Distal colonic Na+ absorption inhibited by luminal P2Y2 receptors

Luminal P2 receptors are ubiquitously expressed in transporting epithelia. In steroid-sensitive epithelia (e.g., lung, distal nephron) epithelial Na⁺ channel (ENaC)-mediated Na⁺ absorption is inhibited via luminal P2 receptors. In distal mouse colon, we have identified that both, a luminal P2Y₂ and a luminal P2Y₄ receptor, stimulate K⁺ secretion. In this study, we investigate the effect of luminal adenosine triphosphate/uridine triphosphate (ATP/UTP) on electrogenic Na⁺ absorption in distal colonic mucosa of mice treated on a low Na⁺ diet for more than 2 weeks. Transepithelial electrical parameters were recorded in an Ussing chamber. Baseline parameters: transepithelial voltage (V _te): −13.7 ± 1.9 mV (lumen negative), transepithelial resistance (R _te): 24.1 ± 1.8 Ω cm², equivalent short circuit current (I _sc): −563.9 ± 63.8 μA/cm² (n = 21). Amiloride completely inhibited I _sc to −0.5 ± 8.5 μA/cm². Luminal ATP induced a slowly on-setting and persistent inhibition of the amiloride-sensitive I _sc by 160.7 ± 29.7 μA/cm² (n = 12, NMRI mice). Luminal ATP and UTP were almost equipotent with IC₅₀ values of 10 μM and 3 μM respectively. In P2Y₂ knock-out (KO) mice, the effect of luminal UTP on amiloride-sensitve Na⁺ absorption was absent. In contrast, in P2Y₄ KO mice the inhibitory effect of luminal UTP on Na⁺ absorption remained present. Semiquantitative polymerase chain reaction did not indicate regulation of the P2Y receptors under low Na⁺ diet, but it revealed a pronounced axial expression of both receptors with highest abundance in surface epithelia. Thus, luminal P2Y₂ and P2Y₄ receptors and ENaC channels co-localize in surface epithelium. Intriguingly, only the stimulation of the P2Y₂ receptor mediates inhibition of electrogenic Na⁺ absorption.     

Stimulatory actions of di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS), voltage-sensitive dye, on the BKCa channel in pituitary tumor (GH3) cells

Di-8-ANEPPS (4-{2-[6-(dibutylamino)-2-naphthalenyl]-ethenyl}-1-(3-sulfopropyl)pyridinium inner salt) has been used as a fast-response voltage-sensitive styrylpyridinium probe. However, little is known regarding the mechanism of di-8-ANEPPS actions on ion currents. In this study, the effects of this dye on ion currents were investigated in pituitary GH₃ cells. In whole-cell configuration, di-8-ANEPPS (10 μM) reversibly increased the amplitude of Ca²⁺-activated K⁺ current. In inside-out configuration, di-8-ANEPPS (10 μM) applied to the intracellular surface of the membrane caused no change in single-channel conductance; however, it did enhance the activity of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels with an EC₅₀ value of 7.5 μM. This compound caused a left shift in the activation curve of BK_Ca channels with no change in the gating charge of these channels. A decrease in mean closed time of the channels was seen in the presence of this dye. In the cell-attached mode, di-8-ANEPPS applied on the extracellular side of the membrane also activated BK_Ca channels. However, neither voltage-gated K⁺ nor ether-à-go-go-related gene (erg)-mediated K⁺ currents in GH₃ cells were affected by di-8-APPNES. Under current-clamp configuration, di-8-ANEPPS (10 μM) decreased the firing of action potentials in GH₃ cells. In pancreatic βTC-6 cells, di-8-APPNES (10 μM) also increased BK_Ca-channel activity. Taken together, this study suggests that during the exposure to di-8-ANEPPS, the stimulatory effects on BK_Ca channels could be one of potential mechanisms through which it may affect cell excitability.     

Adenine nucleotides and intracellular Ca2+ regulate a voltage-dependent and glucose-sensitive potassium channel in neurosecretory cells

Effects of membrane potential, intracellular Ca²⁺ and adenine nucleotides on glucose-sensitive channels from X organ (XO) neurons of the crayfish were studied in excised inside-out patches. Glucose- sensitive channels were selective to K⁺ ions; the unitary conductance was 112 pS in symmetrical K⁺, and the K⁺ permeability (P _K) was 1.3 × 10⁻¹³ cm ⋅s⁻¹. An inward rectification was observed when intracellular K⁺ was reduced. Using a quasi-physiological K⁺ gradient, a non-linear K⁺ current/voltage relationship was found showing an outward rectification and a slope conductance of 51 pS. The open-state probability (P _o) increased with membrane depolarization as a result of an enhancement of the mean open time and a shortening of the longer period of closures. In quasi-physio- logical K⁺ concentrations, the channel was activated from a threshold of about −60 mV, and the activation midpoint was −2 mV. P _o decreased noticeably at 50 μM internal adenosine 5′-triphosphate (ATP), and single-channel activity was totally abolished at 1 mM ATP. Hill analysis shows that this inhibition was the result of simultaneous binding of two ATP molecules to the channel, and the half-blocking concentration of ATP was 174 μM. Internal application of 5′-adenylylimidodiphosphate (AMP-PNP) as well as glibenclamide also decreased P _o. By contrast, the application of internal ADP (0.1 to 2 mM) activated this channel. An optimal range of internal free Ca²⁺ ions (0.1 to 10 μM) was required for the activation of this channel. The glucose--sensitive K⁺ channel of XO neurons could be considered as a subtype of ATP-sensitive K⁺ channel, contributing substantially to macroscopic outward current. Received: 13 November 1995/Received after revision and accepted: 13 December 1995     

Identification of an ATP-sensitive K+ channel in rat cultured cortical neurons

To determine whether membranes of mammalian central neurons contain an ATP-sensitive K⁺ (K_ATP) channel similar to that present in pancreatic cells, the patch-clamp technique was applied to cultured neurons prepared from the neonatal rat cerebral cortex and hippocampus. In whole-cell experiments with hippocampal neurons, extracellular application of 0.5 mM diazoxide (a K_ATP channel activator) elicited a hyperpolarization concomitant with an increase in membrane conductance, whereas application of 0.5 mM tolbutamide (a K_ATP channel blocker) induced a depolarization with a decrease in conductance. Similar results were obtained with cortical neurons. In outside-out patch experiments with cortical neurons, a K⁺ channel sensitive to these drugs was found. The channel was completely blocked by 0.5 mM tolbutamide and activated by 0.5 mM diazoxide. The single-channel conductance was 65 pS under symmetrical 145 mM K⁺ conditions and 24 pS in a physiological K⁺ gradient. In inside-out patch experiments, this channel was demonstrated to be inhibited by an application of 0.2–1 mM ATP to the cytoplasmic surface of the patch membrane. These results indicate that the membranes of rat cortical neurons contain a K_ATP channel that is quite similar to that found in pancreatic cells. It is also suggested that the same or a similar K⁺ channel may exist in membranes of hippocampal neurons.     

Metabolic inhibition and low internal ATP activate K-ATP channels in rat dopaminergic substantia nigra neurones

The patch-clamp technique was used to study whole-cell currents of acutely dissociated rat substantia nigra (SN) neurones. In perforated-patch current-clamp recordings, inhibition of mitochondrial metabolism by rotenone (5 M) produced a hyperpolarisation and inhibited electrical activity. These effects were reversed by the sulphonylureas tolbutamide (0.5 mM) or glibenclamide (0.5 M). Under voltageclamp conditions, rotenone induced a timeand voltage-independent K⁺ current which was selectively blocked by sulphonylureas. The glibenclamide-sensitive current reversed at –81.7±2.7 mV (n=5) and showed marked inward rectification. Intracellular dialysis with 0.3 mM adenosine 5-triphosphate (ATP), but not 2 mM or 5 mM ATP, in standard whole-cell recordings also resulted in activation of a sulphonylurea-sensitive K⁺ current with similar properties (reversal potential, –81.9±2.5 mV, n=5). The close similarity in the properties of the ATP-sensitive K⁺ current observed in whole-cell recordings and the K⁺ current activated by metabolic inhibition in perforated-patch recordings suggest that they both result from activation of the same type of ATP-sensitive K⁺ channel. Sulphonylureas had no effect on electrical activity or membrane currents in the absence of rotenone in perforated-patch recordings, or in cells dialysed with 5 mM ATP, indicating that in SN neurones these drugs are selective for the ATP-sensitive K⁺ current.     

Membrane currents and cytoplasmic sodium transients generated by glutamate transport in Bergmann glial cells

Effects of glutamate and kainate (KA) on Bergmann glial cells were investigated in mouse cerebellar slices using the whole-cell configuration of the patch-clamp technique combined with SBFI-based Na⁺ microfluorimetry. l-Glutamate (1 mM) and KA (100 μM) induced inward currents in Bergmann glial cells voltage-clamped at −70 mV. These currents were accompanied by an increase in intracellular Na⁺ concentration ([Na⁺]_i) from the average resting level of 5.2 ± 0.5 mM to 26 ± 5 mM and 33 ± 7 mM, respectively. KA-evoked signals (1) were completely blocked in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM), an antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/KA ionotropic glutamate receptors; (2) reversed at 0 mV, and (3) disappeared in Na⁺-free, N-methyl-D-glucamine (NMDG⁺)-containing solution, but remained almost unchanged in Na⁺-free, Li⁺-containing solution. Conversely, l-glutamate-induced signals (1) were marginally CNQX sensitive (∼10% inhibition), (2) did not reverse at a holding potential of +20 mV, (3) were markedly suppressed by Na⁺ substitution with both NMDG⁺ and Li⁺, and (4) were inhibited by d,l-threo-β-benzyloxyaspartate. Further, d-glutamate, l-, and d-aspartate were also able to induce Na⁺-dependent inward current. Stimulation of parallel fibres triggered inward currents and [Na⁺]_i transients that were insensitive to CNQX and MK-801; hence, we suggested that synaptically released glutamate activates glutamate/Na⁺ transporter in Bergmann glial cells, which produces a substantial increase in intracellular Na+ concentration.     

cAMP-dependent inward rectifier current in neurons of the rat suprachiasmatic nucleus

Electrophysiological properties of the inward rectification of neurons in the rat suprachiasmatic nucleus (SCN) were examined by using the single-electrode voltage-clamp method, in vitro. Inward rectifier current (I _H) was produced by hyperpolarizing step command potentials to membrane potentials negative to approximately −60 mV in nominally zero-Ca²⁺ Krebs solution containing tetrodotoxin (1 μM), tetraethylammonium (40 mM), Cd²⁺ (500 μM) and 4-aminopyridine (1 mM).I _H developed during the hyperpolarizing step command potential with a duration of up to 5 s showing no inactivation with time.I _H was selectively blocked by extracellular Cs⁺ (1 mM). The activation of the H-channel conductance (G _H) ranged between −55 and −120 mV. TheG _H was 80–150 pS (n=4) at the half-activation voltage of −84±7 mV (n=4). The reversal potential ofI _H obtained by instantaneous current voltage (I/V) relations was −41±6mV (n=4); it shifted to −51±8mV (n=3) in low-Na⁺ (20 mM) solution and to −24±4 mV (n=4) in high-K⁺ (20 mM) solution. Forskolin (1–10 μM) produced an inward current and increased the amplitude ofI _H. Forskolin did not change the half-activation voltage ofG _H. 8-Bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP, 0.1–1 mM) and dibutyryl-cAMP (0.1–1 mM) enhancedI _H. 3-Isobutyl-1-methylxanthine (IBMX, 1 mM) also enhancedI _H. The results suggest that the inward rectifier cation current is regulated by the basal activity of adenylate cyclase in neurons of the rat SCN.     

Identification of an ATP-sensitive K+ channel in spiny neurons of rat caudate nucleus

On the somata of GABAergic spiny neurons in the caudate nucleus of the rat an ATP-sensitive K⁺ channel (K_ATP-channel) was identified. The K_ATP-currents in cell-attached patches were activated both by energy-depleting conditions (200 M cyanide) and by diazoxide (300 M) and were reversibly blocked by tolbutamide (EC₅₀=5 M). In inside-out patch membranes both ATP (1 mM) and its non-hydrolyzable analog AMP-PNP (adenylylimidodiphosphate; EC₅₀=27M) reversibly inhibited channel activity. These results demonstrate that the K_ATP-channel in spiny neurons displays properties characteristic of the K_ATP-channel in hippocampal, neocortical and nigral neurons and in pancreatic ß-cells.     

Localized L-type calcium channels control exocytosis in cat chromaffin cells

Depolarizing 1-s pulses to 0 mV from a holding potential of −70 mV, induced whole-cell currents through Ca²⁺ channels (I _Ca) in patch-clamped cat adrenal medulla chromaffin cells. The dihydropyridine (DHP) furnidipine (3 μM) reduced the peak current by 47% and the late current by 80%. ω-Conotoxin GVIA (CgTx, 1 μM) reduced the peak I _Ca by 42% and the late I _Ca by 55%. Pulses (10 s duration) with 70 mM K⁺/2.5 mM Ca²⁺ solution (70 K⁺/2.5 Ca²⁺), applied to single fura-2-loaded cat chromaffin cells increased the cytosolic Ca²⁺ concentration ([Ca²⁺]_i from 0.1 to 2.21 μM; this increase was reduced by 43.7% by furnidipine and by 42.5% by CgTx. In the perfused cat adrenal gland, secretion evoked by 10-s pulses of 70 K⁺/2.5 Ca²⁺ was reduced by 25% by CgTx and by 96% by furnidipine. Similar results were obtained when secretion from superfused isolated cat adrenal chromaffin cells was studied and when using a tenfold lower [Ca²⁺]_o. The results are compatible with the existence of DHP-sensitive (L-type) as well as CgTx-sensitive (N-type) voltage-dependent Ca²⁺ channels in cat chromaffin cells. It seems, howevever, that though extracellular Ca²⁺ entry through both channel types leads to similar increments of averaged [Ca²⁺]_i, the control of catecholamine release is dominated only by Ca²⁺ entering through L-type Ca²⁺ channels. This supports the idea of a preferential segregation of L-type Ca²⁺ channels to localized “hot spots” in the plasmalemma of chromaffin cells where exocytosis occurs.     

Small-conductance Ca2+-activated K+ channels in bovine chromaffin cells

Simultaneous whole-cell patch-clamp and fura-2 fluorescence [Ca²⁺]_i measurements were used to characterize Ca²⁺-activated K⁺ currents in cultured bovine chromaffin cells. Extracellular application of histamine (10 M) induced a rise of [Ca²⁺]_i concomitantly with an outward current at holding potentials positive to –80 mV. The activation of the current reflected an increase in conductance, which did not depend on membrane potential in the range –80 mV to –40 mV. Increasing the extracellular K⁺ concentration to 20 mM at the holding potential of –78 mV was associated with inwardly directed currents during the [Ca²⁺]_i elevations induced either by histamine (10 M) or short voltage-clamp depolarizations. The current reversal potential was close to the K⁺ equilibrium potential, being a function of external K⁺ concentration. Current fluctuation analysis suggested a unit conductance of 3–5 pS for the channel that underlies this K⁺ current. The current could be blocked by apamin (1 M). Whole-cell current-clamp recordings snowed that histamine (10 M) application caused a transient hyperpolarization, which evolved in parallel with the [Ca²⁺]_i changes. It is proposed that a small-conductance Ca²⁺-activated K⁺ channel is present in the membrane of bovine chromaffin cells and may be involved in regulating catecholamine secretion by the adrenal glands of various species.     

Electrophysiological properties of membrane currents in single myometrial cells isolated from pregnant rats

The whole-cell voltage-clamp method was applied to single smooth muscle cells prepared from the longitudinal layer of the pregnant rat myometrium (17–20 days of gestation). It was found that the transient inward current mainly consists of Ca²⁺ current, because the removal of Ca²⁺ ions from the external medium and 10 M nifedipine eliminated this inward current. Its steady-state inactivation curve was obtained by the standard method, in which the membrane potential of half inactivation and the slope factor were estimated to be –58.0±4.9 mV (n=11) and 8.9±1.4 mV (n=11), respectively. In a small number of preparations (in 2 out of 30 preparations), there remained a very fast inward current in Ca²⁺-free medium containing Mg²⁺. Tetrodotoxin (TTX, 10 M) can abolish this current, suggesting that the channel for this current is equivalent to the Na⁺ channel in nerve cells. Two major phases of outward currents were identified by voltage jumps from negative holding levels to more positive levels. The first phase was a fast transient outward current. This current remained intact after external tetraethylammonium (TEA, 20 mM) was added. Following the transient current, a large delayed rectified outward current reached its peak over a period of 50 ms and then decayed. The reversal potential for this outward current was determined by observing the change of polarity of the tail currents with the change in extracellular K⁺ concentration ([K⁺]₀). The slope for the change of reversal potential per ten-fold change in [K⁺]₀ is 57.7 mV at more than 23.2 mM [K⁺]_o, indicating that this current is mostly carried by K⁺ ions. Voltage-dependent inactivation of the delayed rectified outward current was determined by the standard method. The membrane potential for half inactivation and the slope factor were estimated to be –42.8±3.9 mV (n=3) and 10.1±1.5 mV (n=3), respectively. External TEA (20 mM) effectively eliminated the delayed rectified outward currents. Nifedipine (10 M) suppressed not only Ca²⁺ current but also outward K⁺ currents.     

Effect of vinpocetine on various types of high-threshold potassium currents in snail neurons

A high-threshold (−20 mV) K⁺ current was recorded from isolated edible snail neurons by a two-microelectrode voltage clamp technique. This current consisted of three components: fast-inactivating K⁺ currents (I_A), noninactivating K⁺ current (I_KD), and Ca²⁺-dependent K⁺ current (I_K(Ca)). Different cells had one to three components of K⁺ current. Vinpocetine increased I_A, moderately inhibited I_KD (by 30–50%) and strongly suppressed I_K(Ca) (by 60–90%). Inhibition of I_K(Ca) was not related to the effect of vinpocetine on the inward Ca²⁺ current. When K⁺ current consisted of all three components, the effect of vinpocetine on the ionic current amplitude was opposite at different potentials. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 10, pp. 408–411, October, 1998     

An α3β4 subunit combination acts as a major functional nicotinic acetylcholine receptor in male rat pelvic ganglion neurons

We identified major subunits of the nicotinic acetylcholine receptor (nAChR) involved in excitatory postsynaptic potential and intracellular Ca²⁺ ([Ca²⁺] _i ) increase in the major pelvic ganglion (MPG) neurons of the male rat. ACh elicited fast inward currents in both sympathetic and parasympathetic MPG neurons. Mecamylamine, a selective antagonist for α3β4 nAChR, potently inhibited the ACh-induced currents in sympathetic and parasympathetic neurons (IC₅₀; 0.53 and 0.22 μM, respectively). Furthermore, α-conotoxin AuIB (10 μM), a new selective antagonist for α3β4 nAChR, blocked more than 80% of the ACh-induced currents in MPG neurons. Conversely, α-bungarotoxin, α-methyllycaconitine, and dihydro-β-erythroidine, known as blockers of the α7 or α4β2, did not show selective blocking effects on MPG neurons. ACh transiently increased [Ca²⁺] _i which was subsequently abolished in the extracellular Ca²⁺-free environment. Simultaneous recording of [Ca²⁺] _i and ionic currents revealed that ACh increased [Ca²⁺] _i under the conditions of the voltage-clamped (at −80 mV) state, and this resulted from the influx through nAChR itself. ACh-induced [Ca²⁺] _i increase was blocked by mecamylamine (10 μM), but was not affected by atropine (1 μM). RT-PCR analysis showed that, among subunits of nAChR, α3 and β4 were predominantly expressed in MPG. We suggest that activation of α3 and β4 nAChR subunits in MPG neurons induce fast inward currents and [Ca²⁺] _i increase, possibly mediating a major role in pelvic autonomic synaptic transmission.     

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.     

O2 consumption,aerobic glycolysis and tissue phosphagen content during activation of the NA+/K+ pump in rat portal vein

Oxygen consumption, lactate production and tissue contents of ATP, phosphocreatine (PCr) and lactate were measured following readdition of K⁺ to K⁺-depleted rat portal veins, in order to study the energy turnover associated with Na⁺/K⁺ pumping. During incubation in K⁺-free medium at 37° C spontaneous contractions disappeared in 10–20 min. Readdition of K⁺ (5.9 mM) after 40 min K⁺-free incubation caused hyperpolarization of the cell membrane for the first 5–10 min and then gradual depolarization with return of spontaneous action potentials and contractions by 10–20 min. During the first 4–6 min after K⁺ readdition aerobic lactate production was about doubled and then gradually returned to the original level (0.17 mol/min g) at about 20 min. The increase in glycolytic rate was prevented by 1 mM ouabain. In contrast, O₂ consumption (in K⁺-free medium, 0.38 mol/min g) rose by about 10% when K⁺ was added and this increase lasted about 5 min. By 8 min after K⁺ addition the increased glycolysis and oxidative phosphorylation had accounted for each about the same amount of extra ATP generation over that extrapolated from the steady rate before K⁺ addition. The average total increase in ATP turnover in the first 8 min was 15%. During this period there was no change in the cellular content of ATP, PCr, or extractable ADP. The results indicate that Na⁺/K⁺ pumping utilizes a relatively small share of the total energy turnover in the vascular smooth muscle but is to a large extent dependent on aerobic glycolysis and therefore a major site of carbohydrate usage.     

Prostaglandin E2 potentiates the excitability of small diameter trigeminal root ganglion neurons projecting onto the superficial layer of the cervical dorsal horn in rats

The aim of the present study was to investigate how prostaglandin E₂ (PGE₂) affects the excitability of trigeminal root ganglion (TRG) neurons, projecting onto the superficial layer of the cervical dorsal horn, using fluorescence retrograde tracing and perforated patch-clamp techniques. TRG neurons were retrogradely labeled with fluorogold (FG). The cell diameter of FG-labeled neurons was small (< 30 μm). Under the voltage-clamp mode, application of PGE₂ (0.01–10 μM) concentration-dependently increased the magnitude of the peak tetrodotoxin-resistant sodium current (TTX-R I _Na) and this current was maximal at a concentration of 1 μM. One micromolar PGE₂ application caused a hyperpolarizing shift of 8.3 mV in the activation curve for TTX-R I _Na. In the current-clamp mode, the PGE₂ (1 μM) application significantly increased the number of action potentials during the depolarizing step pulses as well as the level of overshoot but had no significant effect on the resting membrane potential. These results suggest that the excitability of small diameter TRG neurons seen after 1 μM PGE₂ application is involved in an increase in the TTX-R I _Na.     

Ca2+-activated K+ channels in airway smooth muscle are inhibited by cytoplasmic adenosine triphosphate

Large-conductance Ca²⁺-activated K⁺ channels were studied in membranes of cultured rabbit airway smooth muscle cells, using the patch-clamp technique. In cell-attached recordings, channel openings were rare and occurred only at very positive potentials. Bradykinin (10 M), an agonist which releases Ca²⁺ from the sarcoplasmic reticulum, transiently increased channel activity. The metabolic blocker 2,4-dinitrophenol (20 M), which lowers cellular adenosine triphosphate (ATP) levels, induced a sustained increase of channel activity in cell-attached patches. In excised patches, these channels had a slope conductance of 155 pS at 0 mV, were activated by depolarization and by increasing the Ca²⁺ concentration at the cytoplasmic side above 10^–7 mol/l. ATP, applied to the cytoplasmic side of the patches, dose-dependently decreased the channel's open-state probability. An inhibition constant (K _i) of 0.2 mmol/l was found for the ATP-induced inhibition. ATP reduced the Ca²⁺ sensitivity of the channel, shifting the Ca²⁺ activation curve to the right and additionally reducing its steepness. Our results demonstrate that cytoplasmic ATP inhibits a large-conductance Ca²⁺-activated K⁺ channel in airway smooth muscle. This ATP modulation of Ca²⁺-activated K⁺ channels might serve as an important mechanism linking energy status and the contractile state of the cells.     

Comparative roles of ATP-sensitive K channels and Ca-activated BK channels in posthypoxic hyperexcitability and rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro

The aim of this study was to investigate the comparative effects of glibenclamide (GC), a selective blocker of K⁺_ATP channels, and iberiotoxin (IbTX), a selective blocker of BK⁺_Ca channels, on the repeated brief hypoxia-induced posthypoxic hyperexcitability and rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro. The method of field potentials measurement in CA1 region of the rat hippocampal slices was used. In contrast to GC (10 μM), IbTX (10 nM) significantly abolished both posthypoxic hyperexcitability and rapid hypoxic preconditioning induced by brief hypoxic episodes. These effects of IbTX did not depend on its ability to reduce the hypoxia-induced decrease of population spike (PS) amplitude during hypoxic episodes since GC (10 μM), comparatively with IbTX (10 nM), significantly reduced the depressive effect of hypoxia on the PS amplitude during hypoxic episodes but did not abolish both posthypoxic hyperexcitability and rapid hypoxic preconditioning in CA1 pyramidal neurons. Our results indicated that BK⁺_Ca channels, in comparison with K⁺_ATP channels, play a more important role in such repeated brief hypoxia-induced forms of neuroplasticity in hippocampal CA1 pyramidal neurons as posthypoxic hyperexcitability and rapid hypoxic preconditioning.     

Activation of Ca2+-dependent K+ and Cl− currents by UTP and ATP in CFPAC-1 cells

Activation of Cl^– and K⁺ conductances by nucleotide receptor-operated mobilization of intracellular Ca²⁺ was investigated in CFPAC-1 cells with the perforated-patch technique. Adenosine 5-triphosphate (ATP) and uridine 5-triphosphate (UTP) caused a dose-dependent fast and transient membrane hyperpolarization. UTP was more effective than ATP. In voltageclamped cells, two currents with different ionic permeability and kinetics were activated by the nucleotides. The first one was carried by Cl^– ions, peaked in the first few seconds after addition of nucleotides, and lasted for 1±0.3 min. Its amplitude was about 2.7 nA at –100 mV with 100 mol/l of either ATP or UTP. The second current was carried by K⁺ ions and was blocked by Cs⁺. This current peaked more slowly and had a mean duration of 4.6±0.7 min. Its amplitude was 0.9 nA and 0.5 nA at –20 mV with 100 umol/l UTP and ATP, respectively. Activation of the nucleotide receptor caused a transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) that was similar in the presence or absence of extracellular Ca²⁺. The ED₅₀ for UTP was 24 umol/l and that for ATP was 94 mol/l. Depletion of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ store by thapsigargin prevented both the nucleotide-induced [Ca²⁺]_i increase and the activation of membrane currents. Addition of 2 mmol/l Ca²⁺ to thapsigargin-treated cells produced a sustained increase of Cl^– and K⁺ currents, which was reversed by Ca²⁺ removal. The present study demonstrates that CFPAC-1 cells respond to nucleotide receptor activation with a transient increase in [Ca²⁺]_i that stimulates Ca²⁺-dependent Cl^– and K⁺ currents. This phenomenon is probably mediated by inositol 1,4,5-trisphosphate-dependent Ca²⁺ stores.     

Pinacidil activates the ATP-sensitive K+ channel in inside-out and cell-attached patch membranes of guinea-pig ventricular myocytes

Patch-clamp techniques were used to study the effects of pinacidil on the adenosine-5-triphosphate (ATP)-sensitive K⁺ channel current in guinea-pig ventricular myocytes. In inside-out patches, the ATP-sensitive K⁺ channel current could be recorded at an internal ATP concentration of 0.5 mM or less and almost complete inhibition was achieved by raising the concentration to 2 mM. Application of pinacidil (10–30 M) in the presence of 2 mM ATP restored the current, whereas 5 mM ATP antagonized the effect of pinacidil. The conductance of the channel at symmetrical K⁺ concentrations of 140 mM was 75 pS with a slight inward rectification at voltages positive to +40 mV. There was no significant change in the conductance after application of pinacidil. In 0.5 mM ATP, at –80 mV, both the distributions of the open time and the life-time of bursts could be fitted by a single exponential. An increase in ATP concentration decreased the mean life-time of bursts, whereas pinacidil increased it with little increase in the mean open time. Closed time distributions of the channel were fitted by at least two exponentials, with a fast and a slow time constant. An increase in ATP concentration markedly increased the slow time constant associated with a decrease in the number of bursts, whereas the effect of pinacidil was opposite to that of increased ATP. These results indicate that pinacidil increases the open-state probability of the ATP-sensitive K⁺ channel. In cell-attached patches, application of pinacidil (100–200 M) to the extracellular solution reversibly induced the channel activity, which showed similar properties to those of the ATP-sensitive K⁺ channel recorded in cell-free patches.