evidence for a cytoskeletal defect involving acetylcholine receptors and calcium influx in cultured dystrophic (mdx) myotubes.

Single channel events that exhibited the conductance, event duration, and ion selectivity characteristics of calcium leakage activity (CLA) were recorded in association with acetylcholine receptor (AChR) activity in cultured nondystrophic myotubes. The CLA was observed in the presence or absence of acetylcholine (ACh), and at normal or elevated concentrations of calcium. In contrast to results from nondystrophic myotubes, cell-attached patches from several cultured dystrophic (mdx) myotubes exhibited 100% CLA with no AChR activity, even though ACh was present in the pipette solution. Acquisition of an inside-out patch from these membrane areas produced a profound decrease in CLA and the appearance of AChR events exhibiting typical conductance and event duration characteristics. These results suggest that CLA in dystrophic muscle is produced, in part, by unusual physical interactions between AChRs and the dystrophic cytoskeleton that are mediated by the action of intracellular modulators responsible for aggregating and stabilizing AChRs.

A baseline detection method for analyzing transient electrophysiological events.

A baseline detection method has been developed that identifies and defines event transitions for whole-cell voltage or current ('transient') events that are produced by the activity of ion channel ensembles. The method utilizes a variety of iterative techniques that independently determine, for each event, several output parameters that are ultimately referrable to the mean and variance of each pre-event baseline. Examination of miniature endplate potentials using the baseline detection method provided the following output parameters for each transient event: pre-event mean and variance; rise time; peak amplitude and duration; a determination of whether the decay phase was best fit by a one- or two-component negative exponential function; time constants for the slow and/or fast decay components; percent contribution of the slow component to the decay phase; and the predicted peak amplitude determined by extrapolation of the least squares fit to the decay phase. Joint probability density representations involving the rise time and peak amplitudes of miniature endplate potentials indicated the power of this multivariate approach in identifying and isolating specific event classes. The baseline detection method is particularly advantageous for analyzing records containing multiple classes of event amplitudes, and provides a reproducible statistical standard for the analyses of transient events that are characteristic of whole-cell electrophysiological recordings.

Differential influence of electrical blocking agents on embryonic acetylcholine receptor mRNA levels in long-term cultures of aneural mammalian myotubes.

The influence of spontaneous muscle activity on acetylcholine receptor (AChR) expression was examined by exposing long-term cultures of mammalian myotubes to two pharmacological agents that have similar effects on the rate of spontaneous contractile activity but pharmacologically distinct actions on voltage gated Na+ channels. Previous studies by other investigators have shown that tetrodotoxin upregulates and that veratridine downregulates surface AChR expression in short-term mammalian muscle cultures. In order to determine whether these drugs have disparate actions on AChR mRNA levels, myotubes were exposed to either tetrodotoxin or veratridine for a period of 10 days, and measurements of the relative levels of embryonic AChR subunit mRNAs (alpha, beta, gamma, delta) were obtained during and following the period of drug exposure. Veratridine produced a substantial decrease (between 33% and 50% reduction), while tetrodotoxin produced a relatively small increase (between 17% and 23%), in each of the AChR subunit mRNAs after 6 days of drug exposure. At 23 days in culture, spontaneously active myotubes exhibited a decrease in the relative levels of each of the AChR subunit mRNAs. Myotubes previously exposed to either veratridine or tetrodotoxin exhibited elevated levels of beta, gamma, and delta AChR subunit mRNAs 6 days after cessation of drug treatment, thus suggesting that a period of muscle inactivity can induce sustained influences on some AChR mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the endplate accumulation of acetylcholinesterase during synapse elimination in the mouse.

Focal accumulations of acetylcholinesterase (AChE; EC 3.1.1.7), cholinesterase (ChE, EC 3.1.1.8) and total cholinesterase (TChE; AChE+ChE) were examined in developing mouse diaphragm by using a modified Karnovsky/Roots staining method. The lengths of TChE and AChE reaction product accumulations reached significant peaks on postnatal day (PD) 1 (P < 0.05), decreased to a minimum on PD 9 and then increased in proportion to muscle fiber diameter (PD 9 to adult). The normalized area of accumulation (area of accumulation/fiber diameter) for AChE and TChE also decreased by 19% (P < 0.05) between PD 3 and PD 7. In contrast, ChE focal accumulation did not decrease during the period of synapse elimination, but rather increased in proportion to the postnatal growth of the muscle fiber. These results suggest that AChE is more sensitive to neurotrophic influences than ChE; particularly during late embryonic and early postnatal periods of synapse elimination.

Asynaptic expression of the adult nicotinic acetylcholine receptor in long-term cultures of mammalian myotubes.

To determine whether synaptic contact is required to express adult-type nicotinic acetylcholine receptors (A-AChR) in developing mammalian muscle, we have examined single-channel AChR activity in primary muscle cultures maintained for up to 29 days. A-AChRs were first expressed after day 12 in culture (CD12), during a period characterized by the accumulation of embryonic acetylcholine receptors (E-AChR). The highest rate of A-AChR expression was observed between CD15 and 19, during a period of maximal E-AChR accumulation. Although the level of A-AChR expression between individual patches was quite variable during this period, A-AChRs accounted for up to 40% of the events produced by receptors expressed over a 3-day interval. Between CD19 and 29, the density of E-AChRs diminished while the expression of A-AChRs per patch continued to increase but at a lower rate than that observed between CD15 and 19. In 25-29-day cultures, 70.6% of patches exhibited both E-AChR and A-AChR activity, and the percentage of A-AChR events per patch ranged between 0 and 47% with a mean of 11.7 +/- 3.2%. These results demonstrate that endogenous muscle mechanisms promote developmental increases in the expression of A-AChRs in myotubes that have no history of synaptic contact. This conclusion suggests that synaptic imprinting at developing junctions is mediated in part by endogenous muscle mechanisms, and does not require direct neurotrophic activation of epsilon mRNA transcription.

Early postnatal changes in presynaptic potassium sensitivity.

Amplitude histograms of miniature endplate potentials (MEPPs) and the overall frequency of skew-MEPPs and bell-MEPPs were examined in 5 and 15 mM potassium solutions at postnatal day (PD) 3, PD 10 and PD 27 neuromuscular junctions. Temporal non-uniformities in spontaneous release produced clusters of bell-MEPPs at PD 0-PD 3 junctions. PD 3 nerve terminals that preferentially released skew-MEPPs (5 mM potassium) were significantly (P less than 0.01) less sensitive to elevations in potassium than more mature (PD 10) junctions that preferentially released bell-MEPPs. Increases in the potassium concentration at PD 3 junctions increased the frequency of bell-MEPPs and altered the MEPP amplitude distribution profile by significantly (P less than 0.01) reducing the percentage of skew-MEPPs. Although the potassium sensitivity of PD 10 and PD 27 preparations were as expected for adult preparations, there was an increase in overall MEPP frequency in 5 mM potassium between PD 10 and PD 27. These results suggest that early postnatal increases in the number of presynaptic calcium channels establish adult levels of depolarization sensitivity and promote the preferential spontaneous release of bell-MEPPs. Since these changes occur during an early period of synapse elimination, they may play a critical role in synapse stabilization.

A noninvasive procedure to detect muscle weakness in the mdx mouse.

The forward pulling tension exerted by individual mice was measured nearly isometrically in a simple apparatus designed to determine whole body tension (WBT). WBT determinations on control (C57Bl10/SnJ) and experimental (C57Bl10-mdx) mice indicate a muscle weakness which lasts throughout the lifespan of mdx mice. Direct muscle stimulation experiments in vivo also showed significant decreases in peak twitch and tetanic tensions in adult mdx muscle with no obvious alterations in twitch time course or in twitch: tetanus ratios. We suggest that the noninvasive WBT procedure may be used to partially assess various therapies on this murine model of Duchenne muscular dystrophy.