Passive Conductance Research Articles

Endothelial cell shrinkage increases permeability through a Ca2+-dependent pathway in single frog mesenteric microvessels.

1. We tested whether calcium (Ca2+)-dependent mechanisms were essential for our previous observation that a change in the endothelial cell (EC)-extracellular matrix (ECM) attachment caused an increase in microvessel hydraulic permeability (Lp) after exposure to hypertonic solutions in single perfused mesenteric microvessels in pithed frogs (Rana pipiens). 2. In microvessels where integrin-dependent EC-ECM attachments were disrupted by pretreatment with the peptide Gly-Arg-Gly-Asp-Thr-Pro (GRGDTP; 0.3 mmol l-1), we measured microvessel Lp after exposure to hypertonic solutions under experimental conditions that reduced Ca2+ influx into endothelial cells. 3. High K+ solutions (59.7 and 100 mmol l-1 K+) were used to depolarize the endothelial membrane and therefore to reduce the electrochemical driving force for Ca2+ influx through conductive Ca2+ channels. These solutions abolished the increase in Lp caused by hypertonic solutions in the microvessels pretreated with GRGDTP. 4. We previously suggested that the removal of albumin from the perfusate may reduce EC-ECM attachment because hypertonic solutions increased the Lp of microvessels above that due to removal of albumin alone. This additional increase in Lp was attenuated by the 59.7 mmol l-1 K+ solution and was completely abolished by the 100 mmol l-1 K+ solution. 5. Bumetanide, an inhibitor of the Na+-K+-2Cl- co-transporter and one of the mechanisms of regulatory volume increase after exposure to hypertonic solutions in endothelial cells, did not change the response of microvessels to high K+ solutions. 6. Our findings indicate that Ca2+ entry into endothelial cells via passive conductance channels is necessary to increase microvessel Lp after exposure to hypertonic solutions in microvessels where EC-ECM attachments are disrupted.

Acid-base response of bacterial suspensions

The response of suspensions of non-proliferating bacteria to external pH ranging from 3.6 to 9.9 was examined. The acid-base response is affected by the number of viable cells in the suspensions and culture conditions (aerobiosis or anaerobiosis) but not by culture time, composition of the culture medium or the age of suspensions. The results with carbonylcyanide m-chlorophenylhydrazone-and N, N′- dicyclohexylcarbodiimide-treated cells indicated that passive proton conductance and proton translocation by ATP could be involved in this response. pH profiles of suspensions of 18 chemoheterotrophic Gram-negative and Gram-positive bacteria support the hypothesis that the proton gradient generated by an alkali or an acid shift could be dissipated by bacteria with respiratory metabolism.

Estimating the contributions of NMDA and non-NMDA currents to EPSPs in retinal ganglion cells.

Whole-cell recordings were obtained from retinal ganglion cells of the tiger salamander (Ambystoma tigrinum) in a superfused slice preparation to evaluate contributions of NMDA (N-methyl-D-aspartate) and KA/AMPA (kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid) receptors to excitatory postsynaptic potentials (EPSPs) of retinal ganglion cells. Synaptic activation of retinal ganglion cells was achieved through the use of a brief pressure pulse of hyperosmotic Ringer (Ringer + sucrose) delivered through a microelectrode visually placed in the inner plexiform layer while whole-cell recordings were obtained from adjacent cells in the ganglion cell layer. Separation of NMDA and KA/AMPA excitatory postsynaptic currents (EPSCs) was achieved through the application of the antagonists NBQX and D-AP7, while inhibitory currents were blocked by strychnine and picrotoxin. Simple addition of the two independent EPSCs showed, most often, that the sum of the KA/AMPA and NMDA currents was less than the control response, but in some cases the sum of the two currents exceeded the magnitude of the control response. Neither result was consistent with expectations based on voltage-clamp principles and the assumption that the two currents were independent; for this reason, we considered the possibility of nonlinear interactions between KA/AMPA and NMDA receptors. Computer simulations were carried out to evaluate the summation experiments. We used both an equivalent cylinder model and a more realistic, compartmental model of a ganglion cell constrained by a passive leakage conductance, a linear KA/AMPA synaptic current, and a nonlinear NMDA current based on the well-known, voltage-sensitive Mg2+ block. Computer simulation studies suggest that the hypo- and hyper-summation of NMDA and KA/AMPA currents, observed physiologically, can be accounted for by a failure to adequately space clamp the neuron. Clamp failure leads to enhanced NMDA currents as the ion channels are relieved of the Mg2+ block; their contribution is thus exaggerated depending on the magnitude of the conductance change and the spatial location of the synaptic input.

Agonistic effects of xanthines on tetrodotoxin-sensitive sodium channels in the rat dorsal root ganglion neurons

The effects of four xanthine derivatives, caffeine, caffeine benzoate, theophylline, and bromtheophylline, on sodium channels in internally perfused rat dorsal root ganglion neurons were studied under voltage-clamp and whole-cell patch-clamp conditions. Reversible acceleration, enhancement of the amplitude of sodium currents, and shifts of the current-voltage relation (plotted for their maxima), as well as of the steady-state inactivation curve toward more negative potentials, were observed at external applications of the above substances in the concentrations of 0.2–4.0 mM. Under long exposures, inactivation of sodium currents became slower in a part of the cells. Yet, when the exposure to 4 mM or higher concentrations was longer than 10 min, a rise in the passive conductance was obvious, and functional state of the cells became worse. Blocking effects of the xanthine derivatives on transient or delayed potassium currents were not observed. Thus, agonistic action of xanthines on sodium channels has been demonstrated, and it is supposed that a considerable component of their pharmacological effects is provided by the action on Na+/Ca2+ exchange.

Ca2+ entry through conductive pathway modulates receptor-mediated increase in microvessel permeability.

We investigated the relationship between receptor-mediated increases in cytoplasmic Ca2+ concentration ([Ca2+]i) and increased microvessel permeability. In individually perfused venular microvessels of frog mesentery exposed to 10 microM ATP, [Ca2+]i increased from 59 +/- 7 to 172 +/- 21 nM within 1 min and then fell back toward control values. The corresponding peak increase in the hydraulic conductivity (Lp) of the microvessel wall was 5.7 +/- 0.5-fold relative to control. After removal of extracellular Ca2+, there was no significant increase in Lp, and the initial increase in [Ca2+]i was attenuated but not abolished. Depolarization of the endothelial cell membrane with high-K+ Ringer solution reduced the peak increase in [Ca2+]i to 106 +/- 7 nM and attenuated the increase in Lp 1.8 +/- 0.4-fold. The results conform to the hypothesis that Ca2+ entry into endothelial cells is required for acute increase in venular microvessel permeability by inflammatory agents and that the pathway for Ca2+ entry has the properties of a passive conductance pathway. Similar conclusions were reached in previous experiments in frog microvessels exposed to Ca2+ ionophores and perfusates with no plasma proteins. In venular microvessels of hamster mesentery exposed to ATP and bradykinin, a similar pathway for Ca2+ entry was demonstrated in the present experiments. We did not measure permeability changes in hamster microvessels in this study, but these microvessels respond to histamine and ionophores with a transient increase in permeability to macromolecules similar to that measured in frog microvessels [Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H1982-H1991, 1995].

Analogue VLSI ‘integrate-and-fire’ neuron with frequency adaptation

A silicon neuron with separated ‘integrate’ and ‘fire’ representations is presented. The neuron is simple and requires a modest number of transistors, but preserves a number of temporal characteristics of physiological neurons: temporal integration, passive conductance, after-hyperpolarisation, absolute refractory period, and frequency adaptation.

A model study on the influence of a slowly activating potassium conductance on repetitive firing patterns of muscle spindle primary endings

The general mathematical model of Frankenhaeuser and Huxley, which describes the generation of action potentials in myelinated nerve fibres, has been used as a kernel for a model of a sensory nerve ending. Two types of modifications were implemented. First, the four original permeability constants (those of potassium, sodium, non-specific and leak) were changed simultaneously (using an automated tuning algorithm), in order to introduce low-frequency repetitive firing capability (down to 15 Hz), keeping the deviations from the original values as small as possible. Second, a slow potassium conductance was added, in order to model slow processes (like accommodation) with time constants longer than those required to simulate short-lasting action potentials. Sensory stimuli were simulated as changes in passive conductance. The model displayed the following properties, which are typical of many sensory endings in general and of muscle spindle primary endings in particular: (i) The range of sustained repetitive firing was extended into the domain of low discharge rates, so as to span the entire physiological range (from 2 to 700 sec −1). (ii) The relation between firing rate and receptor potential was roughly linear over the full range of firing. (iii) Following a step increase of stimulus, the firing rate showed adaptation with a time constant of about 70 msec. (iv) Sudden reduction of the stimulus was followed by post-release silence. (v) Following a step increase of stimulus, the receptor potential showed a short dynamic peak. (vi) Following a step decrease of stimulus, the receptor potential displayed a post-release undershoot and recovery with a time constant of approximately 100 msec. (vii) With sinusoidal stimuli the receptor potential showed band-pass filter properties with phase advance below and phase lag above 12 Hz and a peak in gain at about 20 Hz. Thus the present equations adequately describe a range of known properties of muscle spindle primary endings. Based on minimal modification and extension of the Frankenhaeuser-Huxley model of action potential generation in myelinated fibres, they constitute a theory of sensory encoding. This theory is further corroborated by the experimental evidence of the presence of calcium-activated potassium channels in numerous sensory—including primary spindle—endings.

Effects of Boron on Proton Transport and Membrane Properties of Sunflower (Helianthus annuus L.) Cell Microsomes.

Boron deficiency and toxicity inhibit ATP-dependent H+ pumping and vanadate-sensitive ATPase activity in sunflower roots and cell suspensions. The effects of boron on H+ pumping and on passive H+ conductance, as well as on fluorescence anisotropy in KI-washed microsomes isolated from sunflower (Helianthus annuus L. cv Enano) cell suspensions, have been investigated. Boron deficiency reduced the total and vanadate-sensitive ATPase activities as well as the vanadate-sensitive ATP-dependent H+ pumping without affecting the amount of antigenic ATPase protein as measured by immunoblotting with an Arabidopsis thaliana plasma membrane anti-H+-ATPase polyclonal antibody. Kinetic studies revealed that boron deficiency reduced Vmax of vanadate-sensitive ATPase activity with little change in the apparent Km for Mg2+-ATP. Proton leakage was greater in microsomal vesicles isolated from cells grown without boron and incubated in reaction medium without added boron, and this effect was reversed by addition of boron to the reaction medium. Fluorescence anisotropy indicated that diphenyl hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene probes were immobilized to a greater extent in microsomes from cells grown without boron than in those from cells grown with 100 [mu]M H3BO3. The apparent decrease of membrane fluidity in microsomes from cells grown without boron was reversed by the addition of boron to the reaction medium. Taken together these data suggest that inhibition of H+ gradient formation in microsomes from sunflower cells grown in the absence of boron could be due to the combined effects of reduced H+-ATPase activity and increased passive conductance across the membrane, possibly resulting from increased membrane rigidity.

Evoked changes of membrane potential in guinea pig sensory neocortical slices: an analysis with voltage-sensitive dyes and a fast optical recording method

Coronal slices from guinea pig visual neocortex were stained with voltage-sensitive fluorescent dyes RH414 or RH795. Activity was evoked by electrical stimulation of either white matter or layer I. Emitted-light intensity changes representing summated changes of membrane potential were recorded by a 10 x 10 photodiode array with a temporal resolution of 0.4 ms and a spatial resolution of 60 microns or 94 microns. Following either stimulation of layer I or of white matter, maximal activity was located close to the respective stimulation electrode, in upper layer III/II, and between layer IV and V. With stimulation of the white matter, additional peak activity was recorded from upper layer VI. Non-synaptic activity was separated from mixed (synaptic and non-synaptic) activity by comparing responses obtained in standard perfusion medium with those obtained in perfusion medium from which the calcium was omitted, such that synaptic transmission was blocked. With stimulation of the white matter, most of the evoked activity in lower cortical layers was of non-synaptic origin. This non-synaptic activity consisted of early and fast potentials, which were predominant in layer VI and probably represented presynaptic fibre activity, and of slower components that were presumably of antidromic origin. Significant postsynaptic activity was only found in upper layer III/II. In contrast, with stimulation of layer I, most of the evoked activity was of postsynaptic origin. Early and fast non-synaptic potentials consisting of presynaptic fibre activity were confined to layer I. Slower non-synaptic activity, that might reflect direct dendritic activation, was minimal and was confined to upper cortical layers. Thus, following either stimulation of layer I or of white matter, the major postsynaptic components were found in upper layer III/II. It is suggested that the postsynaptic response following stimulation of white matter resulted from di- or polysynaptic activation by afferent fibres. The postsynaptic response to stimulation of layer I was presumably a monosynaptic activation of apical dendrites from pyramidal cells by layer I horizontal fibres. Activity following stimulation of white matter spread faster than activity following stimulation of layer I. This might reflect the difference in active conduction along afferent and efferent fibres on the one hand and in passive conductance along the dendritic tree on the other hand.

Modulation of venular microvessel permeability by calcium influx into endothelial cells.

It has been proposed that calcium ion influx into endothelial cells modulates the permeability of venular microvessels via a calcium-dependent contractile process. The results of recent investigations using permeabilized endothelial cell monolayers conform to this hypothesis by demonstrating a calcium-dependent interaction of endothelial actin and myosin during the retraction of adjacent endothelial cells exposed to inflammatory agents. Little is known about the pathway for calcium influx into endothelial cells after exposure to mediators of inflammation, but evidence suggests that the properties of the calcium entry pathways are similar to the calcium entry pathways that regulate the release of endothelium-derived relaxing factor (EDRF). Substances that stimulate EDRF release from arterial endothelium also increase venular microvessel permeability. Recently developed methods to measure cytoplasmic calcium concentration in the endothelial cells forming the walls of individually perfused microvessels enable a direct investigation of the modulation of the permeability of venular microvessels by calcium influx. These experiments demonstrate that the magnitude of the initial increase in the permeability of microvessels after exposure to an agent that increases permeability, such as a calcium ionophore, is determined by the magnitude of calcium ion influx into the endothelial cells. Furthermore, the magnitude of the calcium influx into endothelial cells is modulated by the membrane potential of the endothelial cells. Depolarization of the endothelial cell membrane reduces calcium influx and attenuates increases in permeability whereas hyperpolarization of the endothelial membrane increases calcium influx and potentiates increases in permeability. These data conform to the hypothesis that a passive conductance channel for calcium is a major pathway for calcium ion flux responsible to eliciting an increase in the permeability of the endothelial barrier in microvessels.

ATP‐driven proton pumping in two species of Chara differing in salt tolerance

ABSTRACT Chara corallina is an obligate freshwater alga, while C. buckellii can be grown in salt and freshwater culture. When grown in fresh water, C. buckellii has electrophysiological properties similar to C. corallina, but when cultured in salt water, it has a less negative membrane potential and has a higher conductance. We show in internally perfused, tonoplast‐free cells that the ATP‐dependence of the two species cultured in fresh water is similar, although C. buckellii hyperpolarizes at lower ATP concentrations. We determined the pump parameters in perfused and intact cells. Using both techniques, C. corallina and C. buckellii cultured in fresh water show similar values of Ep, Gp and Ip. However, there is a significant difference between the two techniques: Ep is more negative (–400 to –700 mV) in perfused cells than in intact cells (–220 to –260mV); Gp is lower (0·1–0·2 versus 0·3–0·9 S m−2); and Ip is higher (40–60 versus 10–18 mA m−2). Salt‐cultured C. buckellii was compared with freshwater C. buckellii using intact cells; Gp and Ep were similar, but Ip was much higher in salt‐cultured cells (60 versus 15mA m−2). This higher pump rate is due to the depolarization of the membrane of salt‐cultured algae, which is caused by a higher passive conductance. The significance of the less negative membrane potential and the higher rate of proton pumping is discussed with respect to the banding pattern and salt stress.

Neurons in the rat arcuate nucleus are hyperpolarized by GABAB and mu-opioid receptor agonists: evidence for convergence at a ligand-gated potassium conductance.

Both gamma-aminobutyric acid (GABA) and the endogenous opioid peptides have pervasive effects on neuroendocrine function. This study examined the effects of selective activation of GABAB and/or mu-opioid receptors on neurons of the arcuate nucelus (ARC) of the rat hypothalamus using intracellular recording of cells in a hypothalamic slice. Some recorded neurons were filled with biocytin allowing subsequent identification and immunocytochemical evaluation for the presence of beta-endorphin. ARC neurons exhibited a broad array of active and passive conductances. Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGOL), a mu-opioid receptor agonist, inhibited spontaneous firing, hyperpolarized 68% of ARC cells in a dose-dependent manner and increased cell conductance. Baclofen, a GABAB receptor agonist, hyperpolarized all cells tested. The reversal potentials for both the DAGOL- and baclofen-induced currents were near that of a potassium conductance. Maximal activation by either of the agonists blocked the effects of the other agonist. Identified beta-endorphin cells were inhibited by both DAGOL and baclofen. The results of these in vitro studies suggest that GABAB and mu-opioid receptors are coupled to the same set of potassium channels and that these channels directly and powerfully inhibit most ARC cells, including beta-endorphin neurons. We propose that convergence of inhibitory influences at the ligand-gated potassium conductance described here may be an important site of interaction for opioidergic, GABAergic and other putative neurotransmitter systems in the control of neuroendocrine circuits by the ARC.

Changes in membrane ionic conductances and excitability characteristics of rat skeletal muscle during aging

Membrane electrical properties, component ionic conductances and excitability characteristics of extensor digitorum longus muscle from 3-4, 16 and 29 months old rats were measured "in vitro". Fiber diameter, membrane resistance (Rm) and membrane capacitance, increased with aging, and the increase was significant at 29 months. The increase of Rm was mostly due to a decrease of chloride conductance (GC1), whereas potassium conductance (GK) increased only slightly, at 16 and 29 months. Due to the lowered GC1, the latency of action potential increased at both ages with a consequent prolongation of the duration of action potential. Nevertheless, a decrease in the firing capability was recorded in the aged fibers. Our results indicate, that during aging, the most affected parameter of skeletal muscle fibers is GC1, although changes of this passive conductance alone cannot entirely account for the changes in the excitability characteristics recorded.

Regulation of Electrogenic Proton Pumping by Auxin and Fusicoccin as Related to the Growth of Avena Coleoptiles

The temporal relations between early responses to indoleacetic acid (IAA), proton secretion, hyperpolarization of the membrane potential, and growth change during the incubation of segments of oat (Avena sativa L.) coleoptiles in a low salt medium. When IAA is added after pretreatment of several hours, proton secretion increases after a latency of 7 minutes and reaches its maximum 10 to 15 minutes later. This timing coincides with both the increase in growth of the segments and the hyperpolarization of the membrane potential of parenchyma cells, consistent with the hypothesis that the change in membrane voltage reflects the activity of an electrogenic proton pump. The extent of IAA-induced hyperpolarization is substantially reduced by elevating [KCl](0), most likely because this increases the passive conductance of the membrane. Neither growth nor proton secretion is affected by high [KCl](0) (30 millimolar), indicating that neither process is limited by the magnitude of the membrane potential. These results are consistent with the acid growth hypothesis. Following short incubation times, however, IAA-induced hyperpolarization and growth are detected within 10 minutes, while acidification of the medium is delayed for more than 40 minutes. This result is seemingly in conflict with the acid growth hypothesis, but in freshly cut tissue, the pH of the external medium may not reflect the pH of the epidermal cell walls. The temporal coincidence of auxin-induced growth and hyperpolarization suggests that in freshly isolated segments the hyperpolarization is a more sensitive indication of proton secretion than is acidification of the external aqueous environment.

NMR measurements of intra- and extravesicular sodium in renal microvilli

Previous attempts to separate the nuclear magnetic resonances of intra- and extravesicular Na + in brush-border membrane vesicles (BBMV) were unsuccessful and led to the proposal of rapid exchange of Na + via sodium channels in BBMV. However, passive conductance of Na + in this membrane has been found to be relatively small. This inconsistency prompted us to use a different shift reagent to reassess the issue. In guinea pig renal BBMV (15–30 mg protein/ml) equilibrated with Na + (130 mequiv. 1), using the impermeant Na + shift reagent dysprosium tripolyphosphate (3 mM), the resonances of intra- (3.3%) and extravesicular (96.7%) Na + were resolved by 6 ppm. Increases in Na + conductance induced by gramicidin D did not alter the characteristics of intra- and extravesicular Na + resonances. By contrast, addition of glucose caused a transient increase in the area of the intravesicular Na + resonance. The clear separation between the intra- and the extravesicular Na + resonances allowed us to measure the relaxation times of Na +, which depend on its interactions with its immediate environment. The longitudinal relaxation time of intravesicular Na + (13 ± 1 ms) was much shorter than that of the extravesicular Na + (44.0 ± 0.4 ms). Thus, in intact renal BBMV, as well as in membranes treated with the cationophore gramicidin D, the exchange of Na + between the intra- and the extravesicular compartments is slow on the NMR time scale, consistent with the low Na + channel density of this membrane. In contrast, the increase in intravesicular Na + induced by glucose, is consistent with a significant contribution of the glucose cotransport pathway to Na + flux across these membranes. The short longitudinal relaxation time of Na + in the intravesicular space indicates interaction of Na + with BBMV binding sites or ordering of these ions in the intravesicular compartment.

Oxonol VI as an optical indicator for membrane potentials in lipid vesicles

Experiments with large unilamellar dioleoylphosphatidylcholine vesicles were carried out in order to study the effect of membrane potential on the fluorescence of Oxonol VI. A partition equilibrium of dye between membrane and water was found to exist with a partition coefficient γ c lipid c water of about 19 000 (at zero voltage). In the presence of an inside-positive membrane potential, the negatively charged dye accumulates in the intravesicular aqueous space according to a Nernst equilibrium. This leads to an increased adsorption of dye to the inner lipid monolayer and to a concomitant increase of fluorescence. The fluorescence change can be calibrated as a function of transmembrane voltage by generating a potassium diffusion potential in the presence of valinomycin. The intrinsic fluorescence of the membrane-bound dye is not affected by voltage; the whole influence of voltage on the fluorescence results from voltage-dependent partitioning of the dye between water and membrane. The voltage dependence of the apparent partition coefficient can be quantitatively described by a three-capacitor model in which the dye is assumed to bind to adsorption planes located on the hydrocarbon side of the membrane/solution interface. Oxonol VI was found to be suitable for detecting changes of membrane potential associated with the activity of the (Na + + K +)-ATPase in reconstituted vesicles. When ATP is added to the external medium, pump molecules with the ATP-binding side facing outward become activated; this results in a translocation of net positive charge towards the vesicle interior. Under this condition, fluorescence changes corresponding to (inside-positive) potentials of up to 150–200 mV are observed. After the build-up of the membrane potential, a quasi-stationary state is reached in which the pump current is compensated by a back-flow of charge through passive conductance pathways.

The effect of pH on the passive proton conductance of Bacillus acidocaldarius

The proton conductance and buffering capacity of Bacillus acidocaldarius, an organism that grows optimally at pH 3.5, were measured by an acid-pulse method. Conductance increased as the external pH was lowered from 6.0 to 3.5. The uncoupler CCCP increased the proton conductance, but became less effective as the pH was lowered.

D(-)3-hydroxybutyrate cotransport with Na in rat renal brush border membrane vesicles.

Which are the driving forces for D(-)3-hydroxybutyrate (HB) transport in rat renal brush border membranes (RBB)? Sodium, even in the absence of gradients, accelerates the unidirectional (1-5 s) flux of HB into rat RBB vesicles. Valinomycin (and Ki = Ko) does not significantly alter the NaCl gradient driven HB influx. Thus, the Na-dependent HB influx is driven by the chemical Na+ gradient but it is not driven by changes in the transmembrane electrical potential. Indeed, in valinomycin-treated membranes, vesicle-inside more negative potentials (K-gluconate in-Na-gluconate out) sufficient to accelerate Na-glucose cotransport, did not stimulate HB influx, in the presence of inwardly directed Na+ gradients, and did not significantly inhibit when in the absence of Na+. Thus, cotransport of HB with Na in rat RBB membranes does not involve the net transfer of positive charge and the passive conductance of this membrane for HB- is not large. However, vesicle inside more negative potentials (induced by inwardly directed NaNO3 gradients or by outwardly directed K+ gradients and valinomycin in the presence of inwardly directed Na+ gradients) inhibited HB influx, suggesting that another potential sensitive mechanism, perhaps redistribution of intramembrane charges, may influence HB influx. Acidification (pHi = pHo = 6.4 vs. 7.4) or inwardly directed H+ gradients (pHo/pHi = 6.4/7.4) did not alter HB influx, in the absence of Na+. Thus there is no evidence for a H+ driven HB influx. HB influx is significantly inhibited by high (100 mEq/l) trans concentration of Na+. Also, influx of 2.25 mM 14C-HB was significantly increased by 5-10 mM intravesicular HB under Na-equilibrated conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the driving force for the sodium pump (E Na) and of active and passive conductances (G Na and G sh) in isolated toad skin: Influence of antidiuretic hormone

1. Values of the sodium potential (E Na), active conductance (G Na) and passive conductance (G sh) were measured in the isolated skin of the toad Pleurodema thaul placed in an Ussing chamber, and Isaacson's test was performed with 2,4,6-triminopyrimidine (TAP) and with amiloride. 2. The numerical estimates obtained in the presence of TAP were E Na122.85 ± 15.17 mV, G Na0.493 ± 0.09 mS/cm 2 and G sh1.145 ± 0.23 mS/cm 2. 3. After exposure to ADH these values were as follows: E Na85.76 ± 12.17 mV, G Na1.191 ± 0.20 mS/cm 2 and G sh0.935 ± 0.14 mS/cm 2. 4. Addition of 0.5 × 10 −2 TAP produced a 53.90 ± 5.10% decrease in transepithelial potential and a 37.90 ± 4.90% fall in short-circuit current. 5. Exposure to ADH increased the transepithelial potential difference 34.20 ± 13.20% and the short-circuit current to 78.00 ± 20.50% above the control values. 6. Comparison of the efficiency and mechanism of action of TAP and amiloride in the determination of electrical parameters shows that both agents induce a similar decrease in G sh, a finding which could indicate that TAP blocks toad skin apical membrane Na + channels without affecting tight junction conductance.

Indacrinone (MK-196)--a specific inhibitor of the voltage-dependent Cl- permeability in toad skin.

The effect of indacrinone (MK-196) on Cl- transport through toad (Bufo bufo) skin epithelium was studied by the voltage clamping technique. At the transepithelial potential, V = 50 mV (serosal bath grounded) the unidirectional fluxes, governed by a Cl- self-exchange diffusion pathway, were not affected by 1 mM racemic MK-196 in the outer bath. Likewise at V = o mV, the unidirectional fluxes as well as the active (net) inward flux of Cl- were unaffected by MK-196. Voltage clamping the epithelium in the physiological range of potentials activated a Cl- specific passive conductance that saturated for V less than or equal to -90 mV. The influx and efflux of Cl- through this pathway were inhibited by MK-196, and the (passive) Cl- current was inhibited in a dose-dependent way for [MK-196] greater than or equal to 50 microM with about 70% inhibition for [MK-196] = 1 mM. The maximum Cl- conductance was decreased without shifting the position along the V-axis of the inverted S-shaped conductance-voltage relationship. The time constants for the voltage-stimulated Cl- conductance activation were not affected by MK-196 (50 microM less than or equals [MK-196] less than or equals 1 mM). The (+) and (-) isomers and racemic MK-196 affected the voltage-dependent Cl- conductance in similar ways. It is concluded that MK-196 has the properties of a Cl- channel blocker which is specific for the voltage-dependent Cl- permeability of the epithelium. The time course for development of inhibition exhibited a fast (min) and a slow (h) component.(ABSTRACT TRUNCATED AT 250 WORDS)