Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes.
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Spontaneous transient outward currents (STOCs) were recorded from smooth muscle cells of the guinea pig taenia coli using the whole cell patch-clamp technique. STOCs were resolved at potentials positive to -50 mV. Treating cells with caffeine (1 mM) caused a burst of outward currents followed by inhibition of STOCs. Replacing extracellular Ca(2+) with equimolar Mn(2+) caused STOCs to "run down. " Iberiotoxin (200 nM) or charybdotoxin (ChTX; 200 nM) inhibited large-amplitude STOCs, but small-amplitude "mini-STOCs" remained in the presence of these drugs. Mini-STOCs were reduced by apamin (500 nM), an inhibitor of small-conductance Ca(2+)-activated K(+) channels (SK channels). Application of ATP or 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) increased the frequency of STOCs. The effects of 2-MeS-ATP persisted in the presence of charybdotoxin but were blocked by combination of ChTX (200 nM) and apamin (500 nM). 2-MeS-ATP did not increase STOCs in the presence of pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, a P(2) receptor blocker. Similarly, pretreatment of cells with U-73122 (1 microM), an inhibitor of phospholipase C (PLC), abolished the effects of 2-MeS-ATP. Xestospongin C, an inositol 1,4,5-trisphosphate (IP(3)) receptor blocker, attenuated STOCs, but these events were not affected by ryanodine. The data suggest that purinergic activation through P(2Y) receptors results in localized Ca(2+) release via PLC- and IP(3)-dependent mechanisms. Release of Ca(2+) is coupled to STOCs, which are composed of currents mediated by large-conductance Ca(2+)-activated K(+) channels and SK channels. The latter are thought to mediate hyperpolarization and relaxation responses of gastrointestinal muscles to inhibitory purinergic stimulation. (+info)
The peripheral antinociceptive effect induced by morphine is associated with ATP-sensitive K(+) channels.
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The effect of several K(+) channel blockers such as glibenclamide, tolbutamide, charybdotoxin (ChTX), apamin, tetraethylammonium (TEA), 4-aminopyridine (4-AP) and cesium on the peripheral antinociceptive effect of morphine was evaluated by the paw pressure test in Wistar rats. The intraplantar administration of a carrageenan suspension (250 microg) resulted in an acute inflammatory response and a decreased threshold to noxious pressure. Morphine administered locally into the paw (25, 50, 100 and 200 microg) elicited a dose-dependent antinociceptive effect which was demonstrated to be mediated by a peripheral site up to the 100 microg dose. The selective blockers of ATP-sensitive K(+) channels glibenclamide (20, 40 and 80 microg paw(-1)) and tolbutamide (40, 80 and 160 microg paw(-1)) antagonized the peripheral antinociception induced by morphine (100 microg paw(-1)). This effect was unaffected by ChTX (0. 5, 1.0 and 2.0 microg paw(-1)), a large conductance Ca(2+)-activated K(+) channel blocker, or by apamin (2.5, 5.0 and 10.0 microg paw(-1)), a selective blocker of a small conductance Ca(2+)-activated K(+) channel. Intraplantar administration of the non-specific K(+) channel blockers TEA (160, 320 and 640 microg), 4-AP (10, 50 and 100 microg) and cesium (125, 250 and 500 microg) also did not modify the peripheral antinociceptive effect of morphine. These results suggest that the peripheral antinociceptive effect of morphine may result from activation of ATP-sensitive K(+) channels, which may cause a hyperpolarization of peripheral terminals of primary afferents, leading to a decrease in action potential generation. In contrast, large conductance Ca(2+)-activated K(+) channels, small conductance Ca(2+)-activated K(+) channels as well as voltage-dependent K(+) channels appear not to be involved in this transduction pathway. British Journal of Pharmacology (2000) 129, 110 - 114 (+info)
Involvement of cyclic AMP - PKA pathway in VIP-induced, charybdotoxin-sensitive relaxation of longitudinal muscle of the distal colon of Wistar-ST rats.
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The intracellular mechanism of vasoactive intestinal peptide (VIP)-induced, charybdotoxin (ChTx)-sensitive relaxation of longitudinal muscle of the distal colon of Wistar-ST rats was studied. A single pulse or 100 pulses at 10 Hz of electrical field stimulation (EFS) induced rapid transient relaxation or that with a subsequent contraction of the longitudinal muscle in the presence of atropine and guanethidine, respectively. Rp-8 bromo cAMPS, an inhibitor of cyclic AMP dependent protein kinase (PKA), at 30 microM inhibited the relaxations induced by EFS with a single or 100 pulses maximally by about 80 or 60%, respectively. It also inhibited VIP (300 nM)-induced relaxation by 82%. VIP (100 nM - 1 microM) increased the cyclic AMP content of longitudinal muscle myenteric plexus preparations obtained from the distal colon. ChTx at 100 nM almost completely inhibited 8 bromo cyclic AMP-induced relaxation of the distal segments. EFS with two or three pulses at 10 Hz induced inhibitory junction potentials consisting of two phases, rapid and subsequent slow hyperpolarization in the membrane potential of longitudinal smooth muscle cells. Rp-cAMPS, another inhibitor of PKA, inhibited the delayed slow hyperpolarization. It also inhibited the exogenously added VIP-induced hyperpolarization of the cell membrane. Thus, the present study suggests that activation of PKA via activation of VIP receptors is associated with activation of ChTx-sensitive K(+) channels in relaxation of longitudinal muscle of the distal colon of Wistar-ST rats. British Journal of Pharmacology (2000) 129, 140 - 146 (+info)
Role of gap junctions and EETs in endothelium-dependent hyperpolarization of porcine coronary artery.
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1. The effects of endothelium-derived hyperpolarizing factor (EDHF: elicited using substance P or bradykinin) were compared with those of 11,12-EET in pig coronary artery. Smooth muscle cells were usually impaled with microelectrodes through the adventitial surface. 2. Substance P (100 nM) and 11,12-EET (11,12-epoxyeicosatrienoic acid; 3 microM) hyperpolarized endothelial cells in intact arteries. These actions were unaffected by 100 nM iberiotoxin but were abolished by charybdotoxin plus apamin (each 100 nM). 3. Substance P (100 nM) and bradykinin (30 nM) hyperpolarized intact artery smooth muscle; Substance P had no effect after endothelium removal. 11,12-EET hyperpolarized de-endothelialized vessels by 12.6+/-0.3 mV, an effect abolished by 100 nM iberiotoxin. 4. 11,12-EET hyperpolarized intact arteries by 18.6+/-0.8 mV, an action reduced by iberiotoxin, which was ineffective against substance P. Hyperpolarizations to 11, 12-EET and substance P were partially inhibited by 100 nM charybdotoxin and abolished by further addition of 100 nM apamin. 5. 30 microM barium plus 500 nM ouabain depolarized intact artery smooth muscle but responses to substance P and bradykinin were unchanged. 500 microM gap 27 markedly reduced hyperpolarizations to substance P and bradykinin which were abolished in the additional presence of barium plus ouabain. 6. Substance P-induced hyperpolarizations of smooth muscle cells immediately below the internal elastic lamina were unaffected by gap 27, even in the presence of barium plus ouabain. 7. In pig coronary artery, 11,12-EET is not EDHF. Smooth muscle hyperpolarizations attributed to 'EDHF' are initiated by endothelial cell hyperpolarization involving charybdotoxin- (but not iberiotoxin) and apamin-sensitive K(+) channels. This may spread electrotonically via myoendothelial gap junctions but the involvement of an unknown endothelial factor cannot be excluded. (+info)
Potassium- and acetylcholine-induced vasorelaxation in mice lacking endothelial nitric oxide synthase.
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1. The contribution of an endothelium-derived hyperpolarizing factor (EDHF) was investigated in saphenous and mesenteric arteries from endothelial nitric oxide synthase (eNOS) (-/-) and (+/+) mice. 2. Acetylcholine-induced endothelium-dependent relaxation of saphenous arteries of eNOS(-/-) was resistant to N(omega)-nitro-L-arginine (L-NNA) and indomethacin, as well as the guanylyl cyclase inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a) quinoxalin-1-one(ODQ). 3. Potassium (K(+)) induced a dose-dependent vasorelaxation which was endothelium-independent and unaffected by either L-NNA or indomethacin in both saphenous and mesenteric arteries from eNOS(-/-) or (+/+) mice. 4. Thirty microM barium (Ba(2+)) and 10 microM ouabain partially blocked potassium-induced, but had no effect on acetylcholine-induced vasorelaxation in saphenous arteries. 5. Acetylcholine-induced relaxation was blocked by a combination of charybdotoxin (ChTX) and apamin which had no effect on K(+)-induced relaxation, however, iberiotoxin (IbTX) was ineffective against either acetylcholine- or K(+)-induced relaxation. 6. Thirty microM Ba(2+) partially blocked both K(+)- and acetylcholine-induced relaxation of mesenteric arteries, and K(+), but not acetylcholine-induced relaxation was totally blocked by the combination of Ba(2+) and ouabain. 7. These data indicate that acetylcholine-induced relaxation cannot be mimicked by elevating extracellular K(+) in saphenous arteries from either eNOS(-/-) or (+/+) mice, but K(+) may contribute to EDHF-mediated relaxation of mesenteric arteries. (+info)
Molecular characterization of a voltage-gated potassium channel expressed in rat testis.
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Potassium (K(+)) channels are present in both mammalian testis and spermatozoa. Immunofluorescent detection of sperm-bound biotinylated charybdotoxin, an inhibitor of Ca(2+)-activated and of delayed rectifier K(+) channels, indicated that these ion channels are uniformly distributed over the surface of both heads and tails of unfixed rat epididymal spermatozoa. Reverse transcription-polymerase chain reaction (RT-PCR) analysis on rat testis RNA with PCR primers, based on known nucleotide sequences of different classes of K(+) channels, amplified sequences homologous to delayed rectifier K(+) channels. In-situ RT-PCR on rat testis sections showed that these K(+) channel transcripts are present in the cytoplasm of primary spermatocytes and post-meiotic elongating spermatids. Northern blot analysis of various rat tissues identified multiple K(+) channel transcripts, some of which were observed only in testis. An attempt to obtain a full length rat testis K(+) channel cDNA sequence gave an assembled sequence of 2693 base pairs with >90% homology to a delayed rectifier K(+) channel, Kv1.3. A method for rapid amplification of cDNA ends was employed to amplify the 5' sequences of the rat testis cDNA but a unique sequence could not be obtained. DNA sequencer traces suggest that multiple related K(+) channels which differed at their 5' ends were amplified in rat testis. (+info)
Close association of the N terminus of Kv1.3 with the pore region.
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The Shaker superfamily encodes voltage-gated potassium (Kv) channels. The N termini of Shaker proteins are located intracellularly and contain several domains shown to regulate important aspects of channel function, such as speed of inactivation, channel assembly (T1 domain), and steady state protein level (T0 domain, amino acids 3-39 in rabbit). Mutations and/or deletion of certain amino acids in the T0 domain lead to a 13-fold amplification of Kv current as compared with wild type channels, primarily by increasing the absolute number of channel proteins present in the membrane (Segal, A. S., Yao, X., and Desir, G. V. (1999) Biochem. Biophys. Res. Commun. 254, 54-64). Although T0 mutants have kinetic properties virtually indistinguishable from wild type, they were noted to have a slightly larger single channel conductance, suggesting that the T0 domain might also interact with the pore region. In the present study we show that although T0 does not affect pore selectivity, it does modulate the binding affinity of the pore blocker, charybdotoxin. These results suggest that the N terminus of Kv1.3 is closely associated with the pore region. (+info)
Small conductance Ca2+-activated K+ channels are regulated by Ca2+-calmodulin-dependent protein kinase II in murine colonic myocytes.
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1. Ca2+ regulates the activity of small conductance Ca2+-activated K+ (SK) channels via calmodulin-dependent binding. We investigated whether other forms of Ca2+-dependent regulation might control the open probability of SK channels. 2. Under whole-cell patch-clamp conditions, spontaneous openings of SK channels can be resolved as charybdotoxin-insensitive spontaneous transient outward currents (STOCs). The Ca2+-calmodulin-dependent (CaM) protein kinase II inhibitor KN-93 reduced the occurrence of charybdotoxin-insensitive STOCs. 3. The charybdotoxin-insensitive STOCs are related to spontaneous, local release of Ca2+. KN-93 did not affect spontaneous Ca2+-release events. 4. KN-93 and W-7, a calmodulin inhibitor, decreased the open probability of SK channels in on-cell patches but not in excised patches. 5. Application of autothiophosphorlated CaM kinase II to the cytoplasmic surface of excised patches increased the open probalibity of SK channels. Boiled CaM kinase II had no effect. 6. We conclude that CaM kinase II regulates SK channels in murine coloni myocytes. This mechanism provides a secondary means of regulation, increasing the impact of a given Ca2+ transient on SK channel open probability. (+info)