Synthesis and structure-activity relationships of cetiedil analogues as blockers of the Ca(2+)-activated K+ permeability of erythrocytes.

Cetiedil, [2-cyclohexyl-2-(3-thienyl)ethanoic acid 2-(hexahydro-1H-azepin-1-yl)ethyl ester], which blocks the intermediate calcium-activated potassium ion permeability (IK(Ca)) in red blood cells, was used as a lead for investigating structure-activity relationships with the aim of determining the pharmacophore and of synthesizing agents of greater potency. A series of compounds having structures related to cetiedil was made and tested on rabbit erythrocytes. Channel blocking activity within the series was found to correlate well with octanol-water partition coefficients but not with the specific chemical structure of the acid moiety. However, whereas log P for the compounds spans a range of values over 4 orders of magnitude, potency only increases by 2 orders. This suggests that hydrophobic interactions with an active site on the channel are probably not the main determinants of activity. It seems more likely that increased lipophilicity enhances access to the channel, probably from within the cell membrane. In keeping with this interpretation, cetiedil methoiodide was found to be inactive. Triphenylethanoic was found to be a more effective acid grouping than 2-cyclohexyl-2-(3-thienyl)ethanoic, and its 2-(hexahydro-1H-azepin-l-yl)ethyl ester (11) was approximately 3 times more potent than cetiedil. The 9-benzylfluoren-9-yl carboxylic acid ester (21) was found to be approximately 9 times more active than cetiedil, and replacing -CO(2)- in 21 by an ethynyl (-C identical to C-) linkage (compound 26, UCL 1608) increased potency by some 15-fold over that of cetiedil.

Clotrimazole analogues: effective blockers of the slow afterhyperpolarization in cultured rat hippocampal pyramidal neurones.

1. The pharmacology of the slow afterhyperpolarization (sAHP) was studied in cultured rat hippocampal pyramidal neurones. 2. Clotrimazole, its in vivo metabolite, 2-chlorophenyl-bisphenyl-methanol (CBM) and the novel analogues, UCL 1880 and UCL 2027, inhibited the sI(AHP) with similar IC50s (1-2 microM). 3. Clotrimazole and CBM also inhibited the high voltage-activated (HVA) Ca2+ current in pyramidal neurones with IC50s of 4.7 microM and 2.2 microM respectively. UCL 1880 was a less effective Ca2+ channel blocker, reducing the HVA Ca2+ current by 50% at 10 microM. At concentrations up to 10 microM, UCL 2027 had no effect on the Ca2+ current, indicating that its effects on the sI(AHP) were independent of Ca2+ channel block. 4. Clotrimazole also inhibited both the outward holding current (IC50=2.8 microM) present at a potential of -50 mV and the apamin-sensitive medium AHP (mAHP; IC50 approximately amp;10 microM). The other clotrimazole analogues tested had smaller effects on these two currents. The present work also shows that 100 nM UCL 1848, an inhibitor of apamin-sensitive conductances, abolishes the mAHP. 5. Currents were recorded from HEK293 cells transfected with hSK1 and rSK2. The SK currents were very sensitive to inhibition by UCL 1848 but were not significantly reduced by the sI(AHP) inhibitor, UCL 2027 (10 microM). 10 microM UCL 1880 reduced the hSK1 current by 40%. 6. UCL 2027 appears to be the first relatively selective blocker of the sAHP to be described. Furthermore, the ability of UCL 2027 to block the sAHP with minimal effect on SK1 channel activity questions the role of this channel in the sAHP.

Ca(2+) channels involved in the generation of the slow afterhyperpolarization in cultured rat hippocampal pyramidal neurons.

The advantages of using isolated cells have led us to develop short-term cultures of hippocampal pyramidal cells, which retain many of the properties of cells in acute preparations and in particular the ability to generate afterhyperpolarizations after a train of action potentials. Using perforated-patch recordings, both medium and slow afterhyperpolarization currents (mI(AHP) and sI(AHP), respectively) could be obtained from pyramidal cells that were cultured for 8-15 days. The sI(AHP) demonstrated the kinetics and pharmacologic characteristics reported for pyramidal cells in slices. In addition to confirming the insensitivity to 100 nM apamin and 1 mM TEA, we have shown that the sI(AHP) is also insensitive to 100 nM charybdotoxin but is inhibited by 100 microM D-tubocurarine. Concentrations of nifedipine (10 microM) and nimodipine (3 microM) that maximally inhibit L-type calcium channels reduced the sI(AHP) by 30 and 50%, respectively. However, higher concentrations of nimodipine (10 microM) abolished the sI(AHP), which can be partially explained by an effect on action potentials. Both nifedipine and nimodipine at maximal concentrations were found to reduce the HVA calcium current in freshly dissociated neurons to the same extent. The N-type calcium channel inhibitor, omega-conotoxin GVIA (100 nM), irreversibly inhibited the sI(AHP) by 37%. Together, omega-conotoxin (100 nM) and nifedipine (10 microM) inhibited the sI(AHP) by 70%. 10 microM ryanodine also reduced the sI(AHP) by 30%, suggesting a role for calcium-induced calcium release. It is concluded that activation of the sI(AHP) in cultured hippocampal pyramidal cells is mediated by a rise in intracellular calcium involving multiple pathways and not just entry via L-type calcium channels.

The pharmacology of hSK1 Ca2+-activated K+ channels expressed in mammalian cell lines.

The pharmacology of hSK1, a small conductance calcium-activated potassium channel, was studied in mammalian cell lines (HEK293 and COS-7). In these cell types, hSK1 forms an apamin-sensitive channel with an IC(50) for apamin of 8 nM in HEK293 cells and 12 nM in COS-7 cells. The currents in HEK293 cells were also sensitive to tubocurarine (IC(50)=23 microM), dequalinium (IC(50)=0.4 microM), and the novel dequalinium analogue, UCL1848 (IC(50)=1 nM). These results are very different from the pharmacology of hSK1 channels expressed in Xenopus oocytes and suggest the properties of the channel may depend on the expression system. Our findings also raise questions about the role of SK1 channels in generating the apamin-insensitive slow afterhyperpolarization observed in central neurones.

Dequalinium: a potent inhibitor of apamin-sensitive K+ channels in hepatocytes and of nicotinic responses in skeletal muscle.

The bisquaternary compound dequalinium has been tested for its ability to inhibit the loss of K+ which angiotensin II causes in guinea-pig hepatocytes and which occurs through apamin-sensitive Ca(2+)-activated K+ (SKCa) channels. Dequalinium blocked angiotensin II-evoked K+ loss with an IC50 of 1.5 +/- 0.1 microM and also inhibited 125I-monoiodoapamin binding with a KI of 1.1 +/- 0.1 microM. It is the most active non-peptide SKCa blocker so far described. The neuromuscular blocking agent vecuronium was also tested, and proved to be considerably less effective (IC50, 4.5 +/- 0.3 microM; KI, 3.6 +/- 0.5 microM). Dequalinium was also examined for its actions at nicotinic receptors in skeletal muscle and was found to be a potent, non-competitive antagonist of carbachol contractions of the frog rectus abdominis. In the frog cutaneous pectoris muscle, end-plate depolarizations induced by carbachol became smaller and more transient in the presence of dequalinium at 10 nM. However, contractions of the frog sartorius and rat diaphragm in response to nerve stimulation were inhibited only by concentrations > 1 microM. These apparently discrepant effects of dequalinium on nicotinic responses could be explained either by open channel block of slow onset or by 'stabilization' of the desensitized state of the receptor. The potency of dequalinium will make it a useful agent for the study of nicotinic receptors as well as of SKCa channels.

Effects of cromakalim on the membrane potassium permeability of frog skeletal muscle in vitro.

1. The effects of the potassium channel opener, cromakalim, and its active enantiomer, lemakalim, have been investigated in frog skeletal muscle. 2. Cromakalim (30-300 microM) increased 86Rb efflux from muscles loaded with the isotope, hyperpolarized the fibres and reduced membrane resistance. 3. These effects were inhibited by the sulphonylureas, glibenclamide and tolbutamide. The IC50 for glibenclamide inhibition of 86Rb efflux was ca. 8 nM. 4. Phentolamine (300 microM) (which blocks responses to cromakalim in smooth muscle and inhibits ATP-sensitive K+ channels in pancreatic beta-cells) had no effect on the reduction in membrane resistance caused by 100 microM lemakalim. 5. Diazoxide (600 microM) had no effect on 86Rb efflux. 6. The similarities of the K+ channel activated by cromakalim in frog skeletal muscle to the channel acted on in smooth muscle and to the ATP-sensitive K+ channel of beta-cells are discussed.

Toxins in the characterization of potassium channels.

Several recently characterized toxins (apamin, charybdotoxin, dendrotoxin and noxiustoxin) are proving invaluable for establishing what kinds of potassium channel are expressed in neurones, and what the roles of the channels might be.

Effect of channel blockers on potassium efflux from metabolically exhausted frog skeletal muscle.

1. 86Rb and 42K have been used to assess potassium exchange in frog skeletal muscle which had been metabolically exhausted by electrical stimulation (1 Hz) after treatment with 2 mM-cyanide and 1 mM-iodoacetate. These conditions led to the development of rigor. 2. Poisoning by itself induced a small but variable increase in tracer efflux. Complete mechanical exhaustion subsequent to electrical stimulation was, however, accompanied by a 5-6 fold increase in the rate coefficient for both 86Rb and 42K efflux. In the case of rubidium this was maintained for at least 20 min and often for up to 1 h. 3. The increase in tracer efflux induced by metabolic exhaustion was inhibited by barium (0.03-5 mM) in a reversible and concentration-dependent manner. Inhibition was also observed with glibenclamide (3-100 microM), tolbutamide (0.3-2 mM), TEA (5-100 mM) and the local anaesthetics lignocaine (1-3 mM) and tetracaine (1 mM). Quinine produced a dual response consisting of an inhibitory component which was most clearly seen at low concentrations (0.3 mM) and an enhancement of tracer efflux that became increasingly dominant at higher concentrations (1-10 mM). 4. Both apamin (30 and 100 nM) and Israeli scorpion (Leiurus quinquestriatus) venom (16 micrograms ml-1) produced little or no block of the tracer efflux activated by metabolic exhaustion. Similarly 4-aminopyridine (3 mM) and decamethonium (0.3 mM) were without obvious effect. 5. It is concluded that metabolic exhaustion of frog skeletal muscle leads to an increased permeability to both 42K and 86Rb. Our results with channel blockers suggest that this K+ permeability can be attributed neither to the delayed rectifier nor to an apamin- or charybdotoxin-sensitive calcium-activated K+ permeability (PK(Ca) but may be predominantly due to activation of ATP-sensitive channels similar to those found in the beta-cells of pancreatic islets.

Neuromuscular blocking agents inhibit receptor-mediated increases in the potassium permeability of intestinal smooth muscle.

The neuromuscular blocking agents tubocurarine, atracurium and pancuronium have been tested for their ability to inhibit receptor-mediated increases in the K+ permeability of intestinal smooth muscle. All three agents, as well as the bee venom peptide apamin, reduced both the resting efflux of 86Rb and the increase in efflux caused by the application of either bradykinin (1 microM) or an alpha 1-adrenoceptor agonist, amidephrine (20 microM), to depolarized strips of guinea-pig taenia caeci. This suggested that like apamin, the neuromuscular blocking agents inhibit the Ca2+-dependent K+ permeability (PK(Ca] mechanism which in this tissue is activated by a variety of membrane receptors. The concentrations (IC50S) of atracurium, pancuronium and (+)-tubocurarine which reduced the effect of amidephrine on 86Rb efflux by 50% were 12, 37 and 67 microM respectively. Also in keeping with an ability to block PK(Ca), the neuromuscular blockers and apamin reduced the inhibition by amidephrine and bradykinin of physalaemin-mediated contractions of the taenia caeci. The IC50 values were 15, 31 and 120 microM for atracurium, tubocurarine and pancuronium respectively, and 2.3 nM for apamin. Each of the neuromuscular blockers, and apamin, increased the spontaneous contractions of the rabbit duodenum and blocked the inhibitory effect of amidephrine thereon. It is concluded that the PK(Ca) mechanism in the longitudinal smooth muscle of the intestine It is concluded that the PK(Ca) mechanism in the longitudinal smooth muscle of the intestine resembles that of hepatocytes and sympathetic ganglion cells in its susceptibility to inhibition by neuromuscular blocking agents, as well as by apamin.

Effects of apamin, quinine and neuromuscular blockers on calcium-activated potassium channels in guinea-pig hepatocytes.

The bee venom peptide, apamin, has been radiolabelled with 125I, the monoiodinated derivative purified, and its binding to intact guinea-pig liver cells studied. At 37 degrees C 125I-monoiodoapamin associated with, and dissociated from, guinea-pig hepatocytes remarkably rapidly. The association and dissociation rate constants were 1.4 X 10(8) M-1 s-1 and 0.035 s-1 respectively. Equilibrium binding studies demonstrated a saturable binding component compatible with 1:1 binding to a single class of site and having an equilibrium dissociation constant (KL) of 390 pM. The maximal binding capacity was 1.1 fmol mg-1 dry wt. of tissue. Unlabelled apamin displaced bound 125I-monoiodoapamin with a KI of 380 pM, which is consistent with the concentration of apamin required to inhibit Ca2+-activated K+ permeability (PK(Ca) ) in these cells. Inhibitable binding of 125I-monoiodoapamin to rat hepatocytes was much less than to guinea-pig hepatocytes and could not be reliably quantified. Neither was there any discernible inhibitable binding to human erythrocytes. This is in keeping with the reported lack of apamin-sensitive Ca2+-activated K+ channels in these cell types. Various agents were tested for their ability to inhibit monoiodoapamin binding to, and Ca2+-mediated K+ efflux from, guinea-pig hepatocytes. All compounds tested which inhibited binding also blocked K+ efflux at similar concentrations. TEA and quinine affected hepatocytes only at high concentration (KI = 5.8 and 0.51 mM respectively). 9-aminoacridine, quinacrine and chloroquine were slightly more effective (KI = 70-180 microM). By far the most active compounds (apart from apamin) were the neuromuscular blocking agents; tubocurarine, pancuronium and atracurium (KI = 7.5, 6.8 and 4.5 microM respectively). Gallamine was slightly less effective (KI = 14 microM) and decamethonium and hexamethonium much less so (KI = 620 and 760 microM respectively). 3,4-diaminopyridine, alpha-bungarotoxin and tetrodotoxin were among several compounds which showed little or no affinity for apamin binding sites or inhibition of K+ efflux in guinea-pig hepatocytes. The saturable binding of 125I-monoiodoapamin to guinea-pig hepatocytes corresponds to about 1700 sites per cell. Assuming, tentatively, that binding sites correspond to channels the rate of K+ loss observed following agonist action can readily be explained if these channels have unitary conductances in the range reported for PK(Ca) in other tissues.

Calcium-activated potassium channels in liver cells.

Activation of certain membrane receptors increases the concentration of Ca2+ in the cytosol of hepatocytes. Since in most species these cells possess a PK(Ca) mechanism, the outcome is a rise in PK. This can be blocked by quinine, apamin and certain neuromuscular blocking agents. The binding of labelled apamin to hepatocytes has been studied under physiological conditions, and the relationship between the binding sites and K+ channels is discussed. The physiological role of the PK(Ca) mechanism in hepatocytes is unclear, though it is largely responsible for 'adrenaline hyperkalaemia'.

High affinity binding of [125I]monoiodoapamin to isolated guinea-pig hepatocytes.

The bee venom neurotoxin apamin has been labelled with 125I and its binding to isolated guinea-pig hepatocytes measured under physiological conditions. A single saturable component of [125I]monoiodoapamin binding with a Kd of 350 pM and Bmax of 0.99 fmol/mg dry wt was identified. Native apamin displaced labelled apamin with a Kd of 376 pM which is broadly in keeping with the concentrations found to inhibit K loss from guinea-pig hepatocytes. These observations, together with the binding found in other tissues, suggest that specific binding of labelled apamin is particularly associated with apamin-sensitive, Ca-activated K-channels.

Effects of sympathomimetic amines on 45Ca efflux from liver slices.

The efflux of 45Ca from slices of guinea-pig and rabbit liver is greatly increased by alpha-adrenoceptor agonists. Isoprenaline is much less effective. The effects of these agents on the efflux of 45Ca mirror their actions on 42K loss and suggest that the two may be related. Glucose release from both rabbit and guinea-pig liver slices is increased to a similar extent by either alpha- or beta-receptor agonists. The possible relationship between Ca and K movements and the production of glucose is discussed.

The receptors concerned in the actions of catecholamines on glucose release, membrane potential and ion movements in guinea-pig liver.

1. Experiments have been made to identify the kinds of receptors concerned in the actions of catecholamines in increasing potassium efflux, membrane potential and glucose release in tissue slices prepared from guinea-pig liver.2. Glucose release could be accelerated by activation of either of two distinct receptors, one of which resembles the alpha receptor of Ahlquist's classification, the other being beta-like. Thus both amidephrine and isoprenaline (sympathomimetic amines which selectively activate alpha and beta receptors respectively) elicited the response.3. The effect of isoprenaline on glucose release was inhibited by the beta blocking agent propranolol (1 muM) but not by the alpha blocker phentolamine (10 muM), whereas the converse held for amidephrine. However, the degree of antagonism observed with phentolamine was less than found in smooth muscle, suggesting that the alpha-like receptor in the liver may differ somewhat from that in other tissues, or be less accessible to the antagonist.4. Amidephrine, like noradrenaline, increased the efflux of (42)K from the slices and caused hyperpolarization, suggesting that the increase in potassium permeability underlying these responses is alpha-mediated. This conclusion was supported by the finding that the effect of amidephrine on membrane potential was inhibited by phentolamine but not propranolol (1 muM).5. In keeping with this interpretation, low concentrations (20-50 nM) of isoprenaline, although sufficient to activate the beta receptors (as judged by the effect on glucose release) had little effect on the ionic composition of the tissue, and caused only a small increase in (42)K efflux. Thus the ;ion movement' and ;glucose' responses could be largely dissociated.6. Larger concentrations (1-6 muM) of isoprenaline increased (42)K efflux to a greater extent, although the effect was always less than with the same concentration of noradrenaline.7. Activation of the beta-receptors caused a small and inconsistent hyperpolarization which became more pronounced and also more reproducible on replacing the chloride content of the bathing fluid by the larger anion isethionate.8. Although these results show that activation of either of two distinct adrenergic receptors in guinea-pig liver can cause the same end-response (e.g. an increase in glucose release, or, under some circumstances, hyperpolarization of the cell membrane), the underlying mechanisms cannot be assumed to be identical. This is discussed.

Effects of noradrenaline on potassium reflux, membrane potential and electrolyte levels in tissue slices prepared from guinea-pig liver.

1. Some effects of noradrenaline on potassium efflux, electrolyte levels, membrane potential and current distribution in guinea-pig liver slices have been examined.2. The slices (thickness ca. 300 mum) were prepared from the median lobe of the liver and incubated at 38 degrees C in a mammalian Ringer fluid containing 2 mM pyruvate. After an initial recovery period, the ionic composition of the tissue remained stable for several hours.3. The steady-state contents of sodium, potassium and chloride were 296, 266 and 272 m-equiv/kg dry tissue respectively. The inulin space was 29 ml./100 g wet tissue.4. Most if not all of the tissue potassium was exchangeable. The rate constant for (42)K efflux was 0.019 min(-1).5. Noradrenaline (1 muM) markedly increased the efflux of (42)K and within 2 min caused tissue potassium to fall by 8%. At the same time the sodium content rose.6. Traverses of the slices with micro-electrodes showed many negative-going deflexions of 30-40 mV in amplitude. The evidence suggests that these correspond to the membrane potentials of the parenchymal cells.7. Noradrenaline (1 muM) caused a reversible hyperpolarization of about 10 mV. The response became larger on replacing external chloride by isethionate or methylsulphate, but was little affected by a reduction in external potassium.8. After slices had been bathed in potassium and chloride-free solutions for several min, restoration of external potassium caused the membrane potential to increase by up to 10 mV. This hyperpolarization, but not that caused by noradrenaline, was abolished by ouabain.9. Noradrenaline reduced the amplitude and quickened the time course of electrotonic potentials set up by current pulses from another microelectrode, suggesting that the membrane conductance had risen.10. Although certain mechanisms based on electrogenic active transport processes with unusual properties have not been excluded, the present findings are more simply explained by supposing that noradrenaline increases the potassium permeability of the parenchymal cell membrane.