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R L Barchi

Publications and source records attributed to R L Barchi.

107 records · Page 6Linked to original sources
PubMed1977

Isolation and characterization of muscle membranes using surface-specific labels.

Membranes were purified from rat muscle by a differential centrifugation method that avoids the use of salt extraction or incubations at elevated temperature. Three populations of membrane-limited vesicles were defined having average densities of 1.112 (fraction I), 1.141 (fraction II), and 1.158 (fraction III) g/ml in a continuous sucrose gradient. Lactoperoxidase-catalyzed iodination of intact muscle prior to isolation of membranes resulted in highest specific activity in fraction I, although all fractions could be equally labeled after isolation. 125I-Labeled wheat germ agglutinin incubated at low concentration with intact muscle preferentially labeled fraction I. Parallel studies on previously isolated fractions indicated that fraction I also contained the highest concentration of potential receptors for wheat germ agglutinin. In experiments on whole muscle, concanavalin A bound predominantly to sarcolemma with slight variable binding to T-tubular and nuclear membrane but no binding to sarcoplasmic reticulum or mitochondria. Parallel binding studies with isolated membrane fragments indicated heavy binding of concanavalin A by membranes in fraction I with scattered binding in fractions II and III. Na+K+Mg2+ ATPase was specifically enriched in fraction I but was also present in fraction II in a proportion similar to 125I labeling. Ca2+ ATPase was most active in fraction II but present in significant levels in fraction I. It is concluded from these and other data that fraction I contains predominantly sarcolemma membrane, while T-tubular membrane may represent a significant component of fraction II. Ca2+ ATPase activity in fraction I is intrinsic to the sarcolemma.

Adenosine Triphosphatases↗
PubMed1977

Characteristics of the chloride conductance in muscle fibers of the rat diaphragm.

In muscle fibers from the rat diaphragm, 85% of the resting membrane ion conductance is attributable to Cl-. At 37 degree C and pH 7.0, GCl averages 2.11 mmho/cm2 while residual conductance largely due to K+ averages 0.34 mmho/cm2. The resting GCl exhibits a biphasic temperature dependence with a Q10 of 1.6 between 6 degree C and 25 degree C and a Q10 of nearly 1 between 25 degree C and 40 degree C. Decreasing external pH reversibly reduced GCl; the apparent pK for groups mediating this decrease is 5.5. Increasing pH up to 10.0 had no effect on GCl. Anion conductance sequence and permeability sequence were both determined to be Cl-greater than Br-greater than or equal to I-greater than CH3SO4-. Lowering the pH below 5.5 reduced the magnitude of the measured conductance to all anions but did not alter the conductance sequence. The permeability sequence was likewise unchanged at low pH. Experiments with varying molar ratios of Cl- and I- indicated a marked interaction between these ions in their transmembrane movement. Similar but less striking interaction was seen between Cl- and Br-. Current-voltage relationships for GCl measured at early time-points in the presence of Rb+ were linear, but showed marked rectification with longer hyperpolarizing pulses (greater than 50ms) due to a slow time-and voltage-dependent change in membrane conductance to Cl-. This nonlinear behavior appeared to depend on the concentration of Cl- present but cannot be attributed to tubular ion accumulation. Tubular disruption with glycerol lowers apparent GCl but not GK, suggesting that the transverse tubule (T-tubule) system is permeable to Cl- in this species. Quantitative estimates indicate that up to 80% of GCl may be associated with the T tubules.

Animals↗
PubMed1977

On the inhibition of muscle membrane chloride conductance by aromatic carboxylic acids.

25 aromatic carboxylic acids which are analogs of benzoic acid were tested in the rat diaphragm preparation for effects on chloride conductance (G(Cl)). Of the 25, 19 were shown to reduce membrane G(Cl) with little effect on other membrane parameters, although their apparent K(i) varied widely. This inhibition was reversible if exposure times were not prolonged. The most effective analog studied was anthracene-9-COOH (9-AC; K(i) = 1.1 x 10(-5) M). Active analogs produced concentration-dependent inhibition of a type consistent with interaction at a single site or group of sites having similar binding affinities, although a correlation could also be shown between lipophilicity and K(i). Structure-activity analysis indicated that hydrophobic ring substitution usually increased inhibitory activity while para polar substitutions reduced effectiveness. These compounds do not appear to inhibit G(Cl) by altering membrane surface charge and the inhibition produced is not voltage dependent. Qualitative characteristics of the I-V relationship for Cl(-) current are not altered. Conductance to all anions is not uniformly altered by these acids as would be expected from steric occlusion of a common channel. Concentrations of 9-AC reducing G(Cl) by more than 90 percent resulted in slight augmentation of G(I). The complete conductance sequence obtained at high levels of 9-AC was the reverse of that obtained under control conditions. Permeability sequences underwent progressive changes with increasing 9-AC concentration and ultimately inverted at high levels of the analog. Aromatic carboxylic acids appear to inhibit G(Cl) by binding to a specific intramembrane site and altering the selectivity sequence of the membrane anion channel.

Animals↗
PubMed1976

Membrane-associated thiamin triphosphatase. II. Activation by divalent cations.

Activation of membrane-associated thiamin triphosphatase from rat brain requires a divalent cation (Mg2+, Ca2+, or Mn2+). The optimum concentration of Mg2+ necessary for maximal enzyme activity varies with substrate concentration; conversely, the maximal rate of hydrolysis attainbale by increasing thiamin triphosphate concentration is directly proportional to [Mg2+] for all levels of Mg2+ below that of the substrate. Under appropriate conditions, the Km of the thiamin triphosphatase for Mg2+ and for thiamin triphosphate are shown to be identical. Dissociation constants (Kd) for the binding of Mg2+ to thiamin triphosphate, thiamin diphosphate, and thiamin were determined; kinetic data re-expressed in terms of [Mg2+-thiamin triphosphate] conform to simple single substrate predictions, suggesting that the true enzyme substrate may be the Mg2+-thiamin triphosphate complex. Excess free Mg2+ inhibits thiamin triphosphatase activity competitively while excess free thiamin triphosphate in concentrations up to 10 times Km has no effect on the membrane-bound enzyme.

Animals↗
PubMed1975

Myotonia. An evaluation of the chloride hypothesis.

The muscle membrane in myotonia congenita is characterized by a normal resting potential with a greatly increased resting resistance usually attributed to a decrease in membrane chloride permeability (PC1). In this report, the hypothesis that decreased PC1 alone can account for the repetitive action potentials of myotonia is tested with a mathematical model of the muscle membrane and is shown to be valid. Reduction of Pc1 to 20% of control values will produce myotonic activity in response to a single stimulus. Membrane resistance and potential approximate those found experimentally. The model predicts that increasing external K+ will aggravate myotonia due to a reduction of PC15 while decreasing Kout will prevent repetitive spiking. Further, myotonia can be prevented by reducing peak membrane sodium permeability or by shifting the voltage dependency of the membrane rate constants for sodium in a depolarizing direction. These results are shown to correlate well with clinical observtonia.

Action Potentials↗
PubMedDate not supplied

Physical probes of biological membranes in studies of the muscular dystrophies.

This review deals with recent physical approaches to the study of biomembranes. First, current concepts of the structure and dynamic organization of biological membranes are considered. The theoretical basis and practical application of various physical probe techniques are then discussed with specific reference to their usefulness in studies of these membranes. Particular emphasis is given to electron spin resonance and fluorescence techniques and to the potential difficulties encountered in their use and interpretation. Finally, reports applying these physical probes to various aspects of the human muscular dystrophies are reviewed and our current information in this area is summarized.

Cell Membrane↗
PubMedDate not supplied

Chloride efflux measurements in mammalian skeletal muscle.

A rapid sampling technique has been used to resolve the components of 36Cl efflux from isolated extensor digitorum longus (EDL) muscles of young rats. Four distinct fluxes with apparent rate constants of 4.40 +/- 0.02 min-1 (k1), 1.30 +/- 0.04 min-1 (k2), 0.24 +/- 0.04 min-1 (k3), and 0.048 +/- 0.05 min-1 (k4) at 30 degrees C were identified. Together, these fluxes accounted for the movement of more than 98% of exchangeable muscle chloride (Cl) ions. The muscle compartment associated with the fastest flux (k1) contained 23% +/- 2% of the total muscle Cl corresponding to the extracellular space as determined with inulin or mannitol. The compartment associated with k2 accounted for 71% of the intracellular volume, and k2 was assumed to represent 36Cl efflux across the surface membrane. The rate constant k2 was temperature-dependent with a Q10 of 1.11 between 5 degrees C and 30 degrees C. Aromatic carboxylic acids known to block sarcolemmal Cl conductance (GCl) specifically lowered k2 by 25% at 30 degrees C, as did replacement of external Cl with nitrate.

Animals↗
PubMedDate not supplied

Structure, function and expression of voltage-dependent sodium channels.

Voltage-dependent sodium channels control the transient inward current responsible for the action potential in most excitable cells. Members of this multigene family have been cloned, sequenced, and functionally expressed from various tissues and species, and common features of their structure have clearly emerged. Site-directed mutagenesis coupled with in vitro expression has provided additional insight into the relationship between structure and function. Subtle differences between sodium channel isoforms are also important, and aspects of the regulation of sodium channel gene expression and the modulation of channel function are becoming topics of increasing importance. Finally, sodium channel mutations have been directly linked to human disease, yielding insight into both disease pathophysiology and normal channel function. After a brief discussion of previous work, this review will focus on recent advances in each of these areas.

Action Potentials↗