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A H Weston - One of the best experts on this subject based on the ideXlab platform.
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characterization of a Charybdotoxin sensitive intermediate conductance ca2 activated k channel in porcine coronary endothelium relevance to edhf
2002Co-Authors: Rostislav Bychkov, A H Weston, M P Burnham, Gillian R Richards, Gillian Edwards, Michel Feletou, Paul M VanhoutteAbstract:This study characterizes the K+ channel(s) underlying Charybdotoxin-sensitive hyperpolarization of porcine coronary artery endothelium. Two forms of current-voltage (I/V) relationship were evident in whole-cell patch-clamp recordings of freshly-isolated endothelial cells. In both cell types, iberiotoxin (100 nM) inhibited a current active only at potentials over +50 mV. In the presence of iberiotoxin, Charybdotoxin (100 nM) produced a large inhibition in 38% of cells and altered the form of the I/V relationship. In the remaining cells, Charybdotoxin also inhibited a current but did not alter the form. Single-channel, outside-out patch recordings revealed a 17.1±0.4 pS conductance. Pipette solutions containing 100, 250 and 500 nM free Ca2+ demonstrated that the open probability was increased by Ca2+. This channel was blocked by Charybdotoxin but not by iberiotoxin or apamin. Hyperpolarizations of intact endothelium elicited by substance P (100 nM; 26.1±0.7 mV) were reduced by apamin (100 nM; 17.0±1.8 mV) whereas those to 1-ethyl-2-benzimidazolinone (1-EBIO, 600 μM, 21.0±0.3 mV) were unaffected (21.7±0.8 mV). Substance P, bradykinin (100 nM) and 1-EBIO evoked Charybdotoxin-sensitive, iberiotoxin-insensitive whole-cell perforated-patch currents. A porcine homologue of the intermediate-conductance Ca2+-activated K+ channel (IK1) was identified in endothelial cells. In conclusion, porcine coronary artery endothelial cells express an intermediate-conductance Ca2+-activated K+ channel and the IK1 gene product. This channel is opened by activation of the EDHF pathway and likely mediates the Charybdotoxin-sensitive component of the EDHF response. Keywords: Endothelium, EDHF, hyperpolarization, calcium-activated potassium channels, Charybdotoxin, iberiotoxin, apamin, IK1 gene product Introduction The vascular endothelium controls vessel tone by releasing nitric oxide (Furchgott & Zawadzki, 1980) and prostacyclin (Moncada & Vane, 1979) as well as by a third pathway which involves hyperpolarization of the vascular smooth muscle (reviewed by Busse et al., 2002). This ‘endothelium-dependent hyperpolarizing factor (EDHF)' pathway is inhibited by a combination of the toxins apamin and Charybdotoxin, but not apamin and iberiotoxin (Corriu et al., 1996; Zygmunt & Hogestatt, 1996; Petersson et al., 1997; Chataigneau et al., 1998; Edwards et al., 1998; 2000). Given the specificities of these toxins (reviewed by Garcia et al., 1991; Castle, 1999), small- and intermediate-conductance Ca2+-activated K+ channels (SKCa and IKCa, respectively) but not large-conductance Ca2+-activated K+ channels (BKCa) are implicated in the EDHF pathway. In the vasculature, SKCa and IKCa are expressed in endothelial cells (Sakai, 1990; Marchenko & Sage, 1996; Kohler et al., 2000; Burnham et al., 2002) but not in smooth muscle cells with the contractile phenotype while BKCa are mainly expressed in myocytes (Zygmunt et al., 1997; Neylon et al., 1999; Quignard et al., 2000). Furthermore, the combination of apamin and Charybdotoxin blocks EDHF-mediated vasodilatation if selectively applied to the endothelium and inhibits the hyperpolarization of the endothelial cells produced by acetylcholine or bradykinin (Edwards et al., 1998; 2000; Doughty et al., 1999; Ohashi et al., 1999). Finally, the increase in endothelial intracellular calcium concentration, provoked by the agonist, is not inhibited by the two toxins (Ghisdal & Morel, 2001). Altogether, these experimental results suggest that the hyperpolarization of the endothelial cells is the critical initiating step in the EDHF-mediated responses (Quignard et al., 2000; Edwards et al., 2000; Busse et al., 2002) and that apamin plus Charybdotoxin exert their effects at this site (Edwards et al., 1998). In an earlier study, in porcine coronary artery endothelial cells, an apamin-sensitive K+ channel which is likely to be involved in the EDHF response was proposed to be an SKCa containing the SK3 subunit (Burnham et al., 2002). The purpose of the present study was, in the same cells, to characterize the K+ channels sensitive to Charybdotoxin which are also involved in EDHF-mediated responses.
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further investigation of endothelium derived hyperpolarizing factor edhf in rat hepatic artery studies using 1 ebio and ouabain
1999Co-Authors: Gerald E Edwards, M J Gardener, Michel Feletou, Paul M Vanhoutte, G Brady, A H WestonAbstract:The characteristics of endothelium-dependent hyperpolarization in rat hepatic artery have been further investigated in the presence of inhibitors of cyclo-oxygenase and nitric oxide synthase. Using sharp micro-electrodes, the smooth muscle hyperpolarization induced by acetylcholine, KCl or 1-ethyl-2-benzimidazolinone (1-EBIO) in intact hepatic arteries was abolished by 30 μM barium plus 500 nM ouabain. In vessels without endothelium, the smooth muscle hyperpolarization induced by KCl was not reduced by 30 μM barium alone. However, in the presence of barium the effects of KCl were partially inhibited by 100 nM ouabain and essentially abolished by 500 nM ouabain. Using sharp micro-electrodes, the hyperpolarization of both the smooth muscle and the endothelium induced by 1-EBIO or by acetylcholine was unaffected by 100 nM iberiotoxin. However, in the presence of 100 nM Charybdotoxin, the effects of 1-EBIO were abolished whereas those of acetylcholine were only partially reduced. The hyperpolarization induced by levcromakalim was unaffected by either Charybdotoxin or iberiotoxin. Under whole-cell patch-clamp recording conditions, 1-EBIO induced a voltage-insensitive, Charybdotoxin-sensitive K+ current in cultured endothelial cells but was without effect on K+ currents in smooth muscle cells isolated from hepatic arteries. It is concluded that the endothelium-dependent hyperpolarization of smooth muscle induced by either acetylcholine or by 1-EBIO in rat hepatic artery is initially associated with the opening of endothelial calcium-sensitive K+-channels insensitive to iberiotoxin. The resulting accumulation of K+ in the myoendothelial space activates an isoform of Na+/K+-ATPase which is sensitive to low concentrations of ouabain. British Journal of Pharmacology (1999) 128, 1064–1070; doi:10.1038/sj.bjp.0702916
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k is an endothelium derived hyperpolarizing factor in rat arteries
1998Co-Authors: Gerald E Edwards, K A Dora, M J Gardener, C J Garland, A H WestonAbstract:In arteries, muscarinic agonists such as acetylcholine release an unidentified, endothelium-derived hyperpolarizing factor (EDHF) which is neither prostacyclin nor nitric oxide1,2,3. Here we show that EDHF-induced hyperpolarization of smooth muscle and relaxation of small resistance arteries are inhibited by ouabain plus Ba2+; ouabain is a blocker of Na+/K+ ATPase4 and Ba2+ blocks inwardly rectifying K+ channels5. Small increases in the amount of extracellular K+ mimic these effects of EDHF in a ouabain- and Ba2+-sensitive, but endothelium-independent, manner. Acetylcholine hyperpolarizes endothelial cells and increases the K+ concentration in the myoendothelial space; these effects are abolished by charbdotoxin plus apamin. Hyperpolarization of smooth muscle by EDHF is also abolished by this toxin combination, but these toxins do not affect the hyperpolarizaiton of smooth muscle by added K+. These data show that EDHF is K+ that effluxes through Charybdotoxin- and apamin-sensitive K+ channels on endothelial cells. The resulting increase in myoendothelial K+ concentration hyperpolarizes and relaxes adjacent smooth-muscle cells by activating Ba2+-sensitive K+ channels and Na+/K+ ATPase. These results show that fluctuations in K+ levels originating within the blood vessel itself are important in regulating mammalian blood pressure and flow.
Claudio Vita - One of the best experts on this subject based on the ideXlab platform.
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engineering a cd4 mimetic inhibiting the binding of the human immunodeficiency virus 1 hiv 1 envelope glycoprotein gp120 to human lymphocyte cd4 by the transfer of a cd4 functional site to a small natural scaffold
1998Co-Authors: Eugenia Drakopoulou, Jean Vizzavona, Claudio VitaAbstract:The solvent-exposed CDR2-like region of human CD4 was transferred to the structural scaffold of scorpion Charybdotoxin, as a means to reproduce that site in a native-like conformation. The chimeric mini-protein (33 amino acids long), obtained by solid-phase synthesis, is able to specifically prevent the interaction of HIV-1 gp120 with CD4. This CD4 mimetic may represent a valuable tool in the study of the HIV-cell interactions and as a lead in the development of antiviral drugs.
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consequence of the removal of evolutionary conserved disulfide bridges on the structure and function of Charybdotoxin and evidence that particular cysteine spacings govern specific disulfide bond formation
1998Co-Authors: Eugenia Drakopoulou, Francoise Bouet, Jean Vizzavona, Jacques Neyton, Vincent Aniort, Henri Virelizier, And Andre Menez, Claudio VitaAbstract:Scorpion toxins are miniglobular proteins containing a common structural motif formed by an α-helix on one face, an antiparallel β-sheet on the opposite face, and three disulfide bonds making up most of its internal volume. We have investigated the role of these evolutionary conserved bonds by replacing each couple of bridged cysteine residues of the scorpion Charybdotoxin by a pair of nonbridging l-α-aminobutyric acid (Aba) residues. Three analogues were obtained by solid-phase synthesis, Chab I, Chab II, and Chab III, containing the Aba residues in positions 7 and 28, 13 and 33, 17 and 35, respectively. Circular dichroism analysis showed that the purified Chab II acquired a conformation similar to that of Charybdotoxin, while the Chab I and Chab III possess decreased nativelike characteristics. All analogues block single high-conductance Ca2+-activated K+ channels from rat skeletal muscle inserted into planar lipid bilayers, but with different potencies. Chab II is the most active analogue (KD = 8.0 × 1...
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changing the structural context of a functional β hairpin synthesis and characterization of a chimera containing the curaremimetic loop of a snake toxin in the scorpion α β scaffold
1996Co-Authors: Eugenia Drakopoulou, Andre Menez, Bernard Gilquin, Sophie Zinnjustin, Marc Guenneugues, Claudio VitaAbstract:An approach to obtain new active proteins is the incorporation of all or a part of a well defined active site onto a natural structure acting as a structural scaffold. According to this strategy we tentatively engineered a new curaremimetic molecule by transferring the functional central loop of a snake toxin, sequence 26-37, sandwiched between two hairpins, onto the structurally similar β-hairpin of the scorpion toxin Charybdotoxin, stabilized by a short helix. The resulting chimeric molecule, only 31 amino acids long, was produced by solid phase synthesis, refolded, and purified to homogeneity. As shown by structural analysis performed by CD and NMR spectroscopy, the chimera maintained the expected α/β fold characteristic of scorpion toxins and presented a remarkable structural stability. The chimera competitively displaces the snake curaremimetic toxin α from the acetylcholine receptor at 10-5M concentrations. Antibodies, elicited in rabbits against the chimera, recognize the parent snake toxin and prevent its binding to the acetylcholine receptor, thus neutralizing its toxic function. All these data demonstrate that the strategy of active site transfer to the Charybdotoxin scaffold has general applications in the engineering of novel ligands for membrane receptors and in vaccine design.
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changing the structural context of a functional hairpin synthesis and characterization of a chimera containing the curaremimetic loop of a snake toxin in the scorpion α β scaffold
1996Co-Authors: Eugenia Drakopoulou, Andre Menez, Bernard Gilquin, Sophie Zinnjustin, Marc Guenneugues, Claudio VitaAbstract:Abstract An approach to obtain new active proteins is the incorporation of all or a part of a well defined active site onto a natural structure acting as a structural scaffold. According to this strategy we tentatively engineered a new curaremimetic molecule by transferring the functional central loop of a snake toxin, sequence 26-37, sandwiched between two hairpins, onto the structurally similar β-hairpin of the scorpion toxin Charybdotoxin, stabilized by a short helix. The resulting chimeric molecule, only 31 amino acids long, was produced by solid phase synthesis, refolded, and purified to homogeneity. As shown by structural analysis performed by CD and NMR spectroscopy, the chimera maintained the expected α/β fold characteristic of scorpion toxins and presented a remarkable structural stability. The chimera competitively displaces the snake curaremimetic toxin α from the acetylcholine receptor at 10M concentrations. Antibodies, elicited in rabbits against the chimera, recognize the parent snake toxin and prevent its binding to the acetylcholine receptor, thus neutralizing its toxic function. All these data demonstrate that the strategy of active site transfer to the Charybdotoxin scaffold has general applications in the engineering of novel ligands for membrane receptors and in vaccine design.
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synthesis of Charybdotoxin and of two n terminal truncated analogues structural and functional characterisation
1993Co-Authors: Claudio Vita, H Vatanpour, Alan L. Harvey, Francoise Bouet, Andre Menez, Francoise Bontems, Michel Tauc, Philippe Poujeol, Flavio TomaAbstract:Charybdotoxin and two N-terminal truncated peptides, corresponding to the 2-37 and 7-37 sequences, were obtained by stepwise solid-phase synthesis using N alpha-t-butyloxycarbonyl and benzyltype side-chain protection. While this strategy was generally useful, the S-acetamidomethyl protecting group used for the six cysteines was not completely stable under HF treatment and its subsequent removal by mercury(II) treatment was neither complete nor devoid of side reactions. The completely deprotected native and truncated sequences were folded efficiently in the presence of glutathione and were finally purified by high-pressure liquid chromatography with overall yields of 4.0-5.0%. Each protein was characterised chemically, structurally and functionally. 1H-NMR spectroscopy was used and a complete assignment of all the protons of the three synthetic proteins was achieved. NMR data show that synthetic Charybdotoxin is indistinguishable from the natural protein. The two truncated proteins contain the same elements of secondary structure and a similar overall three-dimensional structure, in agreement with circular dichroic measurements. The shortest analogue, however, may have local structural perturbations and/or higher flexibility. Biological activity on dog epithelial Ca(2+)-activated K+ channels and on rat brain synaptosomal voltage-dependent K+ channels show that synthetic Charybdotoxin was as potent as the natural toxin on both channels. For both channels, deletion of the first amino acid, 5-oxoproline (pyroglutamic acid) decreased only slightly the potency of the inhibitor, while deletion of the entire 1-6 segment reduced potency much more. We conclude that the N-terminal region of Charybdotoxin plays a functional role in tuning the toxin's biological activity but is not essential for the folding and stability of the structure. The structure of the shortest analogue represents an interesting example of how a well organised and stable alpha/beta fold can be engineered with only 31 amino acid residues.
Andre Menez - One of the best experts on this subject based on the ideXlab platform.
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changing the structural context of a functional β hairpin synthesis and characterization of a chimera containing the curaremimetic loop of a snake toxin in the scorpion α β scaffold
1996Co-Authors: Eugenia Drakopoulou, Andre Menez, Bernard Gilquin, Sophie Zinnjustin, Marc Guenneugues, Claudio VitaAbstract:An approach to obtain new active proteins is the incorporation of all or a part of a well defined active site onto a natural structure acting as a structural scaffold. According to this strategy we tentatively engineered a new curaremimetic molecule by transferring the functional central loop of a snake toxin, sequence 26-37, sandwiched between two hairpins, onto the structurally similar β-hairpin of the scorpion toxin Charybdotoxin, stabilized by a short helix. The resulting chimeric molecule, only 31 amino acids long, was produced by solid phase synthesis, refolded, and purified to homogeneity. As shown by structural analysis performed by CD and NMR spectroscopy, the chimera maintained the expected α/β fold characteristic of scorpion toxins and presented a remarkable structural stability. The chimera competitively displaces the snake curaremimetic toxin α from the acetylcholine receptor at 10-5M concentrations. Antibodies, elicited in rabbits against the chimera, recognize the parent snake toxin and prevent its binding to the acetylcholine receptor, thus neutralizing its toxic function. All these data demonstrate that the strategy of active site transfer to the Charybdotoxin scaffold has general applications in the engineering of novel ligands for membrane receptors and in vaccine design.
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changing the structural context of a functional hairpin synthesis and characterization of a chimera containing the curaremimetic loop of a snake toxin in the scorpion α β scaffold
1996Co-Authors: Eugenia Drakopoulou, Andre Menez, Bernard Gilquin, Sophie Zinnjustin, Marc Guenneugues, Claudio VitaAbstract:Abstract An approach to obtain new active proteins is the incorporation of all or a part of a well defined active site onto a natural structure acting as a structural scaffold. According to this strategy we tentatively engineered a new curaremimetic molecule by transferring the functional central loop of a snake toxin, sequence 26-37, sandwiched between two hairpins, onto the structurally similar β-hairpin of the scorpion toxin Charybdotoxin, stabilized by a short helix. The resulting chimeric molecule, only 31 amino acids long, was produced by solid phase synthesis, refolded, and purified to homogeneity. As shown by structural analysis performed by CD and NMR spectroscopy, the chimera maintained the expected α/β fold characteristic of scorpion toxins and presented a remarkable structural stability. The chimera competitively displaces the snake curaremimetic toxin α from the acetylcholine receptor at 10M concentrations. Antibodies, elicited in rabbits against the chimera, recognize the parent snake toxin and prevent its binding to the acetylcholine receptor, thus neutralizing its toxic function. All these data demonstrate that the strategy of active site transfer to the Charybdotoxin scaffold has general applications in the engineering of novel ligands for membrane receptors and in vaccine design.
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neuromuscular effects of some potassium channel blocking toxins from the venom of the scorpion leiurus quinquestriatus hebreus
1994Co-Authors: David L Marshall, Yvon Doljansky, H Vatanpour, Philippe Boyot, Suzanne Pinkasfeld, Alan L. Harvey, Francoise Bouet, Andre MenezAbstract:The scorpion venom Leiurus quinquestriatus hebreus was fractionated by chromatography in order to isolate toxins that affected binding of radiolabelled dendrotoxin to K+ channel proteins on synaptosomal membranes and that facilitated acetylcholine release in chick biventer cervicis nerve-muscle preparations. In addition to the previously characterized Charybdotoxin, three toxins were isolated: 14-2, 15-1 and 18-2. Toxin 14-2 has a blocked N-terminus and because of low quantities, it has not been sequenced; 15-1 is a newly sequenced toxin of 36 residues with some overall homology to Charybdotoxin and noxiustoxin; 18-2 is identical to Charybdotoxin-2. The apparent Ki against dendrotoxin binding were: Charybdotoxin, 3.8 nM; 14-2, 150 nM; 15-1, 50 nM; and 18-2, 0.25 nM. Toxin 14-2 (75 nM-1.5 microM) had a presynaptic facilitatory effect on neuromuscular preparations. Toxin 15-1 augmented responses to direct muscle stimulation, probably because it blocked Ca(2+)-activated K+ currents in muscle fibres. Toxin 18-2 (Charybdotoxin-2) had a potent presynaptic facilitatory action, with less effect on direct muscle stimulation. This contrasts with the relatively weak neuromuscular effects of the highly homologous Charybdotoxin. On a Ca(2+)-activated K+ current in mouse motor nerve endings, Charybdotoxin and toxin 18-2 produced maximal block at around 100 nM, whereas 15-1 was inactive at 300 nM. Charybdotoxin can increase quantal content, but this is more likely to result from block of voltage-dependent K+ channels than Ca(2+)-activated channels: the increase in transmitter release occurred in conditions in which little IKCa would be present; higher concentration of Charybdotoxin and longer exposure times were required to increase transmitter release than those needed to block IKCa, and the facilitatory effects of Charybdotoxin and toxin 18-2 correlated more with their effects on dendrotoxin binding than on block of IKCa.
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synthesis of Charybdotoxin and of two n terminal truncated analogues structural and functional characterisation
1993Co-Authors: Claudio Vita, H Vatanpour, Alan L. Harvey, Francoise Bouet, Andre Menez, Francoise Bontems, Michel Tauc, Philippe Poujeol, Flavio TomaAbstract:Charybdotoxin and two N-terminal truncated peptides, corresponding to the 2-37 and 7-37 sequences, were obtained by stepwise solid-phase synthesis using N alpha-t-butyloxycarbonyl and benzyltype side-chain protection. While this strategy was generally useful, the S-acetamidomethyl protecting group used for the six cysteines was not completely stable under HF treatment and its subsequent removal by mercury(II) treatment was neither complete nor devoid of side reactions. The completely deprotected native and truncated sequences were folded efficiently in the presence of glutathione and were finally purified by high-pressure liquid chromatography with overall yields of 4.0-5.0%. Each protein was characterised chemically, structurally and functionally. 1H-NMR spectroscopy was used and a complete assignment of all the protons of the three synthetic proteins was achieved. NMR data show that synthetic Charybdotoxin is indistinguishable from the natural protein. The two truncated proteins contain the same elements of secondary structure and a similar overall three-dimensional structure, in agreement with circular dichroic measurements. The shortest analogue, however, may have local structural perturbations and/or higher flexibility. Biological activity on dog epithelial Ca(2+)-activated K+ channels and on rat brain synaptosomal voltage-dependent K+ channels show that synthetic Charybdotoxin was as potent as the natural toxin on both channels. For both channels, deletion of the first amino acid, 5-oxoproline (pyroglutamic acid) decreased only slightly the potency of the inhibitor, while deletion of the entire 1-6 segment reduced potency much more. We conclude that the N-terminal region of Charybdotoxin plays a functional role in tuning the toxin's biological activity but is not essential for the folding and stability of the structure. The structure of the shortest analogue represents an interesting example of how a well organised and stable alpha/beta fold can be engineered with only 31 amino acid residues.
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analysis of side chain organization on a refined model of Charybdotoxin structural and functional implications
1992Co-Authors: Francois Bontems, Andre Menez, Christian Roumestand, Bernard Gilquin, Flavio TomaAbstract:The spatial organization of side chains on a refined model of Charybdotoxin is presented. First, the structural role of two groups of well-defined, low-accessible side chains (Thr3, Val5, Val16, Leu20, Cys33 and Leu20, His21, Thr23, Cys17, Cys35) is discussed. These side chains are conserved in three out of the five known scorpion toxins acting on K+ channels. Interestingly, they are not conserved in scyllatoxin which presents a slightly different secondary structure organization. Second, the spatial organization of all positively charged residues is analyzed. Comparison with the results presented by Park and Miller [(1992) Biochemistry (preceding paper in this issue)] shows that all functionally important positive residues are located on the beta-sheet side of the toxin. These results are different from those obtained by Auguste et al. [(1992) Biochemistry 31, 648-654] on scyllatoxin, which blocks a different type of K+ channel. This study shows, in fact, that functionally important positive residues are located on the helix side of the toxin. Thus, Charybdotoxin and scyllatoxin, which present the same global fold, interact with two different classes of K+ channels by two different parts of the motif.
Paul M Vanhoutte - One of the best experts on this subject based on the ideXlab platform.
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characterization of a Charybdotoxin sensitive intermediate conductance ca2 activated k channel in porcine coronary endothelium relevance to edhf
2002Co-Authors: Rostislav Bychkov, A H Weston, M P Burnham, Gillian R Richards, Gillian Edwards, Michel Feletou, Paul M VanhoutteAbstract:This study characterizes the K+ channel(s) underlying Charybdotoxin-sensitive hyperpolarization of porcine coronary artery endothelium. Two forms of current-voltage (I/V) relationship were evident in whole-cell patch-clamp recordings of freshly-isolated endothelial cells. In both cell types, iberiotoxin (100 nM) inhibited a current active only at potentials over +50 mV. In the presence of iberiotoxin, Charybdotoxin (100 nM) produced a large inhibition in 38% of cells and altered the form of the I/V relationship. In the remaining cells, Charybdotoxin also inhibited a current but did not alter the form. Single-channel, outside-out patch recordings revealed a 17.1±0.4 pS conductance. Pipette solutions containing 100, 250 and 500 nM free Ca2+ demonstrated that the open probability was increased by Ca2+. This channel was blocked by Charybdotoxin but not by iberiotoxin or apamin. Hyperpolarizations of intact endothelium elicited by substance P (100 nM; 26.1±0.7 mV) were reduced by apamin (100 nM; 17.0±1.8 mV) whereas those to 1-ethyl-2-benzimidazolinone (1-EBIO, 600 μM, 21.0±0.3 mV) were unaffected (21.7±0.8 mV). Substance P, bradykinin (100 nM) and 1-EBIO evoked Charybdotoxin-sensitive, iberiotoxin-insensitive whole-cell perforated-patch currents. A porcine homologue of the intermediate-conductance Ca2+-activated K+ channel (IK1) was identified in endothelial cells. In conclusion, porcine coronary artery endothelial cells express an intermediate-conductance Ca2+-activated K+ channel and the IK1 gene product. This channel is opened by activation of the EDHF pathway and likely mediates the Charybdotoxin-sensitive component of the EDHF response. Keywords: Endothelium, EDHF, hyperpolarization, calcium-activated potassium channels, Charybdotoxin, iberiotoxin, apamin, IK1 gene product Introduction The vascular endothelium controls vessel tone by releasing nitric oxide (Furchgott & Zawadzki, 1980) and prostacyclin (Moncada & Vane, 1979) as well as by a third pathway which involves hyperpolarization of the vascular smooth muscle (reviewed by Busse et al., 2002). This ‘endothelium-dependent hyperpolarizing factor (EDHF)' pathway is inhibited by a combination of the toxins apamin and Charybdotoxin, but not apamin and iberiotoxin (Corriu et al., 1996; Zygmunt & Hogestatt, 1996; Petersson et al., 1997; Chataigneau et al., 1998; Edwards et al., 1998; 2000). Given the specificities of these toxins (reviewed by Garcia et al., 1991; Castle, 1999), small- and intermediate-conductance Ca2+-activated K+ channels (SKCa and IKCa, respectively) but not large-conductance Ca2+-activated K+ channels (BKCa) are implicated in the EDHF pathway. In the vasculature, SKCa and IKCa are expressed in endothelial cells (Sakai, 1990; Marchenko & Sage, 1996; Kohler et al., 2000; Burnham et al., 2002) but not in smooth muscle cells with the contractile phenotype while BKCa are mainly expressed in myocytes (Zygmunt et al., 1997; Neylon et al., 1999; Quignard et al., 2000). Furthermore, the combination of apamin and Charybdotoxin blocks EDHF-mediated vasodilatation if selectively applied to the endothelium and inhibits the hyperpolarization of the endothelial cells produced by acetylcholine or bradykinin (Edwards et al., 1998; 2000; Doughty et al., 1999; Ohashi et al., 1999). Finally, the increase in endothelial intracellular calcium concentration, provoked by the agonist, is not inhibited by the two toxins (Ghisdal & Morel, 2001). Altogether, these experimental results suggest that the hyperpolarization of the endothelial cells is the critical initiating step in the EDHF-mediated responses (Quignard et al., 2000; Edwards et al., 2000; Busse et al., 2002) and that apamin plus Charybdotoxin exert their effects at this site (Edwards et al., 1998). In an earlier study, in porcine coronary artery endothelial cells, an apamin-sensitive K+ channel which is likely to be involved in the EDHF response was proposed to be an SKCa containing the SK3 subunit (Burnham et al., 2002). The purpose of the present study was, in the same cells, to characterize the K+ channels sensitive to Charybdotoxin which are also involved in EDHF-mediated responses.
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further investigation of endothelium derived hyperpolarizing factor edhf in rat hepatic artery studies using 1 ebio and ouabain
1999Co-Authors: Gerald E Edwards, M J Gardener, Michel Feletou, Paul M Vanhoutte, G Brady, A H WestonAbstract:The characteristics of endothelium-dependent hyperpolarization in rat hepatic artery have been further investigated in the presence of inhibitors of cyclo-oxygenase and nitric oxide synthase. Using sharp micro-electrodes, the smooth muscle hyperpolarization induced by acetylcholine, KCl or 1-ethyl-2-benzimidazolinone (1-EBIO) in intact hepatic arteries was abolished by 30 μM barium plus 500 nM ouabain. In vessels without endothelium, the smooth muscle hyperpolarization induced by KCl was not reduced by 30 μM barium alone. However, in the presence of barium the effects of KCl were partially inhibited by 100 nM ouabain and essentially abolished by 500 nM ouabain. Using sharp micro-electrodes, the hyperpolarization of both the smooth muscle and the endothelium induced by 1-EBIO or by acetylcholine was unaffected by 100 nM iberiotoxin. However, in the presence of 100 nM Charybdotoxin, the effects of 1-EBIO were abolished whereas those of acetylcholine were only partially reduced. The hyperpolarization induced by levcromakalim was unaffected by either Charybdotoxin or iberiotoxin. Under whole-cell patch-clamp recording conditions, 1-EBIO induced a voltage-insensitive, Charybdotoxin-sensitive K+ current in cultured endothelial cells but was without effect on K+ currents in smooth muscle cells isolated from hepatic arteries. It is concluded that the endothelium-dependent hyperpolarization of smooth muscle induced by either acetylcholine or by 1-EBIO in rat hepatic artery is initially associated with the opening of endothelial calcium-sensitive K+-channels insensitive to iberiotoxin. The resulting accumulation of K+ in the myoendothelial space activates an isoform of Na+/K+-ATPase which is sensitive to low concentrations of ouabain. British Journal of Pharmacology (1999) 128, 1064–1070; doi:10.1038/sj.bjp.0702916
Chris R. Triggle - One of the best experts on this subject based on the ideXlab platform.
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the contribution of d tubocurarine sensitive and apamin sensitive k channels to edhf mediated relaxation of mesenteric arteries from enos mice
2012Co-Authors: Xiaoliang Chen, Chris R. Triggle, Morley D. Hollenberg, Hong DingAbstract:: The nature of the potassium channels involved in determining endothelium-derived hyperpolarizing factor-mediated relaxation was investigated in first-order small mesenteric arteries from male endothelial nitric oxide synthase (eNOS-/-)-knockout and control (+/+) mice. Acetylcholine-induced endothelium-dependent relaxation of small mesenteric arteries of eNOS-/- was resistant to N-nitro-L-arginine and indomethacin and the guanylyl cyclase inhibitor, 1H-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one. Apamin and the combination of apamin and iberiotoxin or apamin and Charybdotoxin induced a transient endothelium-dependent contraction of small mesenteric arteries from both eNOS-/- and +/+ mice. Acetylcholine-induced relaxation in eNOS-/- mice was unaffected by Charybdotoxin or apamin alone but significantly inhibited by the combination of these agents. However, the combination of scyllatoxin and iberiotoxin did not mimic the inhibitory effect of the apamin/Charybdotoxin combination. Tubocurarine alone completely blocked acetylcholine-induced relaxation in eNOS-/- mice. Single channel analysis of myocytes from small mesenteric arterioles revealed a large conductance calcium-activated potassium channel that was sensitive to iberiotoxin, Charybdotoxin, and tetraethylammonium. Tubocurarine blocked this channel from the cytosolic side but not when applied extracellularly. Solutions of nitric oxide (NO) gas also relaxed small mesenteric arteries that had been contracted with cirazoline in a concentration-dependent manner, and the sensitivity to NO was reduced by iberiotoxin and the combination of apamin, scyllatoxin, or tubocurarine with Charybdotoxin but not by apamin, Charybdotoxin, scyllatoxin, or tubocurarine alone. These data indicate that acetylcholine-induced endothelium-derived hyperpolarizing factor-mediated relaxation in small mesenteric arteries from eNOS-/- involved the activation of tubocurarine and apamin-/Charybdotoxin-sensitive K-channels. In eNOS+/+ mice, the acetylcholine-induced response was primarily mediated by NO and was sensitive to iberiotoxin and the combination of apamin and Charybdotoxin.
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multiple mechanisms of vascular smooth muscle relaxation by the activation of proteinase activated receptor 2 in mouse mesenteric arterioles
2002Co-Authors: John J Mcguire, Chris R. Triggle, Morley D. Hollenberg, Patricia AndradegordonAbstract:Activation of PAR2 in second-order mesenteric arteriole (MA) rings from C57BL/6J, NOS3 (−/−) and PAR2 (−/−) mice was assessed for the contributions of NO, cyclo-oxygenases, guanylyl cyclase, adenylyl cyclase, and of K+ channel activation to vascular smooth muscle relaxation. PAR2 agonist, SLIGRL-NH2 (0.1 to 30 μM), induced relaxation of cirazoline-precontracted MA from C57BL/6J and NOS3 (−/−), but not PAR2 (−/−) mice. Maximal relaxation (Emax) was partially reduced by a combination of L-GN-nitroarginine methyl ester (L-NAME), 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and indomethacin. An ODQ/L-NAME/indomethacin resistant relaxation was also caused by trypsin (30 nM) in PAR2 (+/+), but not in PAR2 (−/−) mice. Relaxation was endothelium-dependent and inhibited by either 30 mM KCl-precontraction, or pretreatment with apamin, Charybdotoxin, and their combination; iberiotoxin did not substitute for Charybdotoxin nor did scyllatoxin substitute fully for apamin. Tetraethylammonium (TEA), glibenclamide, tetrodotoxin, 17-octadecynoic acid, carboxy-2-phenyl-4,4,5,5,-tetramethyl-imidazoline-1-oxyl-3-oxide, SQ22536, carbenoxolone, arachidonyl trifluoromethyl ketone, 7-nitroindazole, N-(3-(aminomethyl)benzyl)acetamidine (1400W), N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide (NS-398) and propanolol did not inhibit relaxation. 4-aminopyridine significantly increased the potency of SLIGRL-NH2. A combination of 30 μM BaCl2 and 10 μM ouabain significantly reduced the potency for relaxation, and in the presence of L-NAME, ODQ and indomethacin, Emax was reduced. We conclude PAR2-mediated relaxation of mouse MA utilizes multiple mechanisms that are both NO-cGMP-dependent, and -independent. The data are also consistent with a role for endothelium-dependent hyperpolarization of vascular smooth muscle that involves the activation of an apamin/Charybdotoxin-sensitive K+ channel(s) and, in part, may be mediated by K+. British Journal of Pharmacology (2002) 135, 155–169; doi:10.1038/sj.bjp.0704469