Changes In Vm Research Articles

Acetylcholine receptor: characterization of the voltage-dependent regulatory (inhibitory) site for acetylcholine in membrane vesicles from Torpedo californica electroplax.

Evidence for a voltage-dependent regulatory (inhibitory) site on the nicotinic acetylcholine receptor to which acetylcholine binds was obtained in membrane vesicles prepared from the Torpedo californica electric organ. Two rate coefficients, JA and alpha, which pertain to the receptor-controlled ion flux, were measured. A 1000-fold concentration range of acetylcholine was used in a transmembrane voltage (Vm) range from 0 to -48 mV under a voltage-clamped condition at pH 7.4, 1 degrees C. The following observations were made. (i) At low acetylcholine concentrations, the value of JA, the rate coefficient for ion translocation by the active (nondesensitized) state of the receptor, increased with increasing concentration. (ii) JA decreased at high acetylcholine concentrations. (iii) In contrast, alpha, the rate coefficient for receptor desensitization, did not show such a decrease. (iv) When the transmembrane potential of the vesicle membrane was changed to more negative values, the value of KR (the dissociation constant for binding of acetylcholine to the regulatory site) decreased by a factor of approximately 9 for a 25 mV change in Vm, while KI (the dissociation constant for binding of acetylcholine to the receptor site that controls channel opening) did not show such a change and has a value of 80 microM. When Vm is -48 mV, KR has a value of 8 microM. (v) The effect of a transmembrane voltage on the regulatory site was reversible and occurred within the time resolution (5 ms) of the quench-flow technique used in the measurements.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine-induced change in intracellular Cl- activity of the mouse lacrimal acinar cells.

Using double-barreled Cl- -sensitive microelectrodes, intracellular Cl- activity (AiCl) in the mouse lacrimal acinar cells in vitro was determined in both resting and secretory phases. In the resting state AiCl was 31 mmol/l which was 1.4 times higher than that predicted for the passive distribution according to the membrane potential (Vm) of -41 mV. Addition of acetylcholine (ACh, 1 microM) hyperpolarized Vm to -63 mV and decreased AiCl to 20 mmol/l which was still twice the equilibrium activity. A-23178 produced similar changes in Vm and AiCl to those induced by ACh. It was concluded that Cl- was actively accumulated in the acinar cells and, in the secretory phase, Cl- efflux was enhanced by the increased driving force and Ca2+-mediated increase in the Cl- permeability across the cell membrane.

Electronic device for microelectrode recordings in epithelial cells.

A device is described that permits continuous measurement of electrophysiological parameters in epithelial tissues in the open-circuit mode. Transepithelial potential (VT) and microelectrode (either conventional or ion-selective) potential (VM) are directly measured. Application of transepithelial current pulses allows continuous monitoring of transepithelial resistance (RT) and the ratio between the changes in VM and VT induced by these pulses. Measurement of this ratio, which under some circumstances reflects the apical fractional resistance of the cellular pathway, is important in assessing membrane damage during microelectrode impalement and/or as an index that the microelectrode tip is inside a cell. This is particularly useful when the change in VM during impalement is small. Application of 0.5-nA current pulses through open-tip microelectrodes allows continuous recording of the microelectrode resistance (RM). In epithelia where the individual cells are electrically coupled this permits acceptable impalements (RM remains nearly constant) to be distinguished from those affected by tip potential artifacts due to plugging of the microelectrode tip (RM increases after penetration of the cell membrane). The device provides compensation for the IR voltage drop in the solution between the potential measuring salt bridges and the epithelial surfaces. The microelectrode electrometer has an input impedance greater than 10(15) and is provided with stray capacitance neutralization.

Physiological basis of a steady endogenous current in rat lumbrical muscle.

In an attempt to determine the mechanism by which rat skeletal muscle endplates generate a steady outward current, we measured the effects of several drugs (furosemide, bumetanide, 9-anthracene carboxylic acid [9-AC]) and changes in external ion concentration (Na+, K+, Cl-, Ba++) on resting membrane potential (Vm) and on the steady outward current. Each of the following treatments caused a 10-15-mV hyperpolarization of the membrane: replacement of extracellular Cl- with isethionate, addition of furosemide or bumetanide, and addition of 9-AC. These results suggest that Cl- is actively accumulated by the muscle fibers and that the equilibrium potential of Cl- is more positive than the membrane potential. Removal of external Na+ also caused a large hyperpolarization and is consistent with evidence in other tissues that active Cl- accumulation requires external Na+. The same treatments greatly reduced or abolished the steady outward current, with a time course that paralleled the changes in Vm. These results cannot be explained by a model in which the steady outward current is assumed to arise as a result of a nonuniform distribution of Na+ conductance, but they are consistent with models in which the steady current is produced by a nonuniform distribution of GCl or GK. Other treatments (Na+-free and K+-free solutions, and 50 microM BaCl2) caused a temporary reversal of the steady current. Parallel measurements of Vm suggested that in none of these cases did the electrochemical driving force for K+ change sign, which makes it unlikely that the steady current arises as a result of a nonuniform distribution of GK. All of the results, however, are consistent with a model in which the steady outward current arises as a result of a nonuniform distribution of Cl- conductance, with GCl lower near the endplate than in extrajunctional regions.

Human sperm motility after migration into, and incubation in, synthetic media

AbstractTen frames/sec microcinematography (“film”), 1 second timed‐exposure photomicrography (“photo”), and laser Doppler velocimetry (LDV) were used to analyse the swimming patterns of human spermatozoa after migration (1 h at 37°C) into an overlying layer of either BWW or Menezo's B2 media. The upper layer of medium was carefully removed and further incubated at 37°C for either 4 h (B2) or 5 h (BWW) and the sperm motility analysed again. Five experiments were performed using semen from different donors. Film and photo analyses gave the relative incidence of nonprogressive and progressively motile spermatozoa plus, for the progressive spermatozoa, the velocities of progression (Vp) and amplitudes of lateral head displacement (Ah). LDV gave the percentage of motile spermatozoa and the modal instantaneous velocity (Vm). All postmigration sperm populations showed large significant increases in the percentage of motile spermatozoa, with good survival during incubation. The progressive postmigration spermatozoa generally moved with greater Vp and Ah than in the initial seminal plasma‐diluted material; Vm was also increased. There were further increases in both Vp and Ah during incubation, but no change in Vm was detected. While the majority of spermatozoa were progressive, some showed a highly active pattern of movement which resulted in no net forward progression. The possible homology between these spermatozoa and the “hyperactivated” motility of capacitated spermatozoa in other mammalian species is discussed. Apparent discrepancies between the three methods used for motility analysis were seen, the possible causes and significances of which are also discussed.

Normalization of end-systolic pressure-volume relation and Emax of different sized hearts.

A shift of the ventricular end-systolic pressure-volume (P-V) relation and a change in its slope Emax reasonably reflect a change in contractility in a given ventricle. However, comparison of Emax's of different sized hearts may be difficult without an appropriate normalization. We attempted to normalize Emax of different sized hearts to the force-length (F-L) relation of unit mass of myocardium in the ventricular wall. We formulated the end-systolic P-V relation as (end-systolic pressure) = Emax (end-systolic volume Vi - Vd), where Vd = volume axis intercept of the end-systolic P-V relation line. As a first step, both thick wall sphere and cylinder models of the ventricle with a wall volume of Vm were used. Circumferential F-L relation of unit myocardium in different ventricular wall layers were formulated as functions of Emax, Vd, and Vm. We found that as long as the product of Emax and Vd remains constant, the F-L relation in the midwall layer and the average F-L relation in the wall remain relatively unchanged regardless of wide changes in Vm and Vi. The elevation of the F-L relation curve, which represents myocardial contractility, was found to change in proportion to Emax Vd, largely independent of Vm and Vi, or the size of the ventricle.

The intracellular pH of frog skeletal muscle: its regulation in hypertonic solutions.

Intracellular pH (pHi) was followed with micro-electrodes in frog semitendinosus muscle, superfused at 22 degrees C with hypertonic solutions (external pH, pHo, 7.35) containing 2.5, 15 or 50 mM-K. Tonicity was doubled by addition of 250 mM-mannitol or, in a few cases, 125 mM-extra NaCl. Tripling of tonicity was accomplished by adding 500 mM-mannitol. Because of the ability of hypertonicity to minimize contracture, the course of pHi could be followed from the start of depolarization. The pHi of fibres after about 40 min in Ringer solution (2.5 mM-K, HEPES buffer) of twice normal tonicity was 7.40 +/- 0.04 (S.E. of mean) (n = 17), about 0.2 higher than at normal tonicity. The membrane potential, Vm, was -87.7 +/- 1.3 mV. When the muscle was depolarized in 50 mM-K to about -30 mV, the pHi rapidly fell by 0.3-0.5 unit (n = 9), and then promptly returned. This recovery was followed by a much slower and progressive rise to above control. Removing Na from the medium did not affect the degree of acidification, but the pHi recovered at a slightly slower rate, did not reach control value and showed no progressive rise. A less pronounced transient acidification was also observed when the muscle was depolarized in 15 mM-K to about -60 mV. When contracture was prevented either by 1-2 mM-tetracaine under isotonic conditions or by raising tonicity 3-fold, 50 mM-K produced no transient acidification. When the pHi of resting fibres in Ringer solution (2.5 mM-K) of twice normal tonicity was reduced by 5% CO2 from 7.40 to 7.12 +/- 0.07 (n = 3), it recovered at a slow rate (0.06 +/- 0.03 delta pHi h-1). Depolarization by 15 or 50 mM-K enhanced recovery rate 4-6-fold. These solutions of twice normal tonicity, as compared to those of normal tonicity, shifted the curve relating pHi recovery rate and membrane potential along the potential axis in the direction of hyperpolarization. This shift may be due to increased ionic shielding of fixed negative charges at the inner membrane surface. At twice normal tonicity, the very slow pHi recovery of resting fibres from CO2-induced acidification, as well as the more rapid recovery in depolarized fibres, could be abolished by 1 mM-amiloride or by removing Na. The application of amiloride during pHi recovery in 50 mM-K was not associated with an observable change in Vm. SITS had no significant effect on recovery.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of potassium and chloride permeability over the surface and T-tubule membranes of mammalian skeletal muscle

The distribution of K and Cl permeability, PK and PCl, over the surface and T-tubule membranes of red rat sternomastoid fibers has been determined. Membrane potential, Vm, was recorded with 3-M KCl-filled glass microelectrodes. Changes in Vm with changes in [K]o or [Cl]o were used to estimate PCl/PK in normal and detubulated preparations. The results show that the T-tubule membrane has a high PCl and is therefore different from the T-tubule membrane of amphibian fibers. Analysis of the time course of depolarization when [K]o was raised (in SO4 solutions) showed that PK was distributed over the surface and T-tubule membranes. Two observations suggested that T-tubule PCl was higher than the surface PCl. Firstly, in normal fibers, the depolarization caused by an increase in [K]o was 3.5 times greater in SO4 solutions than in Cl solutions. In marked contrast, the depolarization in glycerol-treated fibers was independent of [Cl]o. Secondly, the rapid change in Vm when [Cl]o was changed was reduced by 80% after glycerol treatment. Both observations suggest that PCl was low in glycerol-treated fibers. PCl/PK was calculated from the Vm data using Goldman, Hodgkin and Katz equations for Na and K or for Na, K, and Cl. In normal fibers PCl/PK = 4.5 and in glycerol-treated fibers PCl/PK = 0.28. Since it is unlikely that glycerol treatment would increase PK, the reduction in the ratio must follow the loss of Cl permeability "channels" in the T-tubule membrane.

The influence of external cations and membrane potential on Ca-activated Na efflux in Myxicola giant axons.

In microinjected Myxicola giant axons with elevated [Na]i, Na efflux was sensitive to Cao under some conditions. In Li seawater, sensitivity to Cao was high whereas in Na seawater, sensitivity to Cao was observed only upon elevation of [Ca]o above the normal value. In choline seawater, the sensitivity of Na efflux to Cao was less than that observed in Li seawater whereas Mg seawater failed to support any detectable Cao-sensitive Na efflux. Addition of Na to Li seawater was inhibitory to Cao-sensitive Na efflux, the extent of inhibition increasing with rising values of [Na]o. The presence of 20 mM K in Li seawater resulted in about a threefold increase in the Cao-activated Na efflux. Experiments in which the membrane potential, Vm, was varied or held constant when [K]o was changed showed that the augmentation of Ca-activated Na efflux by Ko was not due to changes in Vm but resulted from a direct action of K on activation by Ca. The same experimental conditions that favored a large component of Cao-activated Na efflux also caused a large increase in Ca influx. Measurements of Ca influx in the presence of 20 mM K and comparison with values of Ca-activated Na efflux suggest that the Na:Ca coupling ratio may be altered by increasing external [K]o. Overall, the results suggest that the Cao-activated Na efflux in Myxicola giant axons requires the presence of an external monovalent cation and that the order of effectiveness at a total monovalent cation concentration of 430 mM is K + Li greater than Li greater than Choline greater than Na.

Electrical properties and active solute transport in rat small intestine. I. Potential profile changes associated with sugar and amino acid transports.

Addition of D-glucose to the mucosal fluid resulted in a significant depolarization of the mucosal membrane potential (V-m) in rat duodenum, jejunum, and ileum accompanied by an increase in the transepithelial potential difference (PDt). On the other hand, L-glucose did not induce PDt and Vm changes. Glycine applied from the mucosal side also induced Vm-depolarization and PDt-increment in the ileum. Phlorizin added to the mucosal fluid or ouabain added to the serosal fluid inhibited the sugar-dependent changes in PDt and Vm. According to the analysis with an equivalent circuit model for the epithelium, it was concluded that an actively transported solute induced not only a depolarization of the mucosal (brush border) membrane but also a hyperpolarization of the serosal (baso-lateral) membrane of an epithelial cell, so that the origin of solute-induced PDt changes should be attributed to changes in emf's at both membranes. The hyperpolarization of the serosal membrane in the presence of an actively transported solute was attributed to a mechanism of serosal electrogenic sodium pump stimulated by the increase in the extrusion rate of Na+ co-transported into the cell with sugar or amino acid.

β-2-THIENYL-DL-ALANINE (Thi) AS AN INHIBITOR OF PHENYLALANINE HYDROHYLASE (PheH) AND PHENYLALANINE (Phe) INTESTINAL TRANSPORT, IN VITRO AND IN VIVO

Thi inhibits phe tubular resorption and alao causes malabaorption of Phe in animals. Structural similarities between Phe and Thi suggested Thi may be a good inhibitor of PheH and hence reproduce the biochemical lesion in PKU. The effects of Thi on rat liver (Lv) and kidney (Kd) PheH were assessed in vitro and in vivo, and on intestinal transport of Phe. The Km for Lv PheH changed from 0.54 mM in the absence of Thi, to 6.6 mM in the presence of 24 mM Thi with no significant change in Vm. For Kd the respective Km were 0.60 and 3.0 mM, both cases inSicating competitive inhibition. Ki were similar in both tissues: 3.2 and 3.4 mM. Hill coefficients close to 1 showed PheH in Lv and Kd was not an allosteric enzyme. No inhibition of PheH in vivo was observed in Lv and Kd, 1 or 24 hr after an i.p. dose of Thi (2 mmolea/kg). When injections were repeated daily for 4 days, only a marginal inhibition of PheH (14% in Lv and 23% in Kd) was observed. In other experiments, when 24 mM Thi was perfused in vivo together with Phe through a 20 cm jejunal segment, it produced a moderate inhibition of Phe intestinal transport. Kinetic studies also indicated competitive inhibition. The elevated Ki for Thi (80 mM)makes it unlikely that oral Thi could effectively decrease Phe absorption and hence provide an alternative to low Phe diets in the management of PKU.

The effect of synthetic TRH on transmembrane potential and membrane resistance of adenohypophysial cells.

An evaluation of the stimulus-secretion coupling hypothesis was undertaken by the intracarotid administration of synthetic thyroid releasing hormone (TRH) while recording the transmembrane potential (Vm) and membrane resistance (Rm) of cells in the adenohypophysis of propylthiouracil pretreated male rats. Of those 75 cells which responded to TRH administration with a change in membrane parameters, the most predominant effect was a depolarization with a decrease in Rm observed in 21 cells, or no change in Vm but an increase in Rm observed in 23 cells. These results are discussed in terms of the thyrotrophs and other cells comprising the adenohypophysis.