Evidence for two types of potassium current in rat choroid plexus epithelial cells

The whole-cell patch-clamp technique was applied to rat choroid plexus epithelial cells. The resting membrane potential was −53 mV. The whole-cell conductance was mainly K⁺ selective, and the K⁺ current observed appeared to contain two distinct components. Depolarizing voltage pulses (more positive than 0 mV) evoked time-dependent outward currents which resembled delayed-rectifying K⁺ currents in other tissues. The current exhibited time-dependent activation and, at potentials more positive than 40 mV, slower time-dependent inactivation. The reversal potential measured by tail current analysis showed a shift of 43 mV for a tenfold increase in extracellular K⁺ concentration ([K⁺]₀). The current was reduced by extracellular 5 mM Ba²⁺, 5 mM tetraethylammonium (TEA⁺), 5 mM Cs⁺ and 1 mM 4-aminopyridine (4-AP). In contrast, hyperpolarizing voltage pulses evoked time-independent, inward-rectifying currents. The reversal potential measured by voltageramp commands showed a shift of 42 mV for a tenfold increase in [K⁺]₀. The chord conductance did not appear to increase with increasing [K⁺]₀. The current was reduced by extracellular 5 mM Ba²⁺ and 0.5 mM Cs⁺, but not by 5 mM TEA⁺ or 1 mM 4-AP. These data suggest that two populations of K⁺ channel contribute to the conductance of choroid plexus epithelial cells.     

Extracellular ionic and volume changes: The role in glia—Neuron interaction

Activity-related changes in extracellular K⁺ concentration ([K⁺]_e), pH (pH_e) and extracellular volume were studied by means ofion-selective microelectrodes in the adult rat spinal cord in vivo and in neonatal rat spinal cords isolated from pups 3–14 days of age (P3–P14). Concomitantly with the ionic changes, the extracellular space (ECS) volume fraction (α), ECS tortuosity (λ) and non-specific uptake (k′), three parameters affecting the diffusion of substances in nervous tissue, were studied in the rat spinal cord gray matter. In adult rats, repetitive electrical nerve stimulation (10–100 Hz) elicited increases in [K⁺]_e of about 2.0–3.5 mm, followed by a post-stimulation K⁺-undershoot and triphasic alkaline-acid-alkaline changes in pH_e with a dominating acid shift. The ECS volume in the adult rat occupies about 20% of the tissue, α = 0.20 ± 0.003, λ = 1.62 ± 0.02 and k′ = 4.6 ± 0.4 × 10⁻³s⁻¹ (n = 39). In contrast, in pups at P3–P6, the [K⁺]_e increased by as much as 6.5 mm at a stimulation frequency of 10 Hz, i.e. K⁺ ceiling level was elevated, and there was a dominating alkaline shift. An increase in [K⁺]_e as large as 1.3–2.5 mm accompanied by an alkaline shift was evoked by a single electrical stimulus. The K⁺ ceiling level and alkaline shifts decreased with age, while an acid shift, which was preceded by a small initial alkaline shift, appeared in the second postnatal week. In pups at P1–P2, the spinal cord was X-irradiated to block gliogenesis. The typical decrease in [K⁺]_e ceiling level and the development of the acid shift in pH_e at P10–P14 were blocked by X-irradiation. Concomitantly, continuous development of glial fibrillary acidic protein positive reaction was disrupted and densely stained astrocytes in gray matter at P10–P14 revealed astrogliosis.The alkaline, but not the acid, shift was blocked by Mg²⁺ and picrotoxin (10⁻⁶m). Acetazolamide enhanced the alkaline but blocked the acid shift. Furthermore, the acid shift was blocked, and the alkaline shift enhanced, by Ba²⁺, amiloride and SITS. Application of glutamate or gamma-aminobutyric acid evoked an alkaline shift in the pH_e baseline at P3–P14 as well as after X-irradiation. The results suggest that the activity-related acid shifts in pH_e are related to membrane transport processes in mature glia, while the alkaline shifts have a postsynaptic origin and are due to activation of ligand-gated ion channels.At P4–P6, the ECS volume was almost double that in adult rats, α = 0.37 ± 0.01 (n = 17), the ECS tortuosity was significantly higher, λ = 1.78 ± 0.02, while the non-specific uptake was not significantly different, k′ = 3.61 ± 0.56 × 10⁻³ s⁻¹. The α gradually decreased to about 24% at P12. In adult rats, electrical or adequate stimulation evoked a shrinkage of the extracellular space by 20–50%, while no significant changes in ECS volume were found in P3–P6. We conclude that the [K⁺]_e ceiling level, character of the pH_e transients, the size of the ECS volume and the activity-related ECS shrinkage are closely related to gliogenesis.     

Role for ionic fluxes on cell death and apoptotic volume decrease in cultured cerebellar granule neurons

Recent evidence suggests a major role for ionic fluxes in apoptotic cell death and apoptotic volume decrease. Cerebellar granule neurons (CGN) undergo apoptosis when they are treated with staurosporine or camptothecin (CPT) or when cells are transferred from high extracellular potassium (25 mM KCl [K⁺]_e, K25) to low potassium concentration (5 mM KCl [K⁺]_e, K5). In this study we described that all three apoptotic conditions induced apoptotic volume decrease in CGN and that two different potassium channel blockers, cesium (Cs⁺) and tetraethylammonium (TEA⁺), prevented the apoptotic volume decrease, caspase-3 activation, nuclear condensation and cell death induced by K5 and CPT, but not by staurosporine. Cs⁺ and TEA⁺ also blocked membrane currents generated in K5 conditions in CGN. On the other hand, non specific Cl⁻ channel blockers such as 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS) prevented loss of cell volume induced by K5 or staurosporine. Only the Cl⁻ channels blocker but not the K⁺ channels blockers protected from staurosporine-induced death of CGN. These data suggest that ionic fluxes play a key role in the activation of the apoptotic volume decrease and apoptotic death of CGN, but the fine mechanism seems to depend on the apoptotic condition.     

Contraluminal transport of organic cations in the proximal tubule of the rat kidney

In order to study the characteristics of contraluminal organic cation transport from the blood site into proximal tubular cells the stopped-flow capillary perfusion method was applied. The disappearance of N ¹-[³H]methylnicotinamide (NMeN⁺) and [³H]tetraethylammonium (TEA⁺) at different concentrations and contact times was measured and the following parameters evaluated: K _m,NMeN = 0.54 mmol/l, J _max,NMeN = 0.4 pmol s^–1 cm^–1; K _m,TEA = 0.16 mmol/l, J _max,TEA = 0.8 pmol s^–1 cm^–1. TEA⁺ inhibited NMeN⁺ transport and NMeN⁺ the uptake of TEA⁺. Thereby, the K _i values for inhibition correspond closely to the K _m values for uptake. Similar inhibitory potencies of ten organic cation against TEA⁺ and NMeN⁺ transport provide further evidence for a common transport system. Omission of HCO ₃ ^– , or Na⁺ and addition of K⁺ (with or without Ba²⁺) reduce NMeN⁺ transport, while omission of K⁺ (with or without valinomycin) or addition of thiocyanate has no effect. Since the manoeuvres that depolarize contraluminal electrical potential difference reduce NMeN⁺ transport, cell-negative electrical potential difference is suggested as a driving force for contraluminal organic cation transport from the interstitium into the cell. Furthermore, the inhibitory potency (app. K _i values) of homologous series of primary, secondary, tertiary and hydroxy amines as well as of mono- and bisquarternary ammonium compounds against NMeN⁺ transport was tested. The inhibitory potency increased in the sequence methyl < ethyl < propyl < butyl and primary < secondary < tertiary amines < quarternary ammonium compounds. With the amines a reversed correlation between K _i,NMeN and the octanol/water partition coefficient (log octanol) is seen. With quarternary ammonium compounds the inhibitory potency decreases with increasing molecular size: tetrabutyl- > tetrapentyl- > tetrahexyl- > tetraheptyl > tetraoctylammonium. Introducing two OH groups into triethylamine reduces the inhibitory potency while introduction of two OH groups into diethylamine or three OH groups into triethylamine abolishes the inhibitory potency as a result of reduced hydrophobicity. With choline (trimethylethanolamine) and its analogues the reversed correlation between K _i,NMeN and log octanol was also seen. Molecules with a similar hydrophobic moiety to those of the monoammonium compounds, but with two ammonium groups, showed only a small or no inhibitory potency against NMeN⁺ transport. The data indicate that (a) hydrophobic moieties are important for the interaction with the contraluminal organic cation transporter, and (b) the size of the molecule can be a limiting factor. The reduced or missing interaction of the bisquarternary compound might be caused either by the second charge and/or reduced hydrophobicity and/or too large size of a molecule.     

Two Ca-dependent K-channels classified by the application of tetraethylammonium distribute to smooth muscle membranes of the rabbit portal vein

Dispersed single smooth muscle cells of rabbit portal vein were prepared by treatment with collagenase and trypsin. The muscle cells were 100–300 m in length, 5–10 m in maximum width and cylindrical in shape. In insideout membrane patches, two different amplitudes of ionic currents were recorded, and these single channel conductances were 273 pS (K_l-channel) and 92 pS (K_s-channel), when both sides of the membrane were exposed to 142 mM K⁺ solution. The channel conductances depended on concentrations of K⁺ on both sides of the membrane. When K⁺ were replaced with Na⁺ or Tris⁺, these single-channel currents were abolished. When the concentration of Ca²⁺ inside the membrane was greater than 10^–7 M, the channel activity was enhanced but there was enhancement when Ca²⁺ was applied to the extracellular membrane surface, in concentrations ranging between 10^–9 and 10^–3 M. During application of tetraethylammonium (TEA⁺; 1–10 mM) to the intracellular membrane surface, amplitudes of the single-channel current of both types of the K-channel were not modified. By contrast application of TEA⁺ (0.1–1 mM) to the extracellular membrane surface, reduced the amplitudes of the current and increased noise levels during the open-state of the K_l-channels, but did not have such an effect on the K_s-channel. We conclude that there are at least two different Ca-dependent K-channels distributed on the smooth muscle membrane of the rabbit portal vein. TEA⁺ applied to the extracellular membrane surface blocks activation of the K_l-channel, but not that of the K_s-channel. These two Ca-dependent K-channels do not seem to be important for maintenance of the resting membrane potential, but do play an important role in the repolarizing stage of the Ca spikes, in the rabbit portal vein.     

Regulatory volume decrease in a renal distal tubular cell line (A6) II. Effect of Na+ transport rate

A6 epithelia, a cell line originating from the distal tubular part of the kidney ofXenopus laevis, were cultured on permeable supports and mounted in an Ussing-type chamber. Cell thickness (T _c), short-circuit current (I _sc) and transepithelial conductance (G _t) were recorded while tissues were bilaterally incubated in NaCl solutions and the transepithelial potential was clamped to zero. Effects of inhibition and stimulation of transepithelial Na⁺ transport on cell volume and on its regulation during a hyposmotic challenge were investigated. Under control conditions a slow spontaneous decrease ofT _c described by a linear baseline was recorded. The reduction of the apical osmolality from 260 to 140 mosmol/kg did not alter cell volume significantly, demonstrating a negligible water permeability of the apical barrier. The inhibition of Na⁺ uptake by replacing apical Na⁺ byN-methyl-d-glucamine (NMDG⁺) did not affect cell volume under isotonic conditions. An increase ofT _c by 12.1% above the control baseline was recorded after blocking active transport with ouabain for 60 min. The activation of Na⁺ transport with insulin or oxytocin, which is known to activate the apical water permeability in other epithelia, did not alter cell volume significantly. The insensitivity of cell volume to alterations in apical Na⁺ uptake or Na⁺ pump rate confirms the close coupling between apical and basolateral transport processes. The blockage of basolateral K⁺ channels by 5 mM Ba²⁺ elicited a significant increase inT _c of 16.3% above control. Quinine, a potent blocker of volume-activated K⁺ channels, did not changeT _c significantly. Basolateral hypotonicity elicited a rapid rise inT _c followed by a regulatory volume decrease (RVD). An RVD was also recorded after blocking apical Na⁺ uptake as well as after stimulating apical Na⁺ uptake with oxytocin or insulin. Inhibition of active transport with ouabain as well as blocking K⁺ efflux at the basolateral side with Ba²⁺ or quinine abolished the RVD. The inhibition of the RVD by ouabain seems to be caused by a depletion of cellular K⁺, whereas the effects of Ba²⁺ and quinine are most likely due to the blockage of the basolateral K⁺ pathway.     

pH dependence of K+ conductances of rat cortical collecting duct principal cells

The K⁺ channels of the principal cells of rat cortical collecting duct (CCD) are pH sensitive in excised membranes. K⁺ secretion is decreased with increased H⁺ secretion during acidosis. We examined whether the pH sensitivity of these K⁺ channels is present also in the intact cell and thus could explain the coupling between K⁺ and H⁺ secretion. Membrane voltages (V _m), whole-cell conductances (g _c), and single-channel currents of K⁺ channels were recorded from freshly isolated CCD cells or isolated CCD segments with the patch-clamp method. Intracellular pH (pH_i) was measured using the pH-sensitive fluorescent dye 2-7-bis(carboxyethyl)-5-6-carboxyfluorescein (BCECF). Acetate (20 mmol/l) had no effect on V _m, g _c, or the activity of the K⁺ channels in these cells. Acetate, however, acidified pH_i slightly by 0.17±0.04 pH units (n=19). V _m depolarized by 12±3 mV (n=26) and by 23±2 mV (n=66) and g _c decreased by 26±5% (n=13) and by 55±5% (n=12) with 3–5 or 8–10% CO₂, respectively. The same CO₂ concentrations decreased pH_i by 0.49±0.07 (n=15) and 0.73±0.11 pH units (n=12), respectively. Open probability (P _o) of all four K⁺ channels in the intact rat CCD cells was reversibly inhibited by 8–10% CO₂. pH_i increased with the addition of 20 mmol/l NH₄ ⁺/NH₃ by a maximum of 0.64±0.08 pH units (n=33) and acidified transiently by 0.37±0.05 pH units (n=33) upon NH₄ ⁺/NH₃ removal. In the presence of NH₄ ⁺/NH₃ V _m depolarized by 16±2 mV (n=66) and g _c decreased by 26±7% (n=16). The activity of all four K⁺ channels was also strongly inhibited in the presence of NH₄ ⁺/NH₃. The effect of NH₄ ⁺/NH₃ on V _m and g _c was markedly increased when the pH of the NH₄ ⁺/NH₃-containing solution was set to 8.5 or 9.2. From these data we conclude that cellular acidification in rat CCD principal cells down-regulates K⁺ conductances, thus reduces K⁺ secretion by direct inhibition of K⁺ channel activity. This pH dependence is present in all four K⁺ channels of the rat CCD. The inhibition of K⁺ channels by NH₄ ⁺/NH₃ is independent of changes in pH_i and rather involves an effect of NH₃.     

Structure and interaction of inhibitors with the TEA/H+ exchanger of rabbit renal brush border membranes

The renal secretion of organic cations (OCs) involves a carrier-mediated exchange of OC for H⁺ in the luminal membrane of proximal cells. To assess the influence of chemical structure on the interaction of potential substrates with this process we examined the effect of a series of quaternary ammonium compounds on the transport of the OC tetraethylammonium (TEA) in a preparation of isolated renal brush-border membrane vesicles. Apparent inhibitory potency varied over a factor of 10⁴, as expressed in inhibitor coefficients (K _i ^TEA) whose approximate values ranged from 0.5 M to 5 mM. The poorest inhibitors of TEA/H⁺ exchange were those molecules with carboxyl or hydroxyl residues, whereas the addition of methylene groups to a parent molecule tended to increase inhibitory potency. A plot of apparent K _i ^TEA versus calculated octanol:water partition coefficient (expressed in terms of a relative lipophilicity factor) showed a clear correlation between these two parameters, although there was considerable variability between apparent lipophilicity and K _i ^TEA for molecules with very different parent structures. For select groups of molecules with similar parent structures (e.g., the n-tetraalkylammoniums or the 4-phenylpyridinium, 3-phenylpyridinium, and quinolinium compounds) the correlation between calculated lipophilicity and apparent K _i ^TEA was more marked. However, even within these groups of closely related parent structures, there appeared to be subtle, but systematic, variations in inhibitory potency that may have been related to the influence of steric factors on the binding of inhibitors to the TEA/H⁺ exchanger. We conclude that the lipophilic nature of a quaternary ammonium compound represents the predominant factor in the binding to, and subsequent inhibition of, luminal TEA/H⁺ exchange. Specific steric factors may influence the binding of substrate to the exchanger, but play a secondary role in this interaction.     

Ion-transporting activity in the murine colonic epithelium of normal animals and animals with cystic fibrosis

Electrogenic ion transport in the isolated co-Ionic epithelium from normal and transgenic mice with cystic fibrosis (CF mice) has been investigated under short-circuit current (I _sc) conditions. Normal tissues showed chloride secretion in response to carbachol or forskolin, which was sensitive to the Na-K-2Cl cotransport inhibitor, frusemide. Responses to both agents were maintained for at least 12 h in vitro, but the responses to carbachol changed in format throughout this period. By contrast CF colons failed to show the normal secretory responses to carbachol and forskolin, most preparations showing a decrease in I _sc that was immediately reversed by frusemide. In CF colons addition of Ba²⁺ ions or tetraethylammonium (TEA⁺) to the apical bathing solution antagonised the reduction in I _sc caused by the secretagogues. It is concluded that the reduction in I _sc in CF colons is due to electrogenic K⁺ secretion and this was confirmed by flux studies using rubidium-86. In normal colons exposed to TEA⁺ the responses to for-skolin were greater, but not significantly so, presumably because the minor K⁺-secretory responses are dominated by major chloride-secretory responses. Again rubidium-86 fluxes showed an increase of K⁺ secretion in normal colons receiving forskolin. Since the amiloride-sensitive current was not different in CF and normal colons there was no evidence that the CF mice were stressed in a way that increased mineralocorticoid levels and hence K⁺ secretion. Knowledge of the phenotype of the colonic epithelium of the CF mouse sets the baseline from which attempts at gene therapy for the gut must be judged.     

Effect of H+ and elevated PCO 2on membrane electrical properties of rat cerebral arteries

Some membrane electrical properties of muscle cells from the middle cerebral artery of the rat were recorded with intracellular microelectrodes. The resting membrane potential (E _m) of this preparation was –63 mV. Reduction of extracellular pH to 7.0 in the face of a constantP _{CO 2}of 40 mm Hg had no significant effect onE _m. Similarly the slope of the steady-state voltage/current curves was not different at pH 7.0 compared to control at pH 7.4. In marked contrast, whenP _{CO 2}was elevated to around 60 to 70 mm Hg there was a rapid hyperpolarization and reduction in the slope of the voltage current curve suggesting an increased conductance for one or more ionic species. In addition elevation ofP _{CO 2}increased the slope of theE _m vs. log[K]₀ curve from 46 mV/decade to 59 m V/decade which is in good agreement with a Nernstian potential for a K⁺ selective membrane. These data suggest that while the smooth muscle cells of rat cerebral arteries are relatively insensitive to a small reduction in extracellular pH; reduction of intracellular pH by elevatingP _{CO 2}induces hyperpolarization by increasing K⁺ conductance (g _k). However, it is not clear from these experiments if theP _{CO 2}effects are mediated entirely by changes in pH or if there is a direct membrane action of CO₂.This work is supported by Grant no. HL27862     

Extracellular potassium accumulation and transmission in frog spinal cord

In the isolated frog spinal cord, intracellular recordings from motoneurons and neuroglia and extracellular recordings from dorsal and ventral roots were used to compare electrophysiological changes produced by tetanic stimulation of dorsal roots with those resulting from increasing extracellular potassium concentration, [K⁺]₀. Many of the after-effects of tetanic dorsal root stimulation could be mimicked by increasing [K⁺]₀. These include the following: depolarization of motoneurons and neuroglia, prolongation and depression of excitatory postsynaptic potentials, depression of dorsal root potentials, facilitation and depression of ventral root potentials, as well as increases in spontaneous synaptic activity and depression of antidromic spikes recorded from motoneurons. The levels of [K⁺]₀ necessary to produce effects comparable to those following maximal dorsal root stimulation are about twice those estimated from measurements with K-selective microelectrodes or glial depolarization, suggesting that both these methods underestimate the amount of potassium which accumulates in the narrow intercellular spaces between neurons and glia in the intermediate region of the spinal cord.It is concluded that, at least within the isolated frog spinal cord, K⁺ accumulation is a significant factor modifying transmission following dorsal root tetanic stimulation and that its distribution is inhomogeneous.     

Potassium kinetics in human muscle interstitium during repeated intense exercise in relation to fatigue

Accumulation of K⁺ in skeletal muscle interstitium during intense exercise has been suggested to cause fatigue in humans. The present study examined interstitial K⁺ kinetics and fatigue during repeated, intense, exhaustive exercise in human skeletal muscle. Ten subjects performed three repeated, intense (61.6±4.1 W; mean±SEM), one-legged knee extension exercise bouts (EX1, EX2 and EX3) to exhaustion separated by 10-min recovery periods. Interstitial [K⁺] ([K⁺]_interst) in the vastus lateralis muscle were determined using microdialysis. Time-to-fatigue decreased progressively (P<0.05) during the protocol (5.1±0.4, 4.2±0.3 and 3.2±0.2 min for EX1, EX2 and EX3 respectively). Prior to these bouts, [K⁺]_interst was 4.1±0.2, 4.8±0.2 and 5.2±0.2 mM, respectively. During the initial 1.5 min of exercise the accumulation rate of interstitial K⁺ was 85% greater (P<0.05) in EX1 than in EX3. At exhaustion [K⁺]_interst was 11.4±0.8 mM in EX1, which was not different from that in EX2 (10.4±0.8 mM), but higher (P<0.05) than in EX3 (9.1±0.3 mM). The study demonstrated that the rate of accumulation of K⁺ in the muscle interstitium declines during intense repetitive exercise. Furthermore, whilst [K⁺]_interst at exhaustion reached levels high enough to impair performance, the concentration decreased with repeated exercise, suggesting that accumulation of interstitial K⁺ per se does not cause fatigue when intense exercise is repeated.     

Intracellular Ca2+ inactivates an outwardly rectifying K+ current in human adenomatous parathyroid cells

We have used whole-cell patch-clamp techniques to study the conductances in the plasma membranes of human parathyroid cells. With a KCl-rich pipette solution containing Ca²⁺ buffered to a concentration of 0.1 mol/l, the zero current potential was –71.1±0.5 mV (n=19) and the whole-cell current/ voltage (I/V) relation had an inwardly rectifying and an outwardly rectifying component. The inwardly rectifying current activated instantaneously on hyperpolarization of the plasma membrane to potentials more negative than –80 mV, and a semi-logarithmic plot of the reversal potential of the inward current (estimated by extrapolation from the range in which it was linear) as a function of extracellular K⁺ concentration ([K⁺]_o) revealed a linear relation with a slope of 64 mV per decade change in [K⁺]_o, which is not significantly different from the Nernstian slope, demonstrating that the current was carried by K⁺ ions. The conductance exhibited a square root dependence on [K⁺]_o as has been observed for inward rectifiers in other tissues. The current was blocked by the presence of Ba²⁺ (1 mmol/l) or Cs⁺ (1.5 mmol/l) in the bath. The outwardly rectifying current was activated by depolarization of the membrane potential to potentials more positive than –20 mV. It was inhibited by replacement of pipette K⁺ with Cs⁺, indicating that it also was a K⁺ current: it was partially (42%) blocked when tetraethylammonium (TEA⁺, 10 mmol/l) was added to the bath. The outwardly rectifying, but not the inwardly rectifying K⁺ current, was regulated by intracellular free Ca²⁺ concentration ([Ca²⁺]_i) such that increasing [Ca²⁺]_i above 10 nmol/l inhibited the outwardly rectifying current, the half-maximum effect being seen at 1 mol/l. Since it is known that increases in [Ca²⁺]_o produce increases in [Ca²⁺]_i, and that they depolarize parathyroid cells by reducing the membrane K⁺ conductance, we suggest that it is the reduction of the outwardly rectifying K⁺ conductance by increases in [Ca²⁺]_i which is responsible for the reduction in K⁺ conductance seen when [Ca²⁺]_o is increased.     

Membrane electrical properties of vascular smooth muscle from the guinea pig superior mesenteric artery

Some electrical membrane properties of an isolated small artery, namely, the superior mesenteric artery of the guinea pig, were studied by intracellular microelectrodes. The mean resting membrane potential (E _m) was –54 mV. The average slope of theE _m vs. log [K]_o curve (between 10 and 100 mM [K]_o) was 32 mV/decade, and the curve extrapolated to a [K]_i of 160 mM. The ratio of Na⁺ permeability to K⁺ permeability (P _Na/P _K) at 4.0 mM [K]_o calculated from the Goldman constant-field equation (assuming Cl^– to be passively distributed) was 0.18 (E _m=–46 mV after a 5 min exposure to ouabain to suppress any electrogenic pump potential). The normal input resistance (R _in) averaged 8.5 m. Choline substitution for Na⁺ or amiloride, an agent known to depressP _Na, hyperpolarized the muscle to about –63 mV without a significant change inR _in. Ba²⁺ (0.5 mM) depolarized the muscle to –37 mV, increasedR _in to 15 m, and produced spontaneous action potentials in this normally quiescent artery; tetraethylammonium (TEA, 5 mM) enabled large overshooting action potentials to be produced upon stimulation. Glutamate of NO ₃ ^– substitution for Cl^– produced an initial depolarization followed by a return to the original resting potential within 10 min; readdition of 25 mM Cl^– transiently hyperpolarized the muscle markedly, followed by a return to the originalE _m. These data indicate that Cl^– is passively distributed and does not contribute to the steady-state resting potential in this vascular muscle. The data also suggest that the relatively lowE _m in this arterial muscle is not due to a low [K]_i, but is due to a highP _Na/P _K ratio, presumably related to a low K⁺ conductance (g _K). Since Ba²⁺ and TEA⁺ are known to decrease restingg _K and K⁺ activation, the data also suggest that K⁺ activation could inhibit action potential generation.     

Relationship between plasma potassium and ventilation during successive periods of exercise in men

Summary During and after two successive incremental cycle ergometer tests (tests A and B), plasma potassium concentration ([K⁺]_p), plasma pH (pH_p), plasma partial pressure of carbon dioxide, blood lactate concentration ([Lac^–]_b) and ventilation (V_E) were measured. While there was a good correlation between the increase in [K⁺]_p and V_E or pH_p, respectively, in test A, in test B a close correlation was found only between the increase in V_E and [K⁺]_p (r>0.9 for nearly all single cases; r was 0.84 and 0.89 for all (pooled) cases in tests A and B, respectively; the correlation coefficients between changes in pH_p and V_E in tests A and B were r=0.74 and r=0.28, respectively, and r=0.89 and r=0.10 between the changes in [Lac^–]_b and V_E in tests A and B). The close relationship for individuals between V_E and [K⁺]_p in tests A and B supported the hypothesis that the extracellular increase in [K⁺] may contribute to the ventilatory drive during exercise. The comparison of the results of tests A and B further indicated that the relationship between pH_p and V_E was dependent on the experimental design, and that pH_p and V_E changes are unlikely to be cause and effect.The study was carried out in the Centre of Physiology, Department of Sports- and Exercise Physiology, Medical School, W-3000 Hannover, Federal Republic of Germany     

Effects of picrotoxin on potassium accumulation and dorsal root potentials in the frog spinal cord.

The effects of picrotoxin on the changes of extracellular potassium concentration (Δ[K⁺]_e), field potentials and dorsal root potentials evoked by afferent stimulation, were studied in the isolated spinal cord of the frog. Δ[K⁺]_e was measured with potassium selective micro-electrodes. In a normal Ringer's solution the Δ[K⁺]_e evoked by a single pulse applied to a dorsal root did not exceed 0.05 mM. In solutions containing picrotoxin (10^?7?10^?5m) the Δ[K⁺]_e increased to 0.06–0.1 mm. At higher concentrations (10^?4?10^?3m) of picrotoxin the Δ[K⁺]_e reached 3–6 mm and spontaneous elevations of [K⁺]_e were observed synchronously with the dorsal root potentials. The latter were depressed by 20–40% and considerably prolonged. The time constant of their ascending phase increased from 9 to 10 ms to 30–40 ms. The second component of the negative field potential, recorded from the intermediate region, increased and its time course corresponded to that of the evoked dorsal root potentials. Impulse activity of motoneurones and interneurones evoked by afferent stimulation was greatly enhanced. Picrotoxin (10^?4?5.10^?4m) was found to have no effect on the ‘asynaptic’ component of evoked dorsal root potentials, which is resistant to 20 mm MgSO₄ and to the absence of Ca²⁺. It is therefore unlikely that the depressant effect of picrotoxin on the evoked dorsal root potentials is produced by its direct action on the potassium conductance of primary afferents.The findings are consistent with a dual mechanism of dorsal root potentials. The fast component of evoked dorsal root potentials which is depressed by picrotoxin is apparently produced by activation of axo-axonic synapses at the primary afferents, while the slow component is due to transient accumulation of extracellular K⁺. The potassium component of the evoked dorsal root potentials becomes dominant in solutions with high concentrations of picrotoxin (10^?4?10^?3m) when impulse transmission is greatly enhanced.     

Test of blockers of AQP1 water permeability by a high-resolution method: no effects of tetraethylammonium ions or acetazolamide

The effects of putative water channel blockers were tested on AQP1-expressing Xenopus laevis oocytes by a fast optical method with a time resolution of 1 s and a volume resolution of 20 pl. The oocytes were exposed to external hyposmolarity and the osmotic water permeability (L _p) derived from the initial 10 s of volume change. For longer durations, the effective osmotic gradient across the membrane was reduced significantly because of dilution of the intracellular contents and of ion transport across the membrane. The latter was monitored by voltage clamp of the oocytes. In contrast to previous reports based on slower and less sensitive assays, we found no effects of tetraethylammonium ions (TEA⁺) and acetazolamide on L _p. We have no single explanation for this, but several factors are considered: (a) If the osmotic gradient is assumed to be constant for periods longer than 10 s, the L _p will be underestimated. (b) Hyposmotic gradients implemented by dilution with water will entail changes in the ionic strength as well; this may enhance loss of salt from the oocyte. (c) By voltage clamping the AQP1-expressing oocytes during hyposmotic challenges, we found that TEA⁺-treated oocytes were more electrically leaky than untreated ones. This may obscure comparisons between the L _p of treated and untreated oocytes. (d) The nature of the ion transport mechanisms in the plasma membrane depends on how oocytes have been prepared for experiments and on their viability as indicated by the membrane potential. These parameters may vary between laboratories.     

Small and maxi K+ channels in the basolateral membrane of isolated crypts from rat distal colon: single-channel and slow whole-cell recordings

The patch-clamp technique was used to characterize K⁺ channel activity in the basolateral membrane of isolated crypts from rat distal colon. In cell-attached patches with KCl in the pipette, channels with conductances ranging from 6 pS to 80 pS appeared. With NaCl in the pipette and KCl in the bath in excised inside-out membrane patches a small-conductance channel with a mean conductance of 12±6 pS (n=18) was observed. The channel has been identified as K⁺ channel by its selectivity for K⁺ over Na⁺ and by its sensitivity to conventional K⁺ channel blockers, Ba²⁺ and tetraethylammonium (TEA⁺). Changes of cytosolic pH did not attenuate channel activity. Activity of the 12-pS channel was increased by membrane depolarization and elevated cytosolic Ca²⁺ concentration. In addition, a maxi K⁺ channel with a mean conductance of 187±15 pS (n=4) in symmetrical KCl solutions was only occasionally recorded. The maxi K⁺ channel could be blocked by Ba²⁺ (5 mmol/l) on the cytosolic side. Using the slow-whole cell recording technique under control conditions, a cell membrane potential of –70±10mV (n=18) was measured. By application of various K⁺ channel blockers such as glibenclamide, charybdotoxin, apamin, risotilide, Ba²⁺ and TEA⁺ in the bath, only Ba²⁺ and TEA⁺ depolarized the cell membrane. The present data suggest that the small K⁺ channel (12 pS) is involved in the maintenance of the cell membrane resting potential.     

Effects of dopamine on ion transport across the rat distal colon

Dopamine (5·10^–6–5·10^–4 M) induced a concentration-dependent decrease in short-circuit current (I_sc) across the rat distal colon. This response was preceded by a transient and inconsistent increase in I_sc. The -adrenoceptor blocker phentolamine and the inhibitors of dopamine-2-like (D₂-like) receptors L-741,626 and L-745,870 inhibited the dopamine response, suggesting a contribution of adrenergic and dopaminergic receptors. The decrease in I_sc evoked by dopamine was inhibited by bumetanide, an inhibitor of the basolateral Na⁺-K⁺-2 Cl^– cotransporter responsible for the uptake of K⁺, and by quinine, a blocker of apical K⁺ channels, indicating that stimulation of K⁺ secretion contributes to the measured change in I_sc. In patch-clamp experiments dopamine hyperpolarized the membrane and increased cellular K⁺ current. This response was not concomitant with a change in the intracellular [Ca²⁺] as demonstrated in parallel fura-2 experiments. These results demonstrate that dopamine, like other catecholamines, stimulates colonic K⁺ secretion.     

H2O2 induced hyperpolarization of pancreatic B-cells

Conventional electrophysiology and the whole-cell patch-clamp technique have been applied to elucidate the effects of H₂O₂ on pancreatic B-cells of the mouse. In these cells, addition of 15 mmol/l glucose leads to depolarization and oscillation of the cell membrane potential. Subsequent addition of H₂O₂ (1 mmol/l) in the presence of glucose was followed by a marked and rapid hyperpolarization of the cell membrane with suppression of the electrical activity. Accordingly, in slow whole-cell patch-clamp experiments (with nystatin in the pipette solution) H₂O₂ induced a marked increase of cell membrane conductance. Tolbutamide, a blocker of K⁺ _ATP channels, only partially blocked the effect of H₂O₂ even at high concentrations. The H₂O₂-induced, tolbutamide-insensitive current component, however, was largely abolished by a high concentration of TEA⁺ (80 mmol/l) or BaCl₂ (10 mmol/l). It is concluded that in B-cells H₂O₂ stimulates a K⁺ current and that this effect leads to marked hyperpolarization and reversal of glucose-induced oscillations of cell membrane potential.