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J P Reuben

Publications and source records attributed to J P Reuben.

At least 37 records · Page 2Linked to original sources
PubMed1986

Two calcium currents in a smooth muscle cell line.

Membrane currents in small cells of a smooth muscle cell line (A10) derived from embryonic rat thoracic aorta were monitored by the patch electrode whole-cell voltage clamp technique. Three currents, two divalent cation currents, and a Ca2+-activated K+ current have been observed. The latter is readily abolished pharmacologically, allowing the characterization of the divalent cation currents. With a holding potential of -50 mV, a single divalent current, which inactivates slowly, is elicited on depolarization of the membrane potential to values positive to ca. -10 mV. The second divalent cation current is only observed when the holding potential is negative to -55 mV and the membrane is pulsed to values positive to ca. -35 mV. This current is rapidly inactivating, peaking in approximately 5 ms and decaying with a t1/2 of ca. 15 ms at 0 mV when conveyed by Ba2+. The rapidly inactivating divalent cation current is depressed by substitution of Ba2+ for Ca2+ in the bathing solution and is highly insensitive to organic Ca2+ channel blockers. The slowly inactivating channel has more typical characteristics of Ca2+ channels; it is more permeable to Ba2+ than to Ca2+ and is sensitive to modulation by dihydropyridines. These data demonstrate the presence of two distinctly different Ca2+ channels in A10 cells.

Animals↗
PubMed1985

Calcium currents in GH3 cultured pituitary cells under whole-cell voltage-clamp: inhibition by voltage-dependent potassium currents.

To isolate inward Ca2+ currents in GH3 rat pituitary cells, an inward Na+ current as well as two outward K+ currents, a transient voltage-dependent current (IKV) and a slowly rising Ca2+-activated current (IKCa), must be suppressed. Blockage of these outward currents, usually achieved by replacement of intracellular K+ with Cs+, reveals sustained inward currents. Selective blockage of either K+ current can be accomplished in the presence of intracellular K+ by use of quaternary ammonium ions. When IKCa and Na+ currents are blocked, the net current elicited by stepping the membrane potential (Vm) from -60 to 0 mV is inward first, becomes outward and peaks in 10-30 msec, and finally becomes inward again. Under this condition, in which both IKV and Ca2+ currents should be present throughout the duration of the voltage step, the Ca2+ current was not detected at the time of peak outward current. That is, plots of peak outward current vs. Vm are monotonic and are not modified by nisoldipine or low external Ca2+ as would be expected if Ca2+ currents were present. However, similar plots at times other than at peak current are not monotonic and are altered by nisoldipine or low Ca2+ (i.e., inward currents decrease and plots become monotonic). When K+ channels are first inactivated by holding Vm at -30 mV, a sustained Ca2+ current is always observed upon stepping Vm to 0 mV. Furthermore, substitution of Ba2+ for Ca2+ causes blockage of IKV and inhibition of this current results in inward Ba2+ currents with square wave kinetics. These data indicate that the Ca2+ current is completely inhibited at peak outward IKV and that Ca2+ conductance is progressively disinhibited as the transient K+ current declines due to channel inactivation. This suggests that in GH3 cells Ca2+ channels are regulated by IKV.

Animals↗
PubMed1984

Characterization of verapamil binding sites in cardiac membrane vesicles.

Specific, saturable, and reversible binding of verapamil has been demonstrated in crude cardiac sarcolemmal membranes. These receptors possess a Kd of approximately 50 nM for verapamil as determined by either equilibrium binding studies, competition binding analysis, or kinetic analysis of on and off rates and display an average density of 1.25 pmol/mg of protein. Specificity of binding is indicated by several criteria. Competition studies with the verapamil analog D-600 indicate that (-)D-600 is 200-fold more potent than the (+)-isomer in displacing bound verapamil. Likewise, several other aryl alkyl amine Ca2+ entry blockers effectively displace bound ligand. In addition, dihydropyridines and diltiazem promote partial (25-35%) displacement of bound verapamil with Ki values similar to the Kd values for their respective receptors. Characterization of nitrendipine binding in this preparation indicates an average density of 0.3 pmol of receptors/mg of protein suggesting that the verapamil:nitrendipine binding site ratio is approximately 4:1. Binding characteristics of verapamil and nitrendipine receptors in highly purified sarcolemmal vesicles are similar to those in the crude preparation except that the ratio of verapamil:nitrendipine sites approaches 1 and nitrendipine and diltiazem promote almost complete displacement of bound verapamil. Fractionation studies of crude sarcolemmal membranes indicate that excess verapamil receptors, insensitive to the action of dihydropyridines or diltiazem, are located in a high-density, nonmitochondrial, non-sarcolemmal membrane fraction. Thus, verapamil receptors exist in two locations in cardiac tissue but only in the sarcolemmal membrane are these receptors coupled to the dihydropyridine receptor.

Animals↗
PubMed1983

Regulation of excitation-secretion coupling by thyrotropin-releasing hormone (TRH): evidence for TRH receptor-ion channel coupling in cultured pituitary cells.

The electrophysiological and secretory properties of a well-studied clonal line of rat anterior pituitary cells (GH3) have been compared with a new line of morphologically distinct cells derived from it (XG-10). The properties of the latter cells differ from the parent cells in that they do not have receptors for thyrotropin-releasing hormone and their basal rate of secretion is substantially higher (ca. three- to fivefold). While both cell types generate Ca++ spikes, the duration of the spike in XG-10 cells (ca. 500 msec) is about 2 orders of magnitude longer than that in GH3 cells (5-10 msec). The current-voltage characteristics of the two cell types are markedly different; the conductance of GH3 cells is at least 20-fold higher than XG-10 cells when cells are depolarized to more positive potentials than the threshold for Ca++ spikes (approximately -35 mV). While treatment of GH3 cells with the secretagogues tetraethylammonium chloride or thyrotropin-releasing hormone decreases the conductance in this voltage region to approximately the same as that for XG-10 cells, the electrophysiological and secretory properties of XG-10 cells are unaffected by treatment with either of these agents. Results of this comparative study suggest that XG-10 cells lack tetraethylammonium-sensitive K+ channels. The parallel loss of thyrotropin-releasing hormone receptor binding activity and of a K+ channel in XG-10 cells implies that the thyrotropin-releasing hormone receptor may be coupled with, or be an integral part of, this channel. Apparently thyrotropin-releasing hormone, like tetraethylammonium chloride, acts by inhibiting K+ channels resulting in a prolongation of the action potential, promoting Ca++ influx and subsequently enhancing hormone secretion.

Action Potentials↗
PubMed1983

Excitation-contraction coupling: role of K-activation within the transverse tubular system.

Long-duration Ca action potentials induced in crustacean muscle fibers after prolonged exposure to quaternary ammonium ions are accompanied by attenuated tensions with unique time courses. The tensions have three phases. The initial phase, correlated with the upstroke of the spike, is a rapid increase in tension followed by relaxation to or near to resting level (on-tension). In the second phase, tension rises slowly as the spike plateau declines. The final phase is another rapid increase and decay in tension that is correlated with termination of the action potential (off-tension). To observe these tensions, fibers must be exposed to 50-100 mM tetrabutylammonium ion for about 1 hr or to lower concentrations for longer periods (e.g., 5 mM for 20-30 hr). To obtain a similar response in fibers treated with tetraethylammonium ion, higher concentrations or longer soaking periods, or both, are required. Because neither caffeine-induced tensions in intact fibers nor contractile protein and sarcoplasmic reticulum function in skinned fibers were modified by quaternary ammonium ions, their site of action appears to be limited to surface or transverse tubular system membranes, or both. The unique tensions can be explained by considering the mode by which quaternary ammonium ions block K channels in conjunction with a scheme in which activation of K channels within the transverse tubular system controls the driving force for influx of Ca ions.

Action Potentials↗
PubMed1982

Calcium and tension regulation in skinned skeletal muscle fibers.

This article reviews the skinned muscle fiber techniques and approaches that have been used in our laboratory in the study of both tension and Ca regulation over the past 15 years. The impetus for using these fibers was the finding that most membrane Cl channels in crayfish muscle are located within the transverse tubules at the junction (diad) with the sarcoplasmic reticulum (SR). This observation led to the postulate that a change in Cl concentration around the SR would cause it to release stored CA. This could be tested by using skinned fibers. Tensions induced by Cl release of SR- Ca or by direct application of Ca are highly dependent on the concentration of substrate (MgATP). A substrate-inhibition formulation accurately summarizes this dependence, but it is not integrated with current biochemical schemes of ATP hydrolysis. To facilitate development of a broad model for substrate regulation of tension, which includes concepts of a cross-bridge cycle, we developed a mammalian skinned fiber preparation and techniques for monitoring regulation in single fibers. Both optical and mechanical techniques are used to investigate contractile protein interactions and Ca binding.

Adenosine Triphosphate↗
PubMed1980

Functional heterogeneity of the sarcoplasmic reticulum within sarcomeres of skinned muscle fibers.

Precipitation of Ca oxalate in the sarcoplasmic reticulum of chemically skinned rabbit psoas fibers caused an increase in light scattering which was proportional to the amount of Ca accumulated per unit fiber volume. The increase in scattering was used to measure net accumulation rates and steady-state Ca capacities of the sarcoplasmic reticulum in single fibers. The data obtained were qualitatively and quantitatively similar to those reported for isolated vesicle preparations. Under conditions in which Ca was not depleted from the medium, Ca accumulation was linear with time over much of its course. Steady-state capacities were independent of the Ca concentration; uptake rates were half-maximal at 0.5 microM Ca++ and saturated above about 1.0 microM. Both rate and capacity varied with the oxalate concentration, being maximal at oxalate concentrations greater than or equal to mM and decreasing in proportion to one another at lower concentrations, with a threshold near 0.25 mM. At the lower loads, electron micrographs showed many sarcoplasmic reticulum elements empty of precipitate alongside others that were full, whereas virtually all were filled in maximally loaded fibers. These data indicate that the Ca oxalate capacity of each fiber varies with the number and volume of elements in which Ca oxalate crystals can form at a given oxalate concentration, and that individual regions of the sarcoplasmic reticulum within each sarcomere differ in their ability to support Ca oxalate precipitation. Our working hypothesis is that this range in ability to form Ca oxalate crystals involves differences in ability to accumulate and retain ionized Ca inside the sarcoplasmic reticulum.

Adenosine Triphosphate↗
PubMed1979

Filament interaction in intact muscle fibers monitored by light scattering.

Measurement of changes in optical properties of intact muscle fibers during contraction has proven to be difficult or, in some cases, impossible due to movement of the muscle relative to the incident beam. In this paper we describe a technique for immobilizing single fibers in clear gelatin, which permits measurement of light scattering signals undistorted by movement artifacts. We also describe the phase and amplitude relationship between changes in intensity of light scattering (at 90 degrees to incident beam) and tensions induced by electrically activating single fibers. With tensions that range up to 50% Po (Po = maximal tension measured by exposure of fibers to 200 mM K+), the maximal increase in light scattering is about 25% of that for resting fibers. The scattering increase precedes tension, and at low temperatures the interval between the two peaks can be 50--100 msec. We interpret these data on intact fibers, as we did our earlier data from studied on skinned fibers, as indicating that increases in light scattering power of muscle are primarily due to attachment of myosin cross-bridges to actin filaments.

Actins↗
PubMed1978

Unusual thick and thin filament packing in a crustacean muscle.

The proximal accessory flexor (PAF) of the myochordotonal organ (MCO) in the meropodite of crayfish walking legs contains two populations of muscle fibers which are distinguishable by their diameters. The large accessory (LA) fibers are 40-80 micrometer in diam and are similar in ultrastructure to other slow crustacean fibers. The small accessory (SA) fibers are 1-12 micrometer in diam and have a unique myofilament distribution at normal body lengths. There is extensive double overlap of thin filaments at these lengths, and some of them form bundles that may extend the length of the sarcomere. In the middle of the sarcomeres, thick and thin filaments are totally segregated from each other. When the fibers are stretched to lengths beyond double overlap length, the myofilament patterns are conventional. The segregated pattern is reestablished when stretched fibers are allowed to shorten passively. The length-tension relationship of the SA fibers is described by a linear ascending branch, a plateau, and a linear descending branch. The ascending branch encompasses normal body lengths from slack length (Ls) with maximum double overlap to the length at which double overlap ceases (1.8 X Ls). The descending phase is comparable to that of other skeletal muscles. That is, tension decreases in proportion with the reduction in thick-thin filament interdigitation (2 X Ls to 3 X Ls).

Animals↗
PubMed1978

Filament interaction monitored by light scattering in skinned fibers.

The intensity of light scattered by chemically skinned rabbit psoas fibers in relaxed, rigor, and activated states was monitored at 90 degrees to the incident beam. In the relaxed state, scattering varied in proportion to the volume of muscle in the beam. Scattering increased to 2.3 times the resting value when rigor was induced by withdrawal of MgATP or when the myofibrils were activated by the caffeine-induced release of Ca from the sarcoplasmic reticulum. The rigor-induced increase in scattering decreased monotonically when MgATP was reintroduced stepwise (0-100 microM). This decrease in scattering was accompanied by an increase in tension up to an optimum MgATP level of approximately 10 microM, and then tension decreased at higher concentrations (10-100 microM). The increase in scattering during both rigor and activation was dependent upon fiber length. At lengths when thick-thin filament overlap was near zero, the light signal due to rigor and activation fell to within 10% of the signal for the relaxed fiber at that length. The signal during rigor increased only minimally (approximately 10%) when stretch (approximately 1%) was applied. This increase in signal was small despite a measured 5- to 10-fold increase in tension and an estimated twofold increase in stiffness. Thus, the increased light scattering caused by rigor and activation depends on filament overlap and not tension, stiffness, or substrate binding.

Adenosine Triphosphate↗
PubMed1978

Duchenne dystrophy: abnormal generation of tension and Ca++ regulation in single skinned fibers.

Skinned, single-fiber preparations from the quadriceps or gastrocneumius muscles of four ambulatory male children with Duchenne dystrophy were tested for theri ability to generate tension and to regulate CA++. To determine the intrinsic strength (P0) of the contractile material, the maximum Ca++ -activated tensions were normalized to the fiber diameters. Sixty-four percent of the Duchenne fibers had P0 values below 1.0 kg per square centimeter--the lowest value observed in control muscle--and the average P0 values of fibers from each Duchenne biopsy were significantly (p less than 0.01) below the average P0 values for control muscle fibers and for muscle fibers obtained from one obligatory carrier of the Duchenne gene. The low tensions in the Duchenne muscle fibers could not be ascribed to altered Ca++ regulation or to substrate sensitivity of the contractile proteins in the fibers, since these were normal. However, ultrastructural abnormalities of the myofilaments, which might reduce the ability of the contractile system to develop tension, were observed. Furthermore, Ca++ regulation by the sarcoplasmic reticulum (SR) was impaired in most of those muscle fibers, from both carriers and Duchenne patients, that did develop normal tension. These results suggest that in Duchenne muscle a functional disorder in the SR may precede loss of the ability of the contractile proteins to generate tension. However, since muscle fibers from Duchenne-gene carriers developed significantly greater tensions than fibers from Duchenne-patients, while yet having similar defects in Ca++ regulation, the SR disorder may not be exclusively responsible for abnormal contractile protein function.

Adult↗
PubMed1977

Regulation of tension in skinned muscle fibers: effect of high concentrations of Mg-ATP.

The control of tension in skinned fibers by Mg-ATP and Ca described in previous publications has been studied at high substrate concentrations over a wide range of temperature and salt concentration. Curves of tension versus pCa shift systemically to the right as [Mg-ATP] increases. The maximum Ca-activated tension of a skinned fiber declines at sufficiently high substrate concentrations. This behavior is described by a generalization of the scheme given in the earlier reports.

Adenosine Triphosphate↗
PubMed1975

Sites of action of D2O in intact and skinned crayfish muscle fibers.

The effect on tension development of replacing 90% of the H2O of the bathing saline with D2O was studied on intact single fibers, and on skinned fibers before and after the latter were treated so as to eliminate Ca-accumulation by the sarcoplasmic reticulum (SR). Excitation-contraction coupling (ECC) of intact fibers is not abolished, but is depressed by D2O so that higher depolarizations are required to elicit a given tension. The reduction in tension at a given level of depolarization is not due to inhibition of the contractile system. The latter showed an enhanced Ca sensitivity; that is, skinned fibers respond to Ca concentrations that are 1-2 orders of magnitude smaller in D2O than in H2O saline. When bathed in D2O saline, intact fibers or skinned fibers with functional SR can still accumulate and release Ca in sufficient quantities to allow repeated induction of maximum tensions. Relaxation is slowed in all three types of preparation, perhaps because of an increased affinity of troponin to Ca in D2O salines.

Animals↗
PubMed1974

Calcium binding and tension development in detergent-treated muscle fibers.

The nonionic detergent Brij 58 eliminates irreversibly the capability of the sarcoplasmic reticulum (SR) of skinned crayfish muscle fibers to sequester Ca and to release it under appropriate stimulation. In contrast to deoxycholate (DOC) which causes an irreversible diminution of tension as well, Brij 58 does not affect the contractile proteins. Comparison of the time-course of tension development before and after Brij treatment demonstrates that Ca is accessible to the contractile proteins more rapidly after the SR is destroyed but, nevertheless, much more slowly than is predicted for free diffusion of Ca in the myoplasm. Slowing apparently results because of the presence of ca 1 mmol/kg fiber of myoplasmic Ca-binding sites that remain after Ca uptake of the SR is eliminated. A theoretical model is presented which allows for the effects of binding sites and of an unstirred layer in the vicinity of the fiber on Ca diffusion into the myoplasm.

Animals↗
PubMed1972

Regulation of tension in the skinned crayfish muscle fiber. II. Role of calcium.

Tension outputs were measured in skinned crayfish muscle fibers exposed to solutions variously buffered for both Mg-adenosine triphosphate (ATP) and Ca. Two types of data are shown, relating tension and substrate concentration with different levels of Ca present, or tension and calcium concentration at different levels of substrate. The data are fitted by curves calculated from a general equation for substrate inhibition. The equation is based on the schema that both tension and relaxation are induced by the substrate and that the relaxing effect of excess substrate is repressed by calcium. The physiological findings of the present work are similar to data obtained by others on biochemical model systems of the contractile proteins.

Actomyosin↗