Central depletion of brain-derived neurotrophic factor in mice results in high bone mass and metabolic phenotype.

Brain-derived neurotrophic factor (BDNF) plays important roles in neuronal differentiation/survival, the regulation of food intake, and the pathobiology of obesity and type 2 diabetes mellitus. BDNF and its receptor are expressed in osteoblasts and chondrocyte. BDNF in vitro has a positive effect on bone; whether central BDNF affects bone mass in vivo is not known. We therefore examined bone mass and energy use in brain-targeted BDNF conditional knockout mice (Bdnf(2lox/2lox)/93). The deletion of BDNF in the brain led to a metabolic phenotype characterized by hyperphagia, obesity, and increased abdominal white adipose tissue. Central BDNF deletion produces a marked skeletal phenotype characterized by increased femur length, elevated whole bone mineral density, and bone mineral content. The skeletal changes are developmentally regulated and appear concurrently with the metabolic phenotype, suggesting that the metabolic and skeletal actions of BDNF are linked. The increased bone development is evident in both the cortical and trabecular regions. Compared with control, Bdnf(2lox/2lox)/93 mice show greater trabecular bone volume (+50% for distal femur, P < 0.001; +35% for vertebral body, P < 0.001) and midfemoral cortical thickness (+11 to 17%, P < 0.05), measured at 3 and 6 months of age. The skeletal and metabolic phenotypes were gender dependent, with female being more affected than male mice. However, uncoupling protein-1 expression in brown fat, a marker of sympathetic tone, was not different between genotypes. We show that deletion of central BDNF expression in mice results in increased bone mass and white adipose tissue, with no significant changes in sympathetic signaling or peripheral serotonin, associated with hyperphagia, obesity, and leptin resistance.

The cardiac glycoside binding site on the Na,K-ATPase alpha2 isoform plays a role in the dynamic regulation of active transport in skeletal muscle.

The physiological significance of the cardiac glycoside-binding site on the Na,K-ATPase remains incompletely understood. This study used a gene-targeted mouse (alpha2(R/R)) which expresses a ouabain-insensitive alpha2 isoform of the Na,K-ATPase to investigate whether the cardiac glycoside-binding site plays any physiological role in active Na(+)/K(+) transport in skeletal muscles or in exercise performance. Skeletal muscles express the Na,K-ATPase alpha2 isoform at high abundance and regulate its transport over a wide dynamic range under control of muscle activity. Na,K-ATPase active transport in the isolated extensor digitorum longus (EDL) muscle of alpha2(R/R) mice was lower at rest and significantly enhanced after muscle contraction, compared with WT. During the first 60 s after a 30-s contraction, the EDL of alpha2(R/R) mice transported 70.0 nmol/g.min more (86)Rb than WT. Acute sequestration of endogenous ligand(s) in WT mice infused with Digibind to sequester endogenous cardiac glycoside(s) produced similar effects on both resting and contraction-induced (86)Rb transport. Additionally, the alpha2(R/R) mice exhibit an enhanced ability to perform physical exercise, showing a 2.1- to 2.8-fold lower failure rate than WT within minutes of the onset of moderate-intensity treadmill running. Their enhanced exercise performance is consistent with their enhanced contraction-induced Na,K-ATPase transport in the skeletal muscles. These results demonstrate that the Na,K-ATPase alpha2 isozyme in skeletal muscle is regulated dynamically by a mechanism that utilizes the cardiac glycoside-binding site and an endogenous ligand(s) and that its cardiac glycoside-binding site can play a physiological role in the dynamic adaptations to exercise.

Concentration, distribution, and comparison of selected trace elements in bed sediment and fish tissue in the South Platte River Basin, USA, 1992-1993.

During August-November 1992 and August 1993, bed sediment and fish liver were sampled in the South Platte River Basin and analyzed for 45 elements in bed sediment and 19 elements in fish liver. The results for aluminum, arsenic, cadmium, chromium, copper, iron, lead,manganese, selenium, silver, uranium, and zinc are presented here. All 12 trace elements were detected in bed sediment, but not all were detected in fish liver or in all species of fish. A background concentration of trace elements in bed sediment was calculated using the cumulative frequency curves of trace element concentrations at all sites. Arsenic, cadmium, copper, lead, manganese, silver, uranium, and zinc concentrations were greater than background concentrations at sites in mining areas or at sites that have natural sources of these elements. Trace element concentrations in fish liver generally did not follow the same patterns as concentrations in bed sediment, although concentrations of aluminum and cadmium were higher in fish liver collected at mountain sites that had been disturbed by mining. Concentrations of aluminum, arsenic, cadmium, chromium, copper, iron, lead, silver, and zinc increased in bed sediments in urban areas. Iron, silver, and zinc concentrations in fish liver also increased in urban areas. Concentrations of cadmium, copper, silver, and zinc in fish liver increased in the agricultural areas of the basin. Downstream changes in trace element concentrations may be the result of geological changes in addition to changes in land use along the river.

Triadic Ca2+ modulates charge movement in skeletal muscle.

The effects of intracellular Ca2+ changes on charge movement in frog skeletal muscle were investigated using high concentrations (10-20 mmol/l) of buffers with different abilities to buffer Ca2+ at distances close to the SR Ca2+ release channels. In BAPTA compared with EGTA perfused fibers, charge movement was attenuated and lacked the characteristic kinetic features (I beta and I gamma) of E-C coupling charge movements. Qmax decreased by 9 nC/microF, Vmid was shifted 1-6 mV to more negative potentials, and the steepness factor increased by 3-5 mV. Results of varying the holding potential suggested that BAPTA decreases the amount of charge available to move upon depolarization. Raising intracellular Ca2+ to micromolar levels at a fixed BAPTA concentration prevented the decline in Qmax, suggesting that intracellular Ca2+ can modulate the amount of charge that is in the resting or available state. The different results obtained with BAPTA and EGTA can be explained by the greater ability of BAPTA to buffer dynamic Ca2+ changes at distances close to the release sites. These results are consistent with the proposals that an intracellular Ca2+ site on or near the dihydropyridine receptor, termed here the 'availability site', modulates the amount of charge available to move upon depolarization and is normally populated by Ca2+ released into the triad junction during activity.

Stimulation-dependent redistribution of charge movement between unavailable and available states.

A previous study (Stroffekova and Heiny 1997) demonstrated that changes in resting, intracellular free Ca2+ can modulate the amount of charge which is available to move upon depolarization and do excitation-contraction-coupling (E-C coupling). Charge movement reflects voltage-driven conformational changes of the dihydropyridine receptor which couple membrane excitation to Ca2+ release from the sarcoplasmic reticulum (SR) and contractile activation (c.f. review: Melzer et al. 1995). The present study demonstrates that dynamic changes in free Ca2+ that occur in the triadic gap during SR Ca2+ release can likewise produce a stimulation-dependent increase in the amount of available charge. Thus this modulation occurs in the physiological range of Ca2+ changes that occur in the triad during normal muscle activity. The modulation of charge movement by intracellular Ca2+ was rapid and maintained; it occurred within 2-3 suprathreshold depolarizations and remained for 5-10 minutes. It could be prevented by intracellular BAPTA and by depleting the SR of Ca2+, but not by EGTA or agents known to alter ion channel phosphorylation. These results are explained by a model in which a Ca2+ binding site on or near the voltage-sensor is normally populated by Ca2+ ions released into the triadic junction during activity and modulates the distribution of voltage sensors between available and unavailable states.

A surface potential change in the membranes of frog skeletal muscle is associated with excitation-contraction coupling.

1. Voltage changes and intramembrane charge movements in the transverse tubule membranes (T-system) of frog fast twitch muscle fibres were compared using the potentiometric dye WW-375 and a Vaseline-gap voltage clamp. As shown previously, the potentiometric dye reports a dynamic surface potential change that occurs on the myoplasmic face of the T-system membranes when the macroscopic potential applied across the surface membrane exceeds the mechanical threshold (about -60 mV). 2. The voltage dependence of the extra surface potential change and charge movement were found to be similar. Both activated with a sigmoid voltage dependence centred around -35 to -40 mV, and saturated at voltages above 0 mV. Both processes inactivated upon sustained depolarization, with a mid-point for inactivation of -40 mV. 3. Pharmacological agents which alter charge movement and excitation-contraction (E-C) coupling altered the non-linear surface potential change in a parallel manner. Perchlorate, which potentiates charge movement and E-C coupling, slowed the activation and deactivation of both charge movement and the non-linear surface potential change at voltages above -40 mV, and shifted the voltage dependence of both processes by 13 14 mV to more negative voltages. Dantrolene, which depresses charge movement and E-C coupling, shifted the voltage dependence of both processes to more positive voltages. Nifedipine, which suppresses charge movement and E-C coupling, reduced the magnitude of both charge movement and the non-linear surface potential change. 4. The non-linear surface potential change remained after the sarcoplasmic reticulum (SR) was depleted of Ca2+, suggesting that it is not a consequence of Ca2+ release. 5. These results suggest that the non-linear surface potential change is closely associated with movements of the voltage sensor (dihydropyridine (DHP) receptor) that control E-C coupling and/or signal transduction across the triadic junction. We propose that the movement of charged intramembrane domains of the DHP receptor which generate charge movement drive a subsequent movement of charged intracellular molecular domains that move within about 1 nm of the T-system membrane to generate a measurable change in surface charge. For example, the postulated mobile surface charges could be on an intracellular domain of the voltage sensor or closely associated protein, or could be a charged molecular domain of a protein that associates/dissociates with T-system membrane or DHP receptor during E-C coupling.

Modulation of pump function by mutations in the first transmembrane region of Na(+)-K(+)-ATPase alpha 1-subunit.

The Cys in the first transmembrane region of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) alpha 1-subunit has been shown to be a critical determinant of cardiac glycoside binding. To study the role of this Cys on ion transport activity, we measured pump currents in HeLa cells expressing wild-type or mutant alpha 1-subunit cDNAs. The endogenous ouabainsensitive Na(+)-K(+)-ATPase was selectively inhibited by growing the cells in 0.1 microM ouabain. A Cys-to-Tyr substituted mutant exhibited decreased sensitivity to digitoxin but not digoxin compared with wild type. The decreased affinity for digitoxin was due to a faster dissociation rate. In contrast, the Cys-to-Ala substitution did not significantly alter the sensitivity to digitoxin or digoxin. Both wild-type and mutant cells displayed marked external K(+)-dependent pump currents; however, the affinity for K+ was reduced by the mutations. The decrease in K+ affinity was due to a slower association rate. The results show that the Cys that interacts with cardiac glycosides also participates in the sensitivity of the pump to external K+.

Beta 2-adrenergic receptor regulation of the cardiac L-type Ca2+ channel coexpressed in a fibroblast cell line.

To characterize the functional coupling of the beta 2-AR to the cardiac Ca2+ channel in a system with a single receptor subtype, we stably cotransfected a Chinese hamster fibroblast (CHW) cell line, which lacks beta 2-ARs and Ca2+ channels, with the rabbit cardiac Ca2+ channel alpha 1 and beta 2 subunits and the human beta 2-AR cDNAs. The effects of beta 2-AR stimulation on the expressed Ca2+ channel current were examined using the whole-cell patch-clamp technique. CHW cells transfected with the Ca2+ channel subunits displayed a voltage-dependent inward current having properties typical of native cardiac L-type Ca2+ channels. The expressed current was increased by a phosphorylation-dependent mechanism. CHW cells cotransfected with the Ca2+ channel subunits and the beta 2-AR were responsive to isoproterenol (Iso) in a dose-dependent manner. Iso (10 microM) increased peak Ca2+ channel current to 172 +/- 5% (n = 17) of control amplitude, indicating that the expressed Ca2+ channels are functionally coupled to the beta 2-AR. The results demonstrate unequivocally that beta 2-ARs can modulate the activity of cardiac Ca2+ channels, independent of beta 1-ARs. The results also demonstrate the usefulness of the CHW heterologous expression system, the first to reconstitute physiological modulation of an L-type Ca2+ channel by the beta 2-AR, for studying receptor subtype-specific regulation of the Ca2+ channel.

Stimulation of the slow calcium current in bullfrog skeletal muscle fibers by cAMP and cGMP.

The effects of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) on slow calcium currents (ICa) were investigated using the Vaseline-gap voltage-clamp technique in bullfrog skeletal muscle cut fibers. Both cAMP and cGMP induced a pronounced increase in the amplitude of ICa when applied to the cut ends of fibers. Both cyclic nucleotides also decreased time to peak current at all membrane potentials. The current-voltage relationship was shifted toward more negative potentials by cAMP as well as cGMP. The potentiating effects of cAMP and cGMP on ICa were additive. 8-Bromo analogues of both nucleotides had similar effects on ICa. The beta-adrenergic agonist isoproterenol, applied extracellularly, also produced an increase in the amplitude of ICa and produced a leftward shift in the current-voltage relationship. These results suggest that both cAMP and cGMP modulate calcium slow channels in bullfrog skeletal muscle fibers, causing stimulation of the ICa. The effect of cyclic nucleotides on ICa in bullfrog skeletal muscle contrasts with that in mammalian cardiac muscle, in which the same nucleotides produce opposite effects on the slow ICa, i.e., in cardiac muscle cAMP stimulates, and cGMP inhibits, the slow ICa.

Optical evidence for a chloride conductance in the T-system of frog skeletal muscle.

T-system action potentials were recorded optically from intact frog skeletal muscle fibers stained with the non-penetrating potentiometric dye NK-2367. The effect of chloride removal on the falling phase of the radially propagating tubular action potential was studied to determine whether a chloride conductance located in the T-system membranes contributes to tubular repolarization during activity. Our results show that, in chloride-free Ringer, repolarization of the tubular action potential is significantly slowed. Moreover, the late phase of tubular repolarization is characterized by a large after-potential, which is highly temperature-dependent and appears as a secondary peak above 10 degrees C. The optical data were compared with predicted T-system action potentials generated from a radial cable equivalent circuit model of the T-system, in which the effects of a distributed tubular leak conductance were tested. Results of this analysis are consistent with the proposal that some of the outward repolarization current during the T-system action potential is drawn across a chloride conductance located in the T-system membranes.

Enantiomeric effects on excitation-contraction coupling in frog skeletal muscle by a chiral phenoxy carboxylic acid.

Aromatic monocarboxylic acids are known to significantly potentiate the mechanical response of skeletal muscle fibers. In this study we investigated the effects of enantiomers of 2-(4-chlorophenoxy)propionic acid, chemically one of the simplest aromatic monocarboxylic acids with chiral properties, on mechanical threshold and charge movement in frog skeletal muscle. The R(+), but not the S(-), enantiomer lowered rheobase mechanical threshold and shifted charge movement to more negative potentials. The R(+) enantiomer also significantly slowed charge movement kinetics, with pronounced delays of the OFF charge transitions. These effects required high temperature for their production. The stereospecific actions of the R(+) enantiomer are interpreted in terms of a specific interaction of this compound at an anion-sensitive site involved in excitation-contraction coupling, most likely on the dihydropryidine-sensitive voltage sensor in the T-system.

A nonlinear electrostatic potential change in the T-system of skeletal muscle detected under passive recording conditions using potentiometric dyes.

Voltage-sensing dyes were used to examine the electrical behavior of the T-system under passive recording conditions similar to those commonly used to detect charge movement. These conditions are designed to eliminate all ionic currents and render the T-system potential linear with respect to the command potential applied at the surface membrane. However, we found an unexpected nonlinearity in the relationship between the dye signal from the T-system and the applied clamp potential. An additional voltage- and time-dependent optical signal appears over the same depolarizing range of potentials where change movement and mechanical activation occur. This nonlinearity is not associated with unblocked ionic currents and cannot be attributed to lack of voltage clamp control of the T-system, which appears to be good under these conditions. We propose that a local electrostatic potential change occurs in the T-system upon depolarization. An electrostatic potential would not be expected to extend beyond molecular distances of the membrane and therefore would be sensed by a charged dye in the membrane but not by the voltage clamp, which responds solely to the potential of the bulk solution. Results obtained with different dyes suggest that the location of the phenomena giving rise to the extra absorbance change is either intramembrane or at the inner surface of the T-system membrane.

Isochannels and blocking modes of voltage-dependent sodium channels.

Our results support the existence of three different Na-channel subtypes or isochannels. These isochannels can be readily distinguished as the predominant Na-channel types in mammalian brain, skeletal muscle, and cardiac muscle. The sensitivity to mu-conotoxin GIIIA and tetrodotoxin is sufficient to classify these channels. The skeletal muscle channel is very sensitive to both tetrodotoxin and mu-conotoxin, the brain channel is sensitive to tetrodotoxin but insensitive to mu-conotoxin, and the heart and denervated muscle channels are insensitive to both toxins. In addition to block at the external receptor site for guanidinium toxins, several other blocking modes can be generalized for batrachotoxin-activated Na channels. One mode is peculiar to certain hydrophobic molecules so far represented by our studies of benzocaine and procaine. These molecules induce discrete blocking events with dwell times that apparently increase with anesthetic concentration and a blocking frequency that increases with negative voltage. This mode is quite distinct from the fast internal block by charged organic molecules that increases with positive voltage. These results imply that it is not possible to ascribe the diverse effects of local anesthetics to a single site in the interior channel mouth, as previously proposed by Hille. Our observations thus support the conclusions of other workers who used mixtures of two local anesthetics to show that the dose-response behavior does not fit single-site behavior, but requires at least two distinct sites. Two additional blocking modes can be distinguished for the interactions of cations at the internal and external mouths of the channel. Organic molecules can apparently enter the electric field from the internal but not the external side of the channel. This result suggests a wide internal entry way to the field and an external constriction that prevents the entry of molecules with a single methyl group but permits entry of divalent inorganic cations such as Ca2+ and Co2+.

Inward rectification in the transverse tubular system of frog skeletal muscle studied with potentiometric dyes.

The non-penetrating potentiometric dyes NK2367 and WW375 were used to investigate the effect of inward rectification on the weighted-average tubular membrane potential in single frog muscle fibres voltage clamped using a three-Vaseline-gap method. In 100 mM-K solution, when inward rectification was activated by hyperpolarization the steady-state amplitude of the transverse tubular system (T-system) optical signal was reduced, and its rise time was faster than that recorded for an equivalent depolarization. The voltage dependence of the optical attenuation followed that of inward rectification, increasing with increasing hyperpolarization. For a voltage-clamp step of -140 mV the optical attenuation was 0.72 which corresponds to a weighted-average T-system potential change of 100 mV. When inward rectification was blocked in a Cs, TEA solution the optical attenuation was also abolished. The voltage dependence of the block of the inward currents in solutions containing low concentrations of Cs was also reflected in the T-system optical signals. Our results were satisfactorily predicted by a radial cable model of the T-system, assuming the same specific inward rectifier conductance in surface and tubular membranes. This analysis predicts that the measured optical attenuation corresponds to a decrease in the tubular space constant, lambda T, from 120 micron under passive conditions to about 40 micron when inward rectification is fully, activated. The voltage dependence of inward rectification measured at the surface membrane was reasonably well predicted by assuming that the specific conductance obeyed a Boltzmann type of voltage dependence; the major effect of tubular decrements was to reduce the steepness of the total (surface + T-system) conductance-voltage relation.

Dichroic behavior of the absorbance signals from dyes NK2367 and WW375 in skeletal muscle fibers.

Absorbance signals were recorded from voltage-clamped single muscle fibers stained with the nonpenetrating potentiometric dyes NK2367 and WW375 and illuminated with quasimonochromatic light from 560 to 800 nm, linearly polarized either parallel (0 degree) or perpendicular (90 degrees) to the fiber long axis. The signals from both dyes depend strongly on the incident polarization. At any wavelength and/or polarization condition, the total absorbance signal is a superposition of the same two signal components previously identified with unpolarized light (Heiny, J. A., and J. Vergara, 1982, J. Gen. Physiol., 80:203)--namely, a fast step signal from the voltage-clamped surface membrane and a signal reflecting the slower T-system potential changes. The 0 degree and 90 degrees spectra of both membranes have similar positive and negative absorbance peaks (720 and 670 nm, respectively, for dye NK2367; 740 and 700 nm for dye WW375); in addition, they have the same dichroic maxima (670 for NK2367; 700 for WW375). However, for the surface membrane, the 0 degrees spectra are everywhere more positive than the 90 degrees spectra, whereas the reverse is true for the T-system, which results in a dichroism of opposite sign for the two membranes. These spectral characteristics were analyzed using a general model for the potential-dependent response of an absorbing dye (Tasaki, I., and A. Warashina, 1976, Photochem. Photobiol., 24:191), which takes into account both the dye response and the membrane geometries. They are consistent with the proposal that the dye responds via a common mechanism in both membranes that consists of a dye reorientation and a change in the absorption maxima.

T-system optical signals associated with inward rectification in skeletal muscle.

The resting potential of many excitable cells, including skeletal muscle, cardiac muscle, nerve cell bodies and egg cells, is determined by a resting potassium conductance which shows inward rectification, allowing potassium ions to move more readily inward across the cell membrane than outward. In skeletal muscle, where inward rectification has been extensively studied, a large part of this conductance is located in the T-system membranes. However, to date, only the kinetic and voltage-dependent properties of this conductance have been studied from analyses of the membrane potential or current recorded at the fibre surface. We report here measurements, obtained using a voltage-sensing dye, of potential changes in the T-system membranes associated with the inwardly rectifying K+ current. Our results show that this conductance alters the time course and significantly attenuates the amplitude of the potential change across the tubular membranes. These optical data provide new evidence for the presence of this conductance in the T-system and, when analysed using a radial cable model for the T-system, provide an estimate of the distribution of the inward rectifier conductance over the surface and T-system which is in agreement with estimates obtained by other techniques.

Optical signals from surface and T system membranes in skeletal muscle fibers. Experiments with the potentiometric dye NK2367.

Absorbance signals were recorded from cut single skeletal muscle fibers stained with the nonpenetrating potentiometric dye NK2367 and mounted in a three-vaseline-gap voltage clamp. The characteristics of the optical signals recorded under current and voltage-clamp conditions were studied at various wavelengths between 500 and 800 nm using unpolarized light. Our results indicate that the absorbance signals recorded with this dye reflect potential changes across both the surface and T system membranes and that the relative contribution of each of these membrane compartments to the total optical change is strongly wavelength dependent. A peak intensity change was detected at 720 nm for the surface membrane signal and at 670 nm for the T system. Evidence for this wavelength-dependent separation derives from an analysis of the kinetics and voltage dependence of the optical signals at different wavelengths, and results obtained in detubulated fibers. The 670-nm optical signal was used to demonstrate the lack of potential control in the T system by the voltage clamp and the effect of a tetrodotoxin (TTX)-sensitive sodium conductance on tubular depolarization.