bluuebunny/crossref_metadata_2025 huggingface dataset:
Ionic mechanisms underlying the responses of off-center bipolar cells in the carp retina. I. Studies on responses evoked by light.
Authors: Saito T , Kaneko A
Published: May 2004

Off-center bipolar cells show hyperpolarizing responses to spot illumination in the receptive field center and depolarization responses to an annulus in the surround. To understand the ionic mechanisms underlying these responses, we examined the current-voltage relationship of these bipolar cells, input resistance changes during their light-evoked responses, and the reversal potentials of these responses. Off-center bipolar cells generally showed inward rectification when they were hyperpolarized and outward rectification when they were strongly depolarized. The membrane potential at which the I-V relationship deviated from linearity varied in individual cells. Hyperpolarizing center responses were generally accompanied by a resistance increase, irrespective of signal inputs either from red-sensitive cones or from rods, and the response polarities reversed at greater than +50 mV. Depolarizing surround responses were accompanied by a resistance decrease with a reversal potential at about +28 mV (one case). From the above observations, it is suggested that the center responses are generated by a decrease in sodium conductance (gNa) and the surround response is generated by an increase in gNa.
Ionic mechanisms underlying the responses of off-center bipolar cells in the carp retina. II. Studies on responses evoked by transretinal current stimulation.
Authors: Kaneko A , Saito T
Published: May 2004

Transretinal current pulses flowing from the receptor side to the vitreous side of the retina cause transient release of transmitter from the photoreceptor terminals, and in off-center bipolar cells they evoke transient depolarizations with a brief (less than 1 ms) synaptic delay. Since it is known that the presence of Na+ in the external medium is not essential for this type of transmitter release, we used this procedure to examine the role of [Na+]o in the generation of light-evoked responses (hyperpolarizing to spot illumination in the receptive field center and depolarizing to an annulus in the surround) of this type of bipolar cell. When the cell membrane was steadily depolarized by current injection through the recording microelectrode, the depolarizing response evoked by the transretinal current pulses decreased in amplitude and reversed its polarity at above +45 mV. Conversely, the response amplitude increased when the cell was steadily hyperpolarized. The reversal potential seems to be lowered in low [Na+]o (28 mM). Removal of Na+ from the superfusate hyperpolarized the cell and both the light-evoked and current-evoked responses disappeared. From these observations, it is hypothesized that the hyperpolarizing center response of the off-center bipolar cells is a result of removal of sustained depolarization produced by sodium permeability increase.
Calcium reduces the sodium permeability of luminal membrane vesicles from toad bladder. Studies using a fast-reaction apparatus.
Authors: Chase H S , Al-Awqati Q
Published: May 2004

Regulation of the sodium permeability of the luminal membrane is the major mechanism by which the net rate of sodium transport across tight epithelia is varied. Previous evidence has suggested that the permeability of the luminal membrane might be regulated by changes in intracellular sodium or calcium activities. To test this directly, we isolated a fraction of the plasma membrane from the toad urinary bladder, which contains a fast, amiloride-sensitive sodium flux with characteristics similar to those of the native luminal membrane. Using a flow-quench apparatus to measure the initial rate of sodium efflux from these vesicles in the millisecond time range, we have demonstrated that the isotope exchange permeability of these vesicles is very sensitive to calcium. Calcium reduces the sodium permeability, and the half-maximal inhibitory concentration is 0.5 microM, well within the range of calcium activity found in cells. Also, the permeability of the luminal membrane vesicles is little affected by the ambient sodium concentration. These results, when taken together with studies on whole tissue, suggest that cell calcium may be an important regulator of transepithelial sodium transport by its effect on luminal sodium permeability. The effect of cell sodium on permeability may be mediated by calcium rather than by sodium itself.
Sodium flux ratio through the amiloride-sensitive entry pathway in frog skin.
Authors: Benos D J , Hyde B A , Latorre R
Published: May 2004

The sodium flux ratio of the amiloride-sensitive Na+ channel in the apical membrane of in vitro Rana catesbeiana skin has been evaluated at different sodium concentrations and membrane potentials in sulfate Ringer solution. Amiloride-sensitive unidirectional influxes and effluxes were determined as the difference between bidirectional 22Na and 24Na fluxes simultaneously measured in the absence and presence of 10(-4) M amiloride in the external bathing solution. Amiloride-sensitive Na+ effluxes were induced by incorporation of cation-selective ionophores (amphotericin B or nystatin) into the normally Na+-impermeable basolateral membrane. Apical membrane potentials (Va) were measured with intracellular microelectrodes. We conclude that since the flux ratio exponent, n', is very close to 1, sodium movement through this channel can be explained by a free-diffusion model in which ions move independently. This result, however, does not necessarily preclude the possibility that this transport channel may contain one or more ion binding sites.
Temperature dependence of ion permeation at the endplate channel.
Authors: Hoffmann H M , Dionne V E
Published: May 2004

The dependence of acetylcholine receptor mean single-channel conductance on temperature was studied at garter snake twitch-muscle endplates using fluctuation analysis. In normal saline under conditions where most of the endplate current was carried by Na+, the channel conductance increased continuously from near 0 degrees C to approximately 23 degrees C with a Q10 of 1.97 +/- 0.14 (mean +/- SD). When 50% of the bath Na+ was replaced by either Li+, Rb+, or Cs+, the Q10 did not change significantly; however, at any temperature the channel conductance was greatest in Cs-saline and decreased with the ion sequence Cs greater than Rb greater than Na greater than Li. The results were fit by an Eyring-type model consisting of one free-energy well on the extracellular side of a single energy barrier. Ion selectivity appeared to result from ion-specific differences in the well and not in the barrier of this model. With a constant barrier enthalpy for different ions, well free-energy depth was greatest for Cs+ and graded identical to the permeability sequence. The correlation between increased well depth (i.e., ion binding) and increased channel conductance can be accounted for by the Boltzmann distribution of thermal energy.
Cyclic AMP regulation of myosin isozymes in mammalian cardiac muscle.
Authors: Winegrad S , McClellan G , Tucker M , Lin L E
Published: May 2004

Hyperpermeable cells from rat heart contain a cAMP-dependent system that can increase the maximum Ca-activated force (contractility) of the contractile proteins. In two different conditions where the relative concentration of the myosin isozymes changes, i.e., hypothyroidism and aging, the size of the increase in contractility from activation of the cAMP-regulated system varies closely with the relative concentration of V1, the isozyme of myosin with the greatest Ca- and actin-activated ATPase activity. The existence of another system for the regulation of the slow isozyme V3 has been demonstrated, and it may be inhibited by beta-adrenergic activity. The possibility of cAMP-dependent myosin regulation of contraction in addition to Ca regulation of troponin is considered. Phosphorylation of the contractile proteins themselves is not required for the increased contractility.
Birefringence signals in mammalian and frog myocardium. E-C coupling implications.
Authors: Weiss R E , Morad M
Published: May 2004

Birefringence signals from mammalian and frog hearts were studied. The period between excitation and the onset of contraction in which optical signals were free of movement artifact was determined by changes in scattered incandescent light and changes in laser diffraction patterns. The birefringence signal preceding contraction was found to behave as a change in retardation and was not contaminated measurably by linear dichroic or isotropic absorption changes. There were two components of the birefringence signal in mammalian heart muscles but only one component in the frog heart. The first component of the birefringence signals in both mammalian and frog hearts had a time course coincident with the action potential upstroke. The second component in mammalian preparations was sensitive to inotropic interventions, such as variation of extracellular Ca2+, stimulation frequency, temperature, and epinephrine, in a manner that correlated with the maximum rate of rise of tension. Caffeine (2-10 mM) not only failed to generate a second component in the frog heart, but also suppressed the second component in the mammalian heart while potentiating twitch tension. The results suggest that the second component of the birefringence signal in the mammalian myocardium is related to Ca2+ release from the sarcoplasmic reticulum.
Depolarizing response of rat parathyroid cells to divalent cations.
Authors: López-Barneo J , Armstrong C M
Published: May 2004

Membrane potentials were recorded from rat parathyroid glands continuously perfused in vitro. At 1.5 mM external Ca++, the resting potential averages -73 +/- 5 mV (mean +/- SD, n = 66). On exposure to 2.5 mM Ca++, the cells depolarize reversibly to a potential of -34 +/- 8 mV (mean +/- SD). Depolarization to this value is complete in approximately 2-4 min, and repolarization on return to 1.5 mM Ca++ takes about the same time. The depolarizing action of high Ca++ is mimicked by all divalent cations tested, with the following order of effectiveness: Ca++ greater than Sr++ greater than Mg++ greater than Ba++ for alkali-earth metals, and Ca++ greater than Cd++ greater than Mn++ greater than Co++ greater than Zn++ for transition metals. Input resistance in 1.5 mM Ca++ was 24.35 +/- 14 M omega (mean +/- SD) and increased by an average factor of 2.43 +/- 0.8 after switching to 2.5 mM Ca++. The low value of input resistance suggests that cells are coupled by low-resistance junctions. The resting potential in low Ca++ is quite insensitive to removal of external Na+ or Cl-, but very sensitive to changes in external K+. Cells depolarize by 61 mV for a 10-fold increase in external K+. In high Ca++, membrane potential is less sensitive to an increase in external K+ and is unchanged by increasing K+ from 5 to 25 mM. Depolarization evoked by high Ca++ may be slowed, but is unchanged in amplitude by removal of external Na+ or Cl-. Organic (D600) and inorganic (Co++, Cd++, and Mn++) blockers of the Ca++ channels do not interfere with the electrical response to Ca++ changes. Our results show remarkable parallels to previous observations on the control of parathormone (PTH) release by Ca++. They suggest an association between membrane voltage and secretion that is very unusual: parathyroid cells secrete when fully polarized, and secrete less when depolarized. The extraordinary sensitivity of parathyroid cells to divalent cations leads us to hypothesize the existence in their membranes of a divalent cation receptor that controls membrane permeability (possibly to K+) and PTH secretion.
Ryanodine modification of cardiac muscle responses to potassium-free solutions. Evidence for inhibition of sarcoplasmic reticulum calcium release.
Authors: Sutko J L , Kenyon J L
Published: May 2004

To test whether ryanodine blocks the release of calcium from the sarcoplasmic reticulum in cardiac muscle, we examined its effects on the aftercontractions and transient depolarizations or transient inward currents developed by guinea pig papillary muscles and voltage-clamped calf cardiac Purkinje fibers in potassium-free solutions. Ryanodine (0.1-1.0 microM) abolished or prevented aftercontractions and transient depolarizations by the papillary muscles without affecting any of the other sequelae of potassium removal. In the presence of 4.7 mM potassium and at a stimulation rate of 1 Hz, ryanodine had only a small variable effect on papillary muscle force development and action potential characteristics. In calf Purkinje fibers, ryanodine (1 nM-1 microM) completely blocked the aftercontractions and transient inward currents without altering the steady state current-voltage relationship. Ryanodine also abolished the twitch in potassium-free solutions, but it enhanced the tonic force during depolarizing voltage-clamp steps. This latter effect was dependent on the combination of ryanodine and potassium-free solutions. The slow inward current was not blocked by 1 microM ryanodine, but ryanodine did appear to abolish an outward current that remained in the presence of 0.5 mM 4-aminopyridine. Our observations are consistent with the hypothesis that ryanodine, by inhibiting the release of calcium from the sarcoplasmic reticulum, prevents the oscillations in intracellular calcium that activate the transient inward currents and aftercontractions associated with calcium overload states.
Spatial and temporal properties of luminosity horizontal cells in the turtle retina.
Authors: Tranchina D , Gordon J , Shapley R
Published: May 2004

Luminosity horizontal cells in the turtle retina respond approximately linearly to visual stimuli with contrast levels spanning a large part of the physiological range. We characterized the response properties of these cells under conditions of low photopic background illumination by measuring their spatial and temporal frequency transfer functions. Our experimental results indicate in two ways that, under these conditions, feedback from luminosity horizontal cells to cones does not play a major role in the mechanisms underlying the spatial and temporal tuning of horizontal cell responses. First, the shape of the spatial transfer function depended only weakly on the temporal frequency with which it was measured. Second, the shape of the temporal transfer function depended only weakly on the spatial frequency with which it was measured.
Hydrogen ion block of the sodium pore in squid giant axons.
Authors: Begenisich T , Danko M
Published: May 2004

The block of squid axon sodium channels by H ions was studied using voltage-clamp and internal perfusion techniques. An increase in the concentration of internal permeant ions decreased the block produced by external H ions. The voltage dependence of the block was found to be nonmonotonic: it was reduced by both large positive and large negative potentials. The ability of internal ions to modify the block by external H+ is explained by a competition among these ions for a binding site within the pore. The nonmonotonic voltage dependence is consistent with this picture if the hydrogen ions are allowed to be permeant.
Rheogenic transport in the renal proximal tubule.
Authors: Sackin H , Boulpaep E L
Published: May 2004

The electrophysiology of the renal Na-K ATPase was studied in isolated perfused amphibian proximal tubules during alterations in bath (serosal) potassium. Intracellular and extracellular ionic activity measurements permitted continuous evaluation of the Nernst potentials for Na+, K+, and Cl- across the basolateral membrane. The cell membrane and transepithelial potential differences and resistances were also determined. Return of K to the basal (serosal) solution after a 20-min incubation in K-free solution hyperpolarized the basolateral membrane to an electrical potential that was more negative than the Nernst potential for either Na, Cl, or K. This constitutes strong evidence that at least under stimulated conditions the Na-K ATPase located at the basolateral membrane of the renal proximal tubule mediates a rheogenic process which directly transfers net charge across the cell membrane. Interpretation of these data in terms of an electrical equivalent circuit permitted calculation of both the rheogenic current and the Na/K coupling ratio of the basolateral pump. During the period between 1 and 3 min after pump reactivation by return of bath K, the basolateral rheogenic current was directly proportional to the intracellular Na activity, and the pump stoichiometry transiently exceeded the coupling ratio of 3Na to 2K reported in other preparations.
Coupling ratio of the Na-K pump in the lobster cardiac ganglion.
Authors: Livengood D R
Published: May 2004

The electrogenic Na-K pump coupling ratio in the large neurons of the lobster cardiac ganglion was determined by two different electrophysiological techniques. A graphical analysis plotting exp(EmF/RT) vs. [K]o after the pump was blocked by ouabain was used to determine values for [K]i, PNa/PK, and the pump coupling ratio. These measurements were made 4-8 h after the cells were penetrated with microelectrodes, and thus represent non-Na-loaded steady state values. The value obtained for the pump coupling ratio under these conditions was 1.44 +/- 0.06 (n = 9) or close to 3 Na for 2 K. The second technique used to measure the coupling ratio was to iontophoretically inject Na ions into the neuron. Neurons were penetrated with three microelectrodes, two of which were filled with 2 M Na-citrate; the third electrode contained either 2 M K-citrate or 3 M KCl. By passing current between the Na salt-containing electrodes, Na was injected into the cell soma. The injection system was calibrated by injecting 24Na-citrate into counting vials from representative microelectrodes (calculated 24Na transport = 0.92). By knowing the Na load injected into the cells, and by measuring the time-current area produced by the Na activation of the Na-K pump, the coupling ratio was calculated to be 1.54 +/- 0.05 (n = 19), which is not significantly different from the value obtained by the first method. This value represents a Na-loaded experimental situation. When Na was removed from the external bathing solution, the coupling ratio shifted to 2 Na to 1 K (2.0 +/- 0.07, n = 4). These results suggest that the pump normally operates with a 3:2 ratio both in steady state and under Na load but that in the absence of external Na, it can operate with less than a full complement (2) of K on the external surface of the pump.
Ca-induced K transport in human red blood cell ghosts containing arsenazo III. Transmembrane interactions of Na, K, and Ca and the relationship to the functioning Na-K pump.
Authors: Yingst D R , Hoffman J F
Published: May 2004

Increasing free intracellular Ca (Cai) from less than 0.1 microM to 10 microM by means of A23187 activated Ca-stimulated K transport and inhibited the Na-K pump in resealed human red cell ghosts. These ghosts contained 2 mM ATP, which was maintained by a regenerating system, and arsenazo III to measure Cai. Ca-stimulated K transport was activated 50% at 2-3 microM free Cai and the Na-K pump was inhibited 50% by 5-10 microM free Cai. Free Cai from 1 to 8 microM stimulated K efflux before it inhibited the Na-K pump, dissociating the effect of Ca on the two systems. 3 microM trifluoperazine inhibited Ca-stimulated K efflux and 0.5 mM quinidine reduced Na-K pumping by 50%. In other studies, incubating fresh intact cells in solutions containing Ca and 0.5 microM A23187 caused the cells to lose K heterogeneously. Under the same conditions, increasing A23187 to 10 microM initiated a homogeneous loss of K. In ATP-deficient ghosts containing Cai equilibrated with A23187, K transport was activated at the same free Cai as in the ghosts containing 2 mM ATP. Neither Cao nor the presence of an inward Ca gradient altered the effect of free Cai on the permeability to K. In these ghosts, transmembrane interactions of Na and K influenced the rate of Ca-stimulated K efflux independent of Na- and K-induced changes in free Cai or sensitivity to Cai. At constant free Cai, increasing Ko from 0.1 to 3 mM stimulated K efflux, whereas further increasing Ko inhibited it. Increasing Nai at constant Ki and free Cai markedly decreased the rate of efflux at 2 mM Ko, but had no effect when Ko was greater than or equal to 20 mM. These transmembrane interactions indicate that the mechanism underlying Ca-stimulated K transport is mediated. Since these interactions from either side of the membrane are independent of free Cai, activation of the transport mechanism by Cai must be at a site that is independent of those responsible for the interaction of Na and K. In the presence of A23187, this activating site is half-maximally stimulated by approximately 2 microM free Ca and is not influenced by the concentration of ATP. The partial inhibition of Ca-stimulated K efflux by trifluoperazine in ghosts containing ATP suggests that calmodulin could be involved in the activation of K transport by Cai.(ABSTRACT TRUNCATED AT 400 WORDS)
Na+-H+ exchange and Na+ entry across the apical membrane of Necturus gallbladder.
Authors: Weinman S A , Reuss L
Published: May 2004

The role of Na+-H+ exchange in Na+ transport across the apical membrane was evaluated in Necturus gallbladder epithelium by means of intracellular Na+ activity (aNai) and 22Na+ uptake measurements. Under control conditions, complete replacement of Na+ in the mucosal solution with tetramethylammonium reduced aNai from 14.0 to 6.9 mM in 2 min (P less than 0.001). Mucosal addition of the Na+-H+ exchange inhibitor amiloride (10(-3) M) reduced aNai from 15.0 to 13.3 mM (P less than 0.001), whereas bumetanide (10(-5) and 10(-4) M) had no effect. Na+ influx across the apical membrane was studied by treating the tissues with ouabain, bathing them in Na-free solutions, and suddenly replacing the mucosal solution with an Na-containing solution. When the mucosal solution was replaced with Na-Ringer's, aNai increased at approximately 11 mM/min. This increase was inhibited by 54% by amiloride (10(-3) M, P less than 0.001) and was unaffected by bumetanide (10(-5) M). Amiloride-inhibitable Na+ fluxes across the apical membrane were also induced by the imposition of pH gradients. Na+ influx was also examined in tissues that had not been treated with ouabain. Under control conditions, 22Na+ influx from the mucosal solution into the epithelium was linear over the first 60 s and was inhibited by 40% by amiloride (10(-3) M, P less than 0.001) and by 19% by bumetanide (10(-5) M, P less than 0.025). We conclude that Na+-H+ exchange is a major pathway for Na+ entry in Necturus gallbladder, which accounts for at least half of apical Na+ influx both under transporting conditions and during exposure to ouabain. Bumetanide-inhibitable Na+ entry mechanisms may account for only a smaller fraction of Na+ influx under transporting conditions, and cannot explain influx in ouabain-treated tissues. These results support the hypothesis that NaCl entry results primarily from the operation of parallel Na+-H+ and Cl--HCO-3 exchangers, and not from a bumetanide-inhibitable NaCl cotransporter.
Properties of an endogenous steady current in rat muscle.
Authors: Caldwell J H , Betz W J
Published: May 2004

A vibrating probe was used to study a steady electric current generated by isolated, whole lumbrical muscles of the rat. Spatial mapping showed that current leaves the muscle in the synaptic region and re-enters in the flanking extrajunctional regions. The point of maximum outward current coincided precisely with the endplate region. As the probe was moved radially away from the endplate region, the current declined monotonically, and the results could be fit with a simple model. As the probe was moved axially away from the endplate region, the current declined and became inward over a distance of approximately 0.5 mm. The physiological mechanism by which the current is generated was also studied. alpha-Bungarotoxin and tetrodotoxin had no significant effect on the current, which suggests that acetylcholine channels and gated sodium channels are not involved in the generation of the current. Ouabain produced a slowly developing, partial inhibition of the current, reducing it by approximately 40% over a period of 30-40 min. Carbachol produced a large inward current at the endplate region. After the carbachol action was terminated with alpha-bungarotoxin, an outward current reappeared, and a transient "overshoot" developed. During the overshoot, which lasted approximately 30-40 min, the outward current was approximately doubled. This overshoot was completely abolished by ouabain. The overshoot is interpreted as reflecting the increased activity of electrogenic sodium pumping in the endplate region, caused by the influx of Na ions during carbachol application. Because of the very different actions of ouabain on the normal current and on the overshoot after carbachol application, we concluded that the normal outward current is not produced by electrogenic sodium pumping in the endplate region.
Changes in apical [K+] produce delayed basal membrane responses of the retinal pigment epithelium in the gecko.
Authors: Griff E R , Steinberg R H
Published: May 2004

We describe here a new retinal pigment epithelium (RPE) response, a delayed hyperpolarization of the RPE basal membrane, which is initiated by the light-evoked decrease of [K+]o in the subretinal space. This occurs in addition to an apical hyperpolarization previously described in cat (Steinberg et al., 1970; Schmidt and Steinberg, 1971) and in bullfrog (Oakley et al., 1977; Oakley, 1977). Intracellular and extracellular potentials and measurements of subretinal [K+]o were recorded from an in vitro preparation of neural retina-RPE-choroid from the lizard Gekko gekko in response to light. Extracellularly, the potential across the RPE, the transepithelial potential (TEP), first increased and then decreased during illumination. Whereas the light-evoked decrease in [K+]o predicted the increase in TEP, the subsequent decrease in TEP was greater than predicted by the reaccumulation of [K+]o. Intracellular RPE recordings showed that a delayed hyperpolarization generated at the RPE basal membrane produced the extra TEP decrease. At light offset, the opposite sequence of membrane potential changes occurred. RPE responses to changes in [K+]o were studied directly in the isolated gecko RPE-choroid. Decreasing [K+]o in the apical bathing solution produced first a hyperpolarization of the apical membrane, followed by a delayed hyperpolarization of the basal membrane, a sequence of membrane potential changes identical to those evoked by light. Increasing [K+]o produced the opposite sequence of membrane potential changes. In both preparations, the delayed basal membrane potentials were accompanied by changes in basal membrane conductance. The mechanism by which a change in extracellular [K+] outside the apical membrane leads to a polarization of the basal membrane remains to be determined.
A parametric study of the stimulation variables affecting the magnitude of the olfactory nerve response.
Authors: Mozell M M , Sheehe P R , Swieck S W , Kurtz D B , Hornung D E
Published: May 2004

The magnitude of olfactory responses can be related to three primary variables [number of odorant molecules (N), sniff volume (V), and sniff duration (T)] and three derived variables [concentration (C = N/V), flow rate (F = V/T), and delivery rate (D = N/T)]. To evaluate the effects of these interdependent variables upon the olfactory response, the summated multiunit discharges were recorded from the olfactory nerves of nine frogs in response to octane presented at two levels (in 2:1 ratio) of each primary variable. This presentation defined eight "sniff" combinations representing three levels of each derived variable. In an ANOVA of the logs of the responses, the effect of each primary variable was highly significant, with no significant interactions. A multiplicative regression model incorporating the effects of the three primary variables represented responses exceedingly well, with positive effects of N and T and a negative effect of V. When, with this model, the effect of each of the derived variables was isolated from the effects of all other variables, the analysis showed a positive effect for C, a near-zero positive effect for D, and a negative effect for F. Placing certain constraints upon the model parameters generates 13 distinct one- and two-variable models (e.g., the [C, T] model requires N and V to have equal but opposite effects). In ranking these reduced models in terms of their ability to predict the neural response, the predictive ability of [F, N] and [C, T] was at least as good as that of the three-variable model.
Intracellular injection of guanyl nucleotides alters the serotonin-induced increase in potassium conductance in Aplysia neuron R15.
Authors: Lemos J R , Levitan I B
Published: May 2004

The effects of the adenylate cyclase inhibitor GDP beta S on the response of Aplysia neuron R15 to serotonin (5HT) were investigated. Previous studies have demonstrated that 5HT causes an increase in K+ conductance in R15 and that the response is mediated by cAMP. At concentrations in the micromolar range, GDP beta S inhibits the stimulation of adenylate cyclase by 5HT in particulate fractions from Aplysia ganglia. When micromolar concentrations of GDP beta S are injected into neuron R15, there is no effect on the resting membrane conductance, but the increase in K+ conductance normally elicited by 5HT is completely inhibited. Furthermore, the decrease in inward current normally elicited by dopamine (DA), which does not appear to involve cAMP, is not affected by micromolar concentrations of GDP beta S. In addition, application of 8-benzylthio cAMP to R15 can evoke an increase in K+ conductance even after the injection of GDP beta S, which indicates that events subsequent to the activation of adenylate cyclase are not inhibited by the GDP analogue. In contrast, when millimolar concentrations of GDP beta S are injected into R15, direct effects on membrane conductance are observed and the response of R15 to 5HT is enhanced. Although these effects of high concentrations of GDP beta S are only poorly understood, the results with micromolar concentrations are consistent with the hypothesis that stimulation of adenylate cyclase is necessary for the 5HT-induced increase in K+ conductance in neuron R15.
The relationship among intracellular sodium activity, calcium, and strophanthidin inotropy in canine cardiac Purkinje fibers.
Authors: Vassalle M , Lee C O
Published: May 2004

The role of sodium and calcium ions in strophanthidin inotropy was studied by measuring simultaneously the electrical, mechanical, and intracellular sodium ion activities in electrically driven cardiac Purkinje fibers under conditions that change the intracellular sodium or calcium level (tetrodotoxin, strophanthidin, high calcium, and norepinephrine). Tetrodotoxin (TTX; 1-5 X 10(-6)M) shifted the action potential plateau to more negative values, shortened the action potential duration, and decreased the contractile tension and the intracellular sodium ion activity (aiNa). The changes in tension and in aiNa caused by TTX appear to be related since they had similar time courses. Strophanthidin (2-5 X 10(-7)M) increased tension and aiNa less in the presence of TTX, and, for any given value of aiNa, tension was less than in the absence of TTX. Increasing extracellular calcium (from 1.8 to 3.3-3.6 mM) or adding norepinephrine (0.5-1 X 10(-6)M) increased tension and decreased aiNa less in the presence than in the absence of TTX. When two of the above procedures were combined, the results were different. Thus, during the increase in aiNa and tension caused by strophanthidin in the presence of TTX, increasing calcium or adding norepinephrine increased tension markedly but did not increase aiNa further. In a TTX-high calcium or TTX-norepinephrine solution, adding strophanthidin increased both tension and aiNa, and the increase in tension was far greater than in the presence of TTX alone. The results indicate that: (a) the contractile force in Purkinje fibers is affected by a change in aiNa; (b) a decrease in aiNa by TTX markedly reduces the inotropic effect of strophanthidin, possibly as a consequence of depletion of intracellular calcium; (c) increasing calcium influx with norepinephrine or high calcium in the TTX-strophanthidin solution produces a potentiation of tension development, even if aiNa does not increase further; and (d) when the calcium influx is already increased by high calcium or norepinephrine, strophanthidin has its usual inotropic effect even in the presence of TTX. In conclusion, the positive inotropic effect of strophanthidin requires that an increase in aiNa be associated with suitable calcium availability.
Excitation-contraction coupling in cardiac Purkinje fibers. Effects of caffeine on the intracellular [Ca2+] transient, membrane currents, and contraction.
Authors: Hess P , Wier W G
Published: May 2004

The effects of caffeine on tension, membrane potential, membrane currents, and intracellular [Ca2+], measured as the light emitted by the Ca2+-activated photoprotein aequorin, were studied in canine cardiac Purkinje fibers. An initial, transient, positive inotropic effect of caffeine was accompanied by a transient increase in the second component of the aequorin signal (L2) but not the first (L1). In the steady state, 4 or 10 mM caffeine always decreased twitch tension and greatly reduced both L1 and L2. At a concentration of 2 mM, caffeine usually reduced but occasionally increased the steady state twitch tension. However, 2 mM caffeine always reduced both L1 and L2. Caffeine eliminated the diastolic oscillations of intracellular [Ca2+] induced by high extracellular [Ca2+]. In voltage-clamp experiments, 10 mM caffeine reduced the transient outward current and the peak tension elicited by step depolarization from a holding potential of -45 mV. In the presence of 20 mM Cs+, 10 mM caffeine reduced slow inward current. However, the time course of this reduction was far slower than that in tension and light observed in separate experiments. The simplest explanation of the results is that caffeine inhibits the sequestration of Ca2+ by the sarcoplasmic reticulum. The results also suggest that in Purkinje fibers caffeine increases the sensitivity of the myofilaments to Ca2+.
The time course of miniature endplate currents and its modification by receptor blockade and ethanol.
Authors: Linder T M , Pennefather P , Quastel D M
Published: May 2004

Miniature endplate currents (MEPCs) recorded from mouse diaphragms with a point voltage clamp, without inhibition of acetylcholinesterase (AChE) and in the absence of any drug, showed in their decay phase consistent deviations from an exponential time course, consisting of (a) "curvature," a progressive increase of decay rate during most of the decay phase, followed by (b) "late" tails. Both phenomena persisted when MEPCs (and channel lifetime) were prolonged by ethanol. Curvature was increased by muscle fiber depolarization and decreased by hyperpolarization. Receptor blockade by (+)-tubocurarine, alpha-bungarotoxin, hexamethonium, or myasthenic IgG accelerated the decay of the main part of MEPCs and eliminated curvature; the time constant of MEPCs became close to the channel time constant. We conclude that curvature arises from repeated action of ACh with cooperativity in ACh-receptor interaction; the voltage sensitivity of curvature follows from the voltage sensitivity of channel closing. Ethanol, in addition to its effect to prolong channel lifetime, enhances the tendency of ACh to act more than once to open channels before being lost to the system. Analysis of the rising phase of the MEPC, in terms of driving functions, also indicated that ethanol promotes channel opening by ACh; this action can account for a substantial increase of MEPC height by ethanol when MEPCs are made small by receptor blockade. Driving functions were also voltage sensitive, in a manner indicating acceleration of channel opening, but reduction of channel conductance, with hyperpolarization. Poisoning or inhibition of AChE prolonged MEPCs without altering the duration of ionic channels. Since ethanol caused further prolongation of MEPCs after poisoning of AChE, with little change in MEPC height, we conclude that the extension of mean channel lifetime by ethanol is accompanied by a similar extension of ACh binding to receptors. After poisoning of AChE, MEPCs became very variable in time course and the decay rate (tau-1) was correlated with MEPC height with a slope of log tau vs. log height of 0.77 for MEPCs of greater than 60% mean size. This slope is larger than expected from cooperativity in ACh-receptor interaction. Correlation of tau and height of MEPCs also exists when AChE is intact; the slope of log tau vs. log height was 0.12 with or without prolongation of MEPCs by ethanol.
Acetylcholine receptor in planar lipid bilayers. Characterization of the channel properties of the purified nicotinic acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayers.
Authors: Labarca P , Lindstrom J , Montal M
Published: May 2004

The properties of the channel of the purified acetylcholine receptor (AChR) were investigated after reconstitution in planar lipid bilayers. The time course of the agonist-induced conductance exhibits a transient peak that relaxes to a steady state value. The macroscopic steady state membrane conductance increases with agonist concentration, reaching saturation at 10(-5) M for carbamylcholine (CCh). The agonist-induced membrane conductance was inhibited by d-tubocurarine (50% inhibition, IC50, at approximately 10(-6) M) and hexamethonium (IC50 approximately 10(-5) M). The single channel conductance, gamma, is ohmic and independent of the agonist. At 0.3 M monovalent salt concentrations, gamma = 28 pS for Na+, 30 pS for Rb+, 38 pS for Cs+, and 50 pS for NH+4. The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of two distinct open states. tau o1 and tau o2, the fast and slow components of the distribution of open times, are independent of the agonist concentration: for CCh this was verified in the range of 10(-6) M less than C less than 10(-3)M. tau 01 and tau o2 are approximately three times longer for suberyldicholine ( SubCh ) than for CCh. tau o1 and tau o2 are moderately voltage dependent, increasing as the applied voltage in the compartment containing agonist is made more positive with respect to the other. At desensitizing concentrations of agonist, the AChR channel openings occurred in a characteristic pattern of sudden paroxysms of channel activity followed by quiescent periods. A local anesthetic derivative of lidocaine ( QX -222) reduced both tau o1 and tau o2. This effect was dependent on both the concentration of QX -222 and the applied voltage. Thus, the AChR purified from Torpedo electric organ and reconstituted in planar lipid bilayers exhibits ion conduction and kinetic and pharmacological properties similar to AChR in intact muscle postsynaptic membranes.
Ionic events during the volume response of human peripheral blood lymphocytes to hypotonic media. I. Distinctions between volume-activated Cl- and K+ conductance pathways.
Authors: Sarkadi B , Mack E , Rothstein A
Published: May 2004

Human peripheral blood lymphocytes (PBL), when placed into hypotonic media, first swell and then shrink back to their original volumes because of a rapid KCl leakage via volume-activated K+ and anion permeation pathways. By using gramicidin, a cation channel-forming ionophore, cation transport through the cell membrane can be shunted so that the salt fluxes and thus the volume changes are limited by the rate of the net anion movements. The "gramicidin method," supplemented with direct measurements of volume-induced ion fluxes, can be used to assess the effects of drugs and of various treatments on cation and anion permeabilities. It is demonstrated that quinine and cetiedil are much more effective blockers of volume-induced K+ transport than of Cl- transport, while dipyridamole, DIDS, and NIP-taurine inhibit only volume-induced Cl- movement. Oligomycins block both cation and anion transport pathways, oligomycin A being more effective in inhibiting K+ transport and oligomycin C preferentially blocking Cl- movement. Ca depletion of PBL abolishes volume-induced K+ transport but has no effect on Cl- transport. Repletion of cell calcium by ionophore A23187 immediately restores rapid K+ transport without significantly affecting volume-induced Cl- transport. These observations, taken together with other reported information, can be best explained by a model in which cell swelling activates independent Cl- and K+ conductance pathways, the latter being similar in properties to the Ca2+-activated K+ transport observed in various cell membranes.
Ionic events during the volume response of human peripheral blood lymphocytes to hypotonic media. II. Volume- and time-dependent activation and inactivation of ion transport pathways.
Authors: Sarkadi B , Mack E , Rothstein A
Published: May 2004

Hypotonic dilution of human peripheral blood lymphocytes (PBL) induces large conductive permeabilities for K+ and Cl-, associated with the capacity of the cells to regulate their volumes. When rapid cation leakage is assured by the addition of the ionophore gramicidin, the behavior of the anion conductance pathway can be independently examined. Using this technique it is demonstrated that the volume-induced activation of Cl- transport is triggered at a threshold of approximately 1.15 X isotonic cell volume. If the volume of a cell is increased to this level or above, the Cl- transport system is activated, whereas if the volume of a swollen cell is decreased below the threshold value, the Cl- transport is inactivated. Activation and inactivation are independent of the relative volume changes and of the actual cellular Na+, K+, or Cl- concentrations, as well as of the changes in membrane potential in PBL. When net salt movement and thus volume change are inhibited by specific blockers of K+ transport (e.g., quinine, or Ca2+ depletion), volume-induced Cl- conductance shows a time-dependent inactivation, with a half-time of 5-8 min. The Cl- conductance, when activated, appears to involve an all-or-none response. In contrast, volume-induced K+ conductance is a graded response, with the increase in K+ flux being roughly proportional to the hypotonicity-induced increase in cell volume. The data indicate that during lymphocyte volume response in hypotonic media, anion conductance increases by orders of magnitude, exceeding the K+ conductance, so that the rate of the volume decrease (KCl efflux) is determined by a graded alteration in K+ conductance. When the cell volume approaches the isotonic value, it is stabilized by the inactivation of the anion conductance pathway.
The water permeability of toad urinary bladder. II. The value of Pf/Pd(w) for the antidiuretic hormone-induced water permeation pathway.
Authors: Levine S D , Jacoby M , Finkelstein A
Published: May 2004

Using the methods described in the preceding paper (Levine et al., 1984) for measuring the magnitude of the water-permeable barriers in series with the luminal membrane, we correct measured values of Pd(w) in bladders stimulated with low doses of antidiuretic hormone (ADH) or 8-bromo cyclic AMP to obtain their true values in the luminal membrane. Simultaneously, we also determine Pf. We thus are able to calculate Pf/Pd(w) for the hormone-induced water permeation pathway in the luminal membrane. Our finding is that Pf/Pd(w) approximately equal to 17. Two channel models consistent both with this value and the impermeability of the ADH-induced water permeation pathway to small nonelectrolytes are: (a) a long (approximately equal to 50 A), small-radius (approximately equal to 2 A) pore through which 17 water molecules pass in single-file array, and (b) a shower-head-like structure in which the stem is long and of large radius (approximately equal to 20 A) and the cap has numerous short, small-radius (approximately equal to 2 A) pores. A third possibility is that whereas the selective permeability to H2O results from small-radius (approximately equal to 2 A) pores, the large value of Pf/Pd(w) arises from their location in the walls of long tubular vesicles (approximately 2 micron in length and 0.1 micron in diameter) that are functionally part of the luminal membrane after having fused with it. Aggregate-containing tubular vesicles of these dimensions have been reported to fuse with the luminal membrane in response to ADH stimulation and have been implicated in the ADH-induced hydroosmotic response.
Phosphorylation of C-protein in intact amphibian cardiac muscle. Correlation between 32P incorporation and twitch relaxation.
Authors: Hartzell H C
Published: May 2004

The molecular mechanisms by which neurotransmitters modulate the force of contraction of cardiac muscle are incompletely understood. Hartzell and Titus (1982. J. Biol. Chem. 257:2111-2120) have recently reported that C-protein, an integral component of the thick filament, is reversibly phosphorylated in response to ionotropic agents. In this communication, C-protein phosphorylation (as measured by isotopic labeling with 32P) is correlated with changes in the rate of relaxation of twitch tension. On the average, isoproterenol simultaneously increases peak systolic tension twofold, decreases twitch relaxation time from a control value of approximately 450 to approximately 300 ms, and increases C-protein phosphorylation two- to threefold, with a maximum effect occurring less than 60 s after addition of 1 microM isoproterenol. Carbamylcholine, in contrast, decreases peak systolic tension more rapidly than it affects relaxation or C-protein phosphorylation. The maximum decrease in peak tension (60%) occurs within 1 min of addition of 0.5 microM carbamylcholine, but relaxation time increases slowly to 800 ms over approximately 6 min. The increase in relaxation time correlates well with the decrease in 32P incorporation into C-protein (r = 0.94). Changing beat frequency between 0.2 and 1/s has no effect on C-protein phosphorylation but does alter relaxation time (relaxation time decreases approximately 100 ms when beat frequency is changed from 0.5 to 1/s) and thus alters the quantitative relationship between C-protein phosphorylation and relaxation rate. These results suggest that two separate processes affect relaxation. It is proposed that the level of C-protein phosphorylation sets the boundaries over which relaxation is regulated by a second process that is dependent upon beat frequency and probably involves changes in intracellular Ca.
Effects of extracellular Ca++, K+, and Na+ on cone and retinal pigment epithelium retinomotor movements in isolated teleost retinas.
Authors: Dearry A , Burnside B
Published: May 2004

We have examined the effects of changes in extracellular ionic composition on cone and retinal pigment epithelium (RPE) retinomotor movements in cultured isolated teleost retinas. In vivo, the myoid portion of teleost cones contracts in the light and elongates in the dark; RPE pigment disperses in the light and aggregates in the dark. In vitro, cones of dark-adapted (DA) retinas cultured in constant darkness contracted spontaneously to their light-adapted (LA) positions if the culture medium contained greater than or equal to 10(-3)M Cao++. DA cones retained their long DA positions in a medium containing less than or equal to 10(-6)M Cao++. Low [Ca++]o (10(-5)-10(-7)M) also permitted darkness to induce cone elongation and RPE pigment aggregation. Light produced cone contraction even in the absence of Cao++, but the extent of contraction was reduced if [Ca++]o was less than 10(-3) M. Thus, full contraction appeared to require the presence of external Ca++. High [K+]o (greater than or equal to 27 mM) inhibited both light-induced and light-independent Ca++-induced cone contraction. However, low [Na+]o (3.5 mM) in the presence of less than or equal to 10(-6)M Cao++ did not mimic light onset by inducing cone contraction in the dark. High [K+]o also promoted dark-adaptive cone and RPE movements in LA retinas cultured in the light. All results obtained in high [K+]o were similar to those observed when DA or LA retinas were exposed to treatments that elevate cytoplasmic cyclic 3',5'-adenosine monophosphate (cAMP) content.