Incomplete dissociation of glibenclamide from wild-type and mutant pancreatic K ATP channels limits their recovery from inhibition.

BACKGROUND AND PURPOSE: The antidiabetic sulphonylurea, glibenclamide, acts by inhibiting the pancreatic ATP-sensitive K(+) (K(ATP)) channel, a tetradimeric complex of K(IR)6.2 and sulphonylurea receptor 1 (K(IR)6.2/SUR1)(4). At room temperature, recovery of channel activity following washout of glibenclamide is very slow and cannot be measured. This study investigates the relation between the recovery of channel activity from glibenclamide inhibition and the dissociation rate of [(3)H]-glibenclamide from the channel at 37 degrees C. EXPERIMENTAL APPROACH: K(IR)6.2, K(IR)6.2DeltaN5 or K(IR)6.2DeltaN10 (the latter lacking amino-terminal residues 2-5 or 2-10 respectively) were coexpressed with SUR1 in HEK cells. Dissociation of [(3)H]-glibenclamide from the channel and recovery of channel activity from glibenclamide inhibition were determined at 37 degrees C. KEY RESULTS: The dissociation kinetics of [(3)H]-glibenclamide from the wild-type channel followed an exponential decay with a dissociation half-time, t(1/2)(D) = 14 min; however, only limited and slow recovery of channel activity was observed. t(1/2)(D) for K(IR)6.2DeltaN5/SUR1 channels was 5.3 min and recovery of channel activity exhibited a sluggish sigmoidal time course with a half-time, t(1/2)(R) = 12 min. t(1/2)(D) for the DeltaN10 channel was 2.3 min; recovery kinetics were again sigmoidal with t(1/2)(R) approximately 4 min. CONCLUSIONS AND IMPLICATIONS: The dissociation of glibenclamide from the truncated channels is the rate-limiting step of channel recovery. The sigmoidal recovery kinetics are in quantitative agreement with a model where glibenclamide must dissociate from all four (or at least three) sites before the channel reopens. It is argued that these conclusions hold also for the wild-type (pancreatic) K(ATP) channel.

KATP channel openers of the benzopyran type reach their binding site via the cytosol.

BACKGROUND AND PURPOSE: ATP-sensitive K+ (KATP) channels are composed of pore-forming subunits (Kir6.x) and of sulphonylurea receptors (SUR). Both sulphonylureas and K(ATP) channel openers act by binding to SUR. Sulphonylureas reach their binding site from the cytosol but it remains unknown whether this holds for openers too. EXPERIMENTAL APPROACH: A poorly membrane-permeant sulphonic acid derivative of the benzopyran-type opener, bimakalim, was synthesized, descyano-bimakalim-6-sulphonic acid (BMSA). Binding of BMSA and bimakalim was compared in membranes and intact cells expressing the Kir6.2/SUR2B channel and channel opening was compared in inside-out patches and whole cells. KEY RESULTS: In membranes, bimakalim and BMSA bound to Kir6.2/SUR2B with Ki values of 61 nM and 4.3 microM, showing that the negative charge decreased affinity 69-fold. In intact cells, however, binding of BMSA was much weaker than in membranes (75-fold) whereas that of bimakalim was unchanged. The Ki value of BMSA decreased with increasing incubation time. In inside-out patches, bimakalim (1 microM) and BMSA (100 microM) opened the Kir6.2/SUR2B channel closed by MgATP to a similar degree whereas in whole-cell experiments, only bimakalim was effective. CONCLUSIONS AND IMPLICATIONS: Despite its negative charge, BMSA is an effective channel opener. The fact that BMSA binds and acts more effectively when applied to the inner side of the cell membrane shows that benzopyran openers reach their binding site at SUR from the cytosol. This suggests that the binding pocket of SUR is only open on the cytoplasmic side.

Selectivity of repaglinide and glibenclamide for the pancreatic over the cardiovascular K(ATP) channels.

AIMS/HYPOTHESIS: Sulfonylureas and glinides close beta cell ATP-sensitive K(+) (K(ATP)) channels to increase insulin release; the concomitant closure of cardiovascular K(ATP) channels, however, leads to complications in patients with cardiac ischaemia. The insulinotrope repaglinide is successful in therapy, but has been reported to inhibit the recombinant K(ATP) channels of beta cells, cardiocytes and non-vascular smooth muscle cells with similar potencies, suggesting that the (patho-)physiological role of the cardiovascular K(ATP) channels may be overstated. We therefore re-examined repaglinide's potency at and affinity for the recombinant pancreatic, myocardial and vascular K(ATP) channels in comparison with glibenclamide. METHODS: K(ATP) channel subunits (i.e. inwardly rectifying K(+) channels [Kir6.x] and sulfonylurea receptors [SURx]) were expressed in intact human embryonic kidney cells and assayed in whole-cell patch-clamp and [(3)H]glibenclamide binding experiments at 37 degrees C. RESULTS: Repaglinide and glibenclamide, respectively, were >or=30 and >or=1,000 times more potent in closing the pancreatic than the cardiovascular channels and they did not lead to complete inhibition of the myocardial channel. Binding assays showed that the selectivity of glibenclamide was essentially based on high affinity for the pancreatic SUR, whereas binding of repaglinide to the SUR subtypes was rather non-selective. After coexpression with Kir6.x to form the assembled channels, however, the affinity of the pancreatic channel for repaglinide was increased 130-fold, an effect much larger than with the cardiovascular channels. This selective effect of coexpression depended on the piperidino substituent in repaglinide. CONCLUSIONS/INTERPRETATION: Repaglinide and glibenclamide show higher potency and efficacy in inhibiting the pancreatic than the cardiovascular K(ATP) channels, thus supporting their clinical use.

Interaction of the sulfonylthiourea HMR 1833 with sulfonylurea receptors and recombinant ATP-sensitive K(+) channels: comparison with glibenclamide.

The novel sulfonylthiourea 1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea (HMR 1883), a blocker of ATP-sensitive K(+) channels (K(ATP) channels), has potential against ischemia-induced arrhythmias. Here, the interaction of HMR 1883 with sulfonylurea receptor (SUR) subtypes and recombinant K(ATP) channels is compared with that of the standard sulfonylurea, glibenclamide, in radioligand receptor binding and electrophysiological experiments. HMR 1883 and glibenclamide inhibited [(3)H]glibenclamide binding to SUR1 with K(i) values of 63 microM and 1.5 nM, and [(3)H]opener binding to SUR2A/2B with K(i) values of 14/44 microM and 0.5/2.8 microM, respectively (values at 1 mM MgATP). The interaction of HMR 1883 with the SUR2 subtypes was more sensitive to inhibition by MgATP and MgADP than that of glibenclamide. In inside-out patches and in the absence of nucleotides, HMR 1883 inhibited the recombinant K(ATP) channels from heart (Kir6.2/SUR2A) and nonvascular smooth muscle (Kir6.2/SUR2B) with IC(50) values of 0.38 and 1.2 microM, respectively; glibenclamide did not discriminate between these channels (IC(50) approximately 0.026 microM). In whole cells, the recombinant vascular K(ATP) channel, Kir6.1/SUR2B, was inhibited by HMR 1883 and glibenclamide with IC(50) values of 5.3 and 0.043 microM, respectively. The data show that the sulfonylthiourea exhibits a selectivity profile quite different from that of glibenclamide with a major loss of affinity toward SUR1 and slight preference for SUR2A. The stronger inhibition by nucleotides of HMR 1883 binding to SUR2 (as compared with glibenclamide) makes the sulfonylthiourea an interesting tool for further investigation.

Chromanol 293B, a blocker of the slow delayed rectifier K+ current (IKs), inhibits the CFTR Cl- current.

The cystic fibrosis transmembrane conductance regulator (CFTR) and the sulphonylurea receptor subunit (SUR) of the KATP channel are both members of the ATP-binding cassette (ABC) protein superfamily. Many compounds that open or block the KATP channel by binding to SUR also inhibit the CFTR Cl- current (ICFTR); an example in point is the chromanol-type KATP channel opener, cromakalim. The structurally related chromanol 293B (trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl-chromane), a blocker of the slow component of the delayed rectifier K+ current (IKs) in the heart, is also a weak inhibitor of KATP. This suggests that 293B may affect also ICFTR- We have addressed this question with human CFTR expressed in Xenopus oocytes. In two-electrode voltage-clamp experiments, 293B inhibited ICFTR with an IC50-value of 19 microM and Hill coefficient of 1.0; the inhibition was weakened by increasing concentrations of isobutyl-methylxanthine (IBMX). Patch-clamp recordings gave an IC50-value of 30 microM but showed a unusual variability in the sensitivity to 293B. The data show that 293B inhibits ICFTR and suggest that the mechanism of inhibition may depend on the phosphorylation state of the CFTR protein. The concentrations required for inhibition of ICFTR are three- to fivefold higher than those reported for inhibition of KvLQT1 + minK expressed in Xenopus oocytes. Since CFTR is expressed also in cardiac myocytes, the effects of 293B in these cells must be analysed with caution.

Characterization of a mutant sulfonylurea receptor SUR2B with high affinity for sulfonylureas and openers: differences in the coupling to Kir6.x subtypes.

ATP-dependent K(+) channels are composed of pore-forming subunits of the Kir6.x family and of sulfonylurea receptors (SURs). SUR1, expressed in pancreatic beta-cells, has a higher affinity for sulfonylureas, such as glibenclamide, than SUR2B, expressed in smooth muscle. This difference is mainly caused by serine 1237 in SUR1 corresponding to tyrosine 1206 in SUR2B. To increase the affinity of SUR2B for glibenclamide, the mutant SUR2B(Y1206S) was constructed. In whole-cell patch-clamp experiments, glibenclamide inhibited the channel formed by coexpression of mutant SUR2B with Kir6.1 or 6.2 in human embryonic kidney cells with IC(50) values of 2.7 and 13 nM, respectively (wild-type, 43 and 167 nM). In intact cells, [(3)H]glibenclamide bound to mutant SUR2B with a K(D) value of 4.7 nM (wild-type, 32 nM); coexpression with Kir6.1 or 6.2 increased affinity by 4- and 8-fold, respectively. Binding of the opener [(3)H]P1075 to SUR2B(Y1206S) was the same as to wild-type and was unaffected by coexpression. In cells, the ratio of glibenclamide:P1075 sites was approximately 1:1; in membranes, it varied with the MgATP concentration. Heterologous competition curves were generally biphasic; the shape of the curve depended on the Kir-subtype. The effects of coexpression were weakened or abolished when binding assays were conducted in membranes. It is concluded that the mutation Y1206S increases the affinity of SUR2B for and the channel sensitivity toward glibenclamide by 7- to 15-fold. The interaction of glibenclamide (but not opener) with mutant SUR2B is modified by coexpression with Kir6.x in a manner depending on the Kir subtype and on the integrity of the cell.

Synthesis and characterization of a novel tritiated KATP channel opener with a benzopyran structure.

The synthesis of a tritiated benzopyran-type opener of the ATP-dependent K+ channel (KATP channel), [3H]-PKF217 - 744 (3S,4R)-N-[3,4-dihydro-2,2-dimethyl-3-hydroxy-6-(2-methyl-4-pyridinyl)-2H-1-benzopyran-4-yl]-3-[2,6-3H]pyridinecarboxamide with a specific activity of 50 Ci mmol(-1) is described. Binding of the ligand was studied in membranes from human embryonic kidney cells transfected with the sulphonylurea receptor isoforms, SUR2B and SUR2A, respectively. PKF217 - 744 was confirmed as being a KATP channel opener by its ability to open the Kir6.1/SUR2B channel, the recombinant form of the vascular KATP channel, and to inhibit binding of the pinacidil analogue, [3H]-P1075, to SUR2B (Ki=26 nM). The kinetics of [3H]-PKF217 - 744 binding to SUR2B was described by rate constants of association and dissociation of 6.9x10(6) M(-1) min(-1) and 0.09 min(-1), respectively. Binding of [3H]-PKF217 - 744 to SUR2B/2A was activated by MgATP (EC50 approximately 3 microM) and inhibited (SUR2B) or enhanced (SUR2A) by MGADP: Binding of [3H]-PKF217 - 744 to SUR2B was inhibited by representatives of the different structural classes of openers and sulphonylureas. Ki values were identical with those obtained using the opener [3H]-P1075 as the radioligand. Glibenclamide accelerated dissociation of the SUR2B-[3H]-PKF217 - 744 complex. The data show that the affinity of [3H]-PKF217 - 744 binding to SUR2B is approximately 6 times lower than that of [3H]-P1075. This is due to a surprisingly slow association rate of the benzopyran-type ligand, suggesting a complex mechanism of opener binding to SUR. The other pharmacological properties of the two opener radioligands are identical.

Potent stimulation and inhibition of the CFTR Cl(-) current by phloxine B.

The effects of the fluoresceine derivative, phloxine B, on the Cl(-) current through the cystic fibrosis transmembrane conductance regulator (CFTR) were examined in Xenopus oocytes expressing human CFTR. In whole oocytes, the CFTR Cl(-) current (I(CFTR)) was activated by superfusion with isobutylmethylxanthine and forskolin. I(CFTR) was stable during activation and deactivated rapidly upon washout of the activation solution. Phloxine B slowed deactivation and, at high concentrations, inhibited I(CFTR) weakly. In excised inside-out macropatches, I(CFTR) was activated by the catalytic subunit of protein kinase A (cPKA) and MgATP. Phloxine B (0.01 - 3 microM), applied after activation, increased I(CFTR) within 30 s followed by a slow decrease which became dominant at high concentrations. Slowing of deactivation of the CFTR was observed at all concentrations. The effect of phloxine B after 30 s had a bell-shaped concentration-dependence with midpoints at 45 and 1600 nM for the stimulatory and the inhibitory limb, respectively; maximum stimulation was about 1.8 times. The slow inhibitory component, measured after 6 min, occurred with an IC(50) value of approximately 1 microM. In the absence of cPKA, phloxine B did not stimulate I(CFTR). In the presence of cPKA and MgATP, the effects of phloxine B were more prominent at low (0.02 mM) than at high ATP (2 mM). The data show that phloxine B modulates I(CFTR) by increasing channel activity and slowing channel deactivation; at high concentrations inhibition dominates. The effects may be mediated by direct interactions with CFTR from the inside of the cell.

Beta(3)-adrenergic stimulation and insulin inhibition of non-selective cation channels in white adipocytes of the rat.

Single-channel currents were recorded from the plasma membrane of white adipocytes of 6-8-week-old male Sprague-Dawley rats. In outside-out patches (high K(+), no Ca(2+) in pipette), a voltage-dependent K-channel (delayed rectifier) with a single-channel conductance (gamma) of 16 pS (24 degrees C) in modified Ringer's was active at a density of 0.5/microm(2). It was blocked by TEA (IC(50)=1.5 mM). A Ca(2+)-activated non-selective cation channel (NSC-channel) appeared at a mean density of 1/microm(2) in inside-out patches ([Ca(2+)](i)=1.2 mM). gamma was 28 pS (24 degrees C). The NSC showed weak voltage dependence and was blocked by mefenamic acid and by internal ATP. In the cell-attached mode spontaneous activity could be blocked reversibly by 100 nM insulin. Noradrenaline (NA, 100 nM) induced a flickering activity of the NSC-channels. Isoproterenol (100 nM) caused activity of the NSC-channel as well. After 1 microM propranolol even 1 microM NA did not induce any activity. The alpha-antagonist phentolamine had no effect on isoproterenol- or on NA-induced currents. The beta(3)-agonists BRL 37344 and BRL 35135A induced activity of the NSC-channel at 100 nM as well. We conclude that white adipocytes express ion channels which are comparable to those in brown adipocytes and that beta-receptor activation opens NSC-channels thus allowing for Na(+) entry into white adipocytes.

Potent inhibition of the CFTR chloride channel by suramin.

After phosphorylation by protein kinase A and in the presence of ATP, the cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel. In this study we have examined the effects of suramin on the CFTR Cl- current (I(CFTR)) in excised inside-out macropatches from Xenopus oocytes expressing human CFTR; glibenclamide, the standard inhibitor of I(CFTR), and some congeners were tested in comparison. I(CFTR) was activated by addition of the catalytic subunit of protein kinase A and MgATP to the bath. Suramin inhibited I(CFTR) with an IC50 value of 1 microM and a Hill coefficient close to 1; the inhibition showed little voltage dependence and was easily reversed upon washout of the drug. In comparison, glibenclamide inhibited I(CFTR) with an IC50 value of approximately 20 microM. When tested against I(CFTR) in whole oocytes, bath application of suramin was ineffective whereas glibenclamide was about four times weaker than in the inside-out patch configuration. The data show that suramin is the most potent inhibitor of CFTR yet described and suggest that the compound approaches its site of action from the cytosol.

Coexpression with the inward rectifier K(+) channel Kir6.1 increases the affinity of the vascular sulfonylurea receptor SUR2B for glibenclamide.

ATP-sensitive K(+) channels are closed by the hypoglycemic sulfonylureas like glibenclamide (GBC) and activated by a class of vasorelaxant compounds, the K(+) channel openers. These channels are octamers of Kir6.x and sulfonylurea receptor (SUR) subunits with 4:4 stoichiometry. The properties of the opener-sensitive K(+) channel in the vasculature are well matched by the SUR2B/Kir6.1 channel; however, the GBC sensitivity of the recombinant channel is unknown. In binding experiments we have determined the affinity of GBC for SUR2B and the SUR2B/Kir6.1 channel and compared the results with the channel blocking potency of GBC. All experiments were performed in whole transfected human embryonic kidney cells at 37 degrees C. The equilibrium dissociation constants (K(D)) of GBC binding to SUR2B and to the SUR2B/Kir6.1 complex were determined to be 32 and 6 nM, respectively; the K(D) value of the opener P1075 (N-cyano-N'-(1, 1-dimethylpropyl)-N"-3-pyridylguanidine) ( approximately 5 nM) was, however, not affected by cotransfection. In whole cell voltage-clamp experiments, GBC inhibited the SUR2B/Kir6.1 channel with IC(50) approximately 43 nM. The data show that, in the intact cell: 1) SUR2B, previously considered to be a low-affinity SUR, has a rather high affinity for GBC; 2) coexpression with the inward rectifier Kir6.1 increases the affinity of SUR2B for GBC; 3) the recombinant channel exhibits the same GBC affinity as the opener-sensitive K(+) channel in vascular tissue; and 4) the K(D) value of GBC binding to the octameric channel is 7 times lower than the IC(50) value for channel inhibition. The latter finding suggests that occupation of all four GBC sites per channel is required for channel closure.

Pharmacological evidence for a KATP channel in renin-secreting cells from rat kidney.

1. Openers of the ATP-sensitive potassium channel (KATP channel) increase and blockers decrease renin secretion. Here we report the effects of levcromakalim (LCRK, a channel opener) and glibenclamide (GBC, a blocker) on membrane potential, whole-cell current and the cytoplasmic Ca2+ concentration of renin-secreting cells (RSC). Studies were performed on afferent arterioles from the kidney of Na+-depleted rats. 2. As monitored with the fluorescent oxonol dye DiBAC4(3), LCRK (0.3 and 1 microM) induced a hyperpolarization of approximately 15 mV which was abolished by GBC (1 microM). 3. Whole-cell current-clamp experiments showed that RSC had a membrane potential of -61 +/- 1 mV (n = 16). LCRK (1 microM) induced a hyperpolarization of 9.9 +/- 0.2 mV (n = 16) which, in the majority of cells, decreased slowly with time. 4. Capacitance measurements showed a strong electrical coupling of the cells in the preparation. 5. At -60 mV, LCRK induced a hyperpolarizing current in a concentration-dependent manner with an EC50 of 152 +/- 31 nM and a maximum current of about 200 pA. 6. Application of GBC (1 microM) produced no effect; however, when applied after LCRK (300 nM), GBC inhibited the opener-induced hyperpolarizing current with an IC50 of 103 +/- 36 nM. 7. LCRK (0.3 and 1 microM) did not significantly affect the cytoplasmic Ca2+ concentration either at rest or after stimulation by angiotensin II. 8. The data show that LCRK induces a GBC-sensitive hyperpolarizing current in rat RSC. This current presumably originates from the activation of KATP channels which pharmacologically resemble those in vascular smooth muscle cells. The stimulatory effect of KATP channel opening on renin secretion is not mediated by a decrease in intracellular Ca2+ concentration.

Binding and effects of KATP channel openers in the vascular smooth muscle cell line, A10.

1. The ATP-sensitive K+ channel (KATP channel) in A10 cells, a cell line derived from rat thoracic aorta, was characterized by binding studies with the tritiated KATP channel opener, [3H]-P1075, and by electrophysiological techniques. 2. Saturation binding experiments gave a KD value of 9.2 +/- 5.2 nM and a binding capacity (BMax) of 140 +/- 40 fmol mg-1 protein for [3H]-P1075 binding to A10 cells; from the BMax value a density of binding sites of 5-10 per microns2 plasmalemma was estimated. 3. KATP channel modulators such as the openers P1075, pinacidil, levcromakalim and minoxidil sulphate and the blocker glibenclamide inhibited [3H]-P1075 binding. The extent of inhibition at saturation depended on the compound, levcromakalim inhibiting specific [3H]-P1075 binding by 85%, minoxidil sulphate and glibenclamide by 70%. The inhibition constants were similar to those determined in strips of rat aorta. 4. Resting membrane potential, recorded with microelectrodes, was -51 +/- 1 mV. P1075 and levcromakalim produced a concentration-dependent hyperpolarization by up to -25 mV with EC50 values of 170 +/- 40 nM and 870 +/- 190 nM, respectively. The hyperpolarization induced by levcromakalim (3 microM) was completely reversed by glibenclamide with an IC50 value of 86 +/- 17 nM. 5. Voltage clamp experiments were performed in the whole cell configuration under a physiological K+ gradient. Levcromakalim (10 microM) induced a current which reversed around -80 mV; the current-voltage relationship showed considerable outward rectification. Glibenclamide (3 microM) abolished the effect of levcromakalim. 6. Analysis of the noise of the levcromakalim (10 microM)-induced current at -40 and -20 mV yielded estimates of the channel density, the single channel conductance and the probability of the channel to be open of 0.14 micron-2, 8.8 pS and 0.39, respectively. 7. The experiments showed that A10 cells are endowed with functional KATP channels which resemble those in vascular tissue; hence, these cells provide an easily accessible source of channels for biochemical and pharmacological studies. The density of binding sites for [3H]-P1075 was estimated to be one order of magnitude higher than the density of functional KATP channels; assuming a plasmalemmal localization of the binding sites this suggests a large receptor reserve for the openers in A10 cells.

Effects of the Na+/H+-exchange inhibitor Hoe 642 on intracellular pH, calcium and sodium in isolated rat ventricular myocytes.

The inhibitors of the Na+/H+-exchange (NHE1) system Hoe 694 and Hoe 642 possess cardioprotective effects in ischaemia/reperfusion. It is assumed that these effects are due to the prevention of intracellular sodium (Nai) and calcium (Cai) overload. The purpose of the present study was to investigate the effects of Hoe 642 on intracellular pH, Na+ and Ca2+ (pHi, Nai and Cai) in isolated rat ventricular myocytes under anoxic conditions or in cells in which oxidative phosphorylation had been inhibited by 1.5 mmol/l cyanide. In cells which were dually loaded with the fluorescent dyes 2, 7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and Fura-2, anoxia caused acidification of the cells (from pHi 7.2 to pHi 6.8) and an increase in Cai from about 50 nmol/l to about 1 micromol/l. The decrease in pHi began before the cells underwent hypoxic (rigor) contracture, whereas Cai only began to rise after rigor shortening had taken place. After reoxygenation, pHi returned to its control value and Cai oscillated and then declined to resting levels. It was during this phase that the cells rounded up (hypercontracture). When 10 micromol/l Hoe 642 was present from the beginning of the experiment, pHi and Cai were not significantly different from control experiments. At reoxygenation, pHi did not recover, but Cai oscillated and returned to its resting level. To monitor Nai, the cells were loaded with the dye SBFI. After adding 1.5 mmol/l cyanide or 100 micromol/l ouabain, Nai increased from the initial 8 mmol/l to approximately 16 mmol/l. Hoe 642 or Hoe 694 (10 micromol/l) did not prevent the increase in Nai. In contrast, the blocker of the persistent Na+ current R56865 (10 micromol/l) attenuated the CN--induced rise in Nai. The substance ethylisopropylamiloride was not used because it augmented considerably the intensity of the 380 nm wavelength of the cell's autofluorescence. In conclusion, the specific NHE1 inhibitor Hoe 642 did not attenuate anoxia-induced Cai overload, nor CN--induced Nai and Cai overload. Hoe 642 prevented the recovery of pHi from anoxic acidification. This low pHi maintained after reoxygenation may be cardioprotective. Other possible mechanisms of NHE1 inhibitors, such as prevention of Ca2+ overload in mitochondria, cannot be ruled out. The increase in Nai during anoxia is possibly due to an influx of Na+ via persistent Na+ channels.

Simultaneous recording of ATP-sensitive K+ current and intracellular Ca2+ in anoxic rat ventricular myocytes. Effects of glibenclamide.

We investigated the temporal relationship between the adenosine triphosphate-sensitive K current (KATP current), hypoxic shortening and Ca accumulation in cardiomyocytes exposed to anoxia or metabolic inhibition. Whole-cell, patch-clamp experiments were performed with nonstimulated isolated rat heart ventricular muscle cells loaded with the Ca-sensitive fluorescent dye 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'- amino-5'-methylphenoxy) ethane-N,N,N',N'-tetraacetic acid (fura-2) via the patch pipette. After approximately 8 min anoxia, the KATP current started to rise and reached a maximum of 21.3 +/- 3.7 nA (n = 5, recorded at 0 mV clamp potential) within 1-3 min. At that time hypoxic contracture also occurred. Resting cytoplasmic free calcium (Cai) did not change significantly before hypoxic shortening. After hypoxic contracture, the KATP current decreased and Cai started to rise, reaching about 1 micromol/l. The presence of glibenclamide (10 micromol/l) in the bath reduced the anoxia-induced KATP current by more than 50%, but did not significantly influence the time dependence of current, hypoxic shortening and Cai, or the magnitude of Cai. Metabolic inhibition with 1.5 mmol/l CN resulted in KATP current increase and hypoxic shortening, occurring somewhat earlier than under anoxia, but all other parameters were comparable. In non-patch-clamped cells loaded with fura-2 AM ester and field-stimulated with 1 Hz, 1 micronol/l glibenclamide had no significant effect on the magnitude of the Cai increase caused by exposure of the cells to 1.5 mmol/l CN-. After CN- wash-out in non-patch-clamped cells, Cai declined, oscillated and finally returned to control values. It can be concluded that glibenclamide inhibits anoxia-induced KATP currents only partially and has no significant effect on anoxia-induced rise in resting Cai.

Kinetic parameters of the ionic currents in myelinated axons: characterization of temperature effects in a hibernator and a nonhibernator.

Na+ and K+ currents were measured by the patch-clamp method in the paranodal region of single sciatic nerve fibres of rats and of warm-adapted and cold-adapted golden hamsters. Kinetic parameters and temperature dependence of the Na+ currents were determined. The time constant for activation (about 0.2 ms for rats and hamsters) as well as the time constant for inactivation (about 1.6 ms for rats and hamsters) at 15 degrees C and at -35 mV compared well with single fibre voltage-clamp data from the rat. Differences amongst the three groups of animals were not significant. The temperature coefficient, Q10, for the activation and the inactivation time constant as well as for the time-to-peak of the Na+ current ranged between 2.3 and 3.1. No data have previously been published on the temperature dependence of the delayed-rectifier K channels of mammalian nerve fibres. Most of the K+ current was carried by intermediate (KI) and fast (KF) K channels. Dendrotoxin block indicated that "approximate"55% of the K+ current was due to KI channels, with no significant difference amongst the three groups of animals tested. The Arrhenius plot of the time constant of K+ current activation, "tau"n, yielded a mean Q10 of 3.3 at -40 mV (4. 0 at + 60 mV). No significant differences of the channel kinetics between rats, warm-adapted hamsters and cold-adapted hamsters were detected. We observed, however, a significant decrease of the Na channel density in the paranodal region of cold-adapted hamsters.

A voltage-dependent and a voltage-independent potassium channel in brown adipocytes of the rat.

Single-channel recordings of a voltage-dependent potassium channel in brown adipocytes of the rat confirm recordings of macroscopic currents. Single-channel conductance (gamma) is 8 pS at 20 degrees C in KF solution inside vs. a modified Ringer's solution outside. With KCl solution outside, gamma is 17 pS for outward currents and 21 pS for inward currents. The majority of the channels inactivate with a time constant around 200 ms; deactivation occurs within milliseconds. The channel is blocked by tetraethylammonium (TEA) with an inhibiting constant of 1.8 mM. The type of block is fast. Selectivity sequence for monovalent cations is K+ > Rb+ >> NH4+ >> Li+ > or = Na+ approximately Cs+. Cs+ at the outside causes a voltage-dependent block of inward currents. This channel is remarkably similar to the delayed rectifier of the F-type in the node of Ranvier. Occasionally, an additional K+ channel was found. This channel is voltage-insensitive, not blocked by 10 mM TEA, and has not been recorded in brown adipocytes before. Physiological relevance of this channel could be the steady-state membrane potential.

Changes of cytoplasmic free Ca(2+) in the green alga Mougeotia scalaris as monitored with indo-1, and their effect on the velocity of chloroplast movements.

The fluorescent calcium-sensitive dye 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2'-amino-5'-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid (indo-1) was loaded by a transplasmalemma pH gradient into filamentous cells and protoplasts of Mougeotia scalaris, such that most of the indo-1 fluorescence originated from the cytoplasm. Incubation of M. scalaris filaments in ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-buffered media (-log [Ca(2+)] (=pCa) 8 versus pCa 3) caused a consistent and significant decrease in the cytoplasmic free [Ca(2+)]. Pulses of the fluorescence excitation light (UV-A 365 nm, 0.7 s) caused an increase in cytoplasmic free [Ca(2+)] in M. scalaris that was nearly independent of the external [Ca(2+)] and of chloroplast dislocation by centrifugation. This calcium flux, highest in UV-A light, compared with blue or red light, probably resulted from a release of Ca(2+) from intracellular stores. Increased cytoplasmic [Ca(2+)] may affect the velocity of chloroplast rotation since UV-A-light-mediated chloroplast movement was faster than in blue or red light. Consistently, the calcium ionophore A23187 and the calcium-channel agonist Bay-K8644 both increased the velocity of the red-light-mediated chloroplast rotation. Based on these and other observations, a Ca(2+)-induced decrease in cytoplasmic viscosity in Mougeotia is presumed to occur.