We have investigated the interaction of charybdotoxin (CTX) with Shaker K channels. We substituted a histidine residue for the wild-type phenylalanine (at position 425) in an inactivation-removed channel. The nature of the imidazole ring of the histidine provides the ability to change the charge on this amino acid side chain with solution hydrogen ion concentration. Wild-type, recombinant CTX blocked wild-type Shaker channels in a bimolecular fashion with a half-blocking concentration (Kd) of 650 nM (at a membrane potential of 0 mV). The F425H mutant channels were much more sensitive to CTX block with an apparent Kd (at pH 7.0) of 75 nM. Block of F425H but not wild-type channels was strongly pH sensitive. A pH change from 7 to 5.5 rendered the F425H channels >200-fold less sensitive to CTX. The pH dependence of CTX block was steeper than expected for inhibition produced by H+ ions binding to identical, independent sites. The data were consistent with H+ ions interacting with subunits of the channel homotetrameric structure. The in situ pK for the imidazole group on the histidine at channel position 425 was determined to be near 6.4 and the dissociation constant for binding of toxin to the unprotonated channel was near 50 nM. We estimate that the binding of a H+ ion to each subunit adds 0.8 kcal/mol or more of interaction energy with CTX. We used mutant toxins to test electrostatic and steric interactions between specific CTX residues and channel position 425. Our results are consistent with a model in which protons on F425H channel subunits interact with three positive charges on CTX at an effective distance 6-7 A from this channel position.

In silico and in vitro studies have made progress in understanding protein-protein complex formation; however, the molecular mechanisms for their dissociation are unclear. Protein-protein complexes, lasting from microseconds to years, often involve induced-fit, challenging computational or kinetic analysis. Charybdotoxin (CTX), a peptide from the Leiurus scorpion venom, blocks voltage-gated K+-channels in a unique example of binding/unbinding simplicity. CTX plugs the external mouth of K+-channels pore, stopping K+-ion conduction, without inducing conformational changes. Conflicting with a tight binding, we show that external permeant ions enhance CTX-dissociation, implying a path connecting the pore, in the toxin-bound channel, with the external solution. This sensitivity is explained if CTX wobbles between several bound conformations, producing transient events that restore the electrical and ionic trans-pore gradients. Wobbling may originate from a network of contacts in the interaction interface that are in dynamic stochastic equilibria. These partially-bound intermediates could lead to distinct, and potentially manipulable, dissociation pathways.

Patch-clamp recordings were made from retinal ganglion cells in the mouse retina. Under dark adaptation, blockage of BK(Ca) channels increases the spontaneous excitatory postsynaptic currents (EPSCs) and light-evoked On-EPSCs, while it decreases the light-evoked Off inhibitory postsynaptic currents (IPSCs). However, under light adaptation it decreases the light-evoked On-EPSCs, the spontaneous IPSCs and the light-evoked On- and Off-IPSCs. Blockage of BK(Ca) channels significantly altered the outputs of RGCs by changing their light-evoked responses into a bursting pattern and increasing the light-evoked depolarization of the membrane potentials, while it did not significantly change the peak firing rates of light-evoked responses.

The mechanism underlying smooth muscle relaxations of cerebral arteries in response to nitric oxide is still not completely understood. The present study was designed to determine the role of soluble guanylate cyclase in the relaxations to a nitric oxide/nucleophile complex, diethylaminodiazen-1-ium-1,2-dioate (DEA-NONOate). Rings of canine middle cerebral arteries without endothelium were suspended in Krebs-Ringer bicarbonate solution for isometric tension recording. The levels of guanosine 3',5'-cyclic monophosphate (cyclic GMP) were measured by radioimmunoassay technique. During contractions to uridine 5'-triphosphate (UTP), DEA-NONOate (10(-10) to 10(-5) M) caused concentration-dependent relaxations. Measurements of cyclic GMP levels in cerebral arterial wall demonstrated that DEA-NONOate is a potent stimulator of guanylate cyclase and subsequent formation of cyclic GMP. Increasing concentrations of a selective soluble guanylate cyclase inhibitor, 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), caused concentration-dependent reduction of both cyclic GMP production and relaxations to DEA-NONOate. Interestingly, in the presence of the highest concentration (3 x 10(-6) M) of ODQ, production of cyclic GMP in response to 10(-6) M of DEA-NONOate was abolished, whereas the same concentration of DEA-NONOate caused almost complete relaxation, suggesting that mechanisms independent of cyclic GMP production may mediate relaxing effect of high concentration of a nitric oxide donor. A selective Ca2+-activated potassium channel blocker charybdotoxin (CTX) significantly reduced relaxations to DEA-NONOate resistant to ODQ, supporting the idea that in cerebral arteries nitric oxide may activate potassium channels independently of cyclic GMP. The results of our study suggest that under physiological conditions, guanylate cyclase is a key mediator of cerebral arterial relaxations to nitric oxide. However, under pathological conditions associated with induction of nitric oxide synthase and increased biosynthesis of nitric oxide (e.g., cerebral ischemia, inflammation, sepsis), mechanisms other than formation of cyclic GMP may be activated.

Charybdotoxin-C (ChTx-C), from the scorpion Leiurus, quinquestriatus hebraeus blocks the calcium-activated potassium channels and causes hyper excitability of the nervous system. Detailed understanding the structure of ChTx-C, conformational stability, and intermolecular interactions are required to select the potential inhibitors of the toxin.

Whereas β2-adrenoceptor (β2-AR) has been reported to reduce GABAergic activity in the prefrontal cortex (PFC), the underlying cellular and molecular mechanisms have not been completely determined. Here, we showed that β2-AR agonist Clenbuterol (Clen) decreased GABAergic transmission onto PFC layer V/VI pyramidal neurons via a presynaptic mechanism without altering postsynaptic GABA receptors. Clen decreased the action potential firing rate but increased the burst afterhyperpolarization (AHP) amplitude in PFC interneurons. Application of L-type Ca2+ channel or charybdotoxin-sensitive Ca2+-activated K+ channel inhibitors blocked Clen-induced decreases in action potential firing rate, spontaneous inhibitory postsynaptic current (sIPSC) frequency and Clen-induced enhancement of AHP amplitude, suggesting that the effects of Clen involves L-type Ca2+ Channels and charybdotoxin-sensitive Ca2+-activated K+ channels. Our results provide a potential cellular mechanism by which Clen controls GABAergic neuronal activity in PFC.

Purified high conductance Ca(2+)-activated K+ (maxi-K) channels from bovine tracheal smooth muscle have been covalently labeled employing monoiodotyrosine charybdotoxin ([125I]ChTX) and different bifunctional cross-linking reagents. [125I]ChTX was specifically incorporated into the beta-subunit, which was thereafter isolated by size exclusion high performance liquid chromatography. Proteolytic fragments of the [125I]ChTX-labeled beta-subunit were generated by digestion with various endoproteinases. Glu-C or Asp-N cleavage yielded a glycosylated [125I]ChTX-labeled fragment of 13-14 kDa. A site-directed antiserum raised against residues 62-75 of the cloned beta-subunit of the maxi-K channel specifically recognizes the beta-subunit in immunostaining experiments and was capable of immunoprecipitating these ChTX-labeled peptides. Lys-C cleavage resulted in two fragments of 16 and 28 kDa, respectively, which were both precipitated by anti-beta (62-75). However, only the 28-kDa fragment was recognized by anti-beta(118-132) and shown to carry double the amount of N-linked carbohydrates. Taken together, these data restrict the site of covalent incorporation of ChTX into the beta-subunit exclusively at Lys69, confirm the predicted topology of this subunit, and indicate that both canonical N-linked glycosylation sites are occupied with complex carbohydrates of 5-6 kDa each. We propose that an extracellularly located portion of the beta-subunit is located within 7.7 A of the ChTX receptor site and could even participate in the formation of this receptor by close apposition of its extracellular domain with structural elements provided by the alpha-subunit.

The present study was performed to determine the characteristics of the endothelium-derived hyperpolarizing factor (EDHF) that mediates the nitric oxide (NO)- and prostacyclin (PGI2)-independent hyperpolarization and relaxation of porcine renal interlobar arteries. Bradykinin-induced changes in isometric force or smooth muscle membrane potential were assessed in rings of porcine renal interlobar artery preconstricted with the thromboxane analogue U46619 in the continuous presence of N(omega)-nitro-L-arginine and diclofenac to inhibit NO synthases and cyclo-oxygenases. 3 Inhibition of NO- and PGI2-production induced a rightward shift in the concentration-relaxation curve to bradykinin without affecting maximal relaxation. EDHF-mediated relaxation was abolished by a depolarizing concentration of KCl (40 mM) as well as by a combination of charybdotoxin and apamin (each 100 nM), two inhibitors of calcium-dependent K+ (K+(Ca)) channels. Charybdotoxin and apamin also reduced the bradykinin-induced, EDHF-mediated hyperpolarization of smooth muscle cells from 13.7+/-1.3 mV to 5.7+/-1.2 mV. 4 In addition to the ubiquitous alpha1 subunit of the Na-K-ATPase, the interlobar artery expressed the gamma subunit as well as the ouabain-sensitive alpha2, alpha3 subunits. A low concentration of ouabain (100 nM) abolished the EDHF-mediated relaxation and reduced the bradykinin-induced hyperpolarization of smooth muscle cells (13.6+/-2.8 mV versus 5.20+/-1.39 mV in the absence and presence of ouabain). Chelation of K+, using cryptate 2.2.2., inhibited EDHF-mediated relaxation, without affecting NO-mediated responses. Elevating extracellular KCl (from 4 to 14 mM) elicited a transient, ouabain-sensitive hyperpolarization and relaxation that was endothelium-independent and insensitive to charybdotoxin and apamin. 6 These results indicate that in the renal interlobar artery, EDHF-mediated responses display the pharmacological characteristics of K+ ions released from endothelial K+(Ca) channels. Smooth muscle cell hyperpolarization and relaxation appear to be dependent on the activation of highly ouabain-sensitive subunits of the Na-K-ATPase.

Mitochondrial volume regulation depends on K+ movement across the inner membrane and a mitochondrial Ca2+-dependent K+ channel (mitoK(Ca)) reportedly contributes to mitochondrial K+ uniporter activity. Here we utilize a novel K(Ca) channel activator, NS11021, to examine the role of mitoK(Ca) in regulating mitochondrial function by measuring K+ flux, membrane potential (DeltaPsi(m)), light scattering, and respiration in guinea pig heart mitochondria. K+ uptake and the influence of anions were assessed in mitochondria loaded with the K+ sensor PBFI by adding either the chloride (KCl), acetate (KAc), or phosphate (KH2PO4) salts of K+ to energized mitochondria in a sucrose-based medium. K+ fluxes saturated at approximately 10 mM for each salt, attaining maximal rates of 172+/-17, 54+/-2.4, and 33+/-3.8 nmol K+/min/mg in KCl, KAc, or KH2PO4, respectively. NS11021 (50 nM) increased the maximal K+ uptake rate by 2.5-fold in the presence of KH2PO4 or KAc and increased mitochondrial volume, with little effect on DeltaPsi(m). In KCl, NS11021 increased K+ uptake by only 30% and did not increase volume. The effects of NS11021 on K+ uptake were inhibited by the K(Ca) toxins charybdotoxin (200 nM) or paxilline (1 microM). Fifty nanomolar of NS11021 increased the mitochondrial respiratory control ratio (RCR) in KH2PO4, but not in KCl; however, above 1 microM, NS11021 decreased RCR and depolarized DeltaPsi(m). A control compound lacking K(Ca) activator properties did not increase K+ uptake or volume but had similar nonspecific (toxin-insensitive) effects at high concentrations. The results indicate that activating K+ flux through mitoK(Ca) mediates a beneficial effect on energetics that depends on mitochondrial swelling with maintained DeltaPsi(m).

Lubricating gut pill (LGP), a traditional Chinese formula, had been conformed to improve the loperamide-induced rat constipation by stimulation of Cl(-) secretion, but its mechanism has not been fully explored. Thus, the purpose of this study was to identify the action sites of LGP-stimulated Cl(-) secretion across rat distal colonic mucosa.

1. We have previously demonstrated that adenosine-5'-O-(2-thiodiphosphate) (ADPbetaS), a potent P2Y-purinoceptor agonist, relaxed pancreatic vasculature not only through prostacyclin (PGI2) and nitric oxide (NO) release from the endothelium but also through other mechanism(s). In this study, we investigated the effects of an inhibitor of the Na+/K+ pump, of ATP-sensitive K+ (K(ATP)) channels and of small (SK(Ca)) or large (BK(Ca)) conductance Ca2+-activated K+ channels. Experiments were performed at basal tone and during the inhibition of NO synthase and cyclo-oxygenase. 2. In control conditions, ADPbetaS (15 microM) induced an initial transient vasoconstriction followed by a progressive and sustained vasodilatation. In the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME, 200 microM) the transient vasoconstriction was reversed into a one minute vasodilator effect, which was then followed by a progressive and sustained vasodilatation similar to that observed with ADPbetaS alone. The addition of indomethacin (10 microM) did not significantly modify the profile of ADPbetaS-induced vasodilatation. 3. Ouabain (100 microM) decreased basal pancreatic flow rate and did not modify ADPbetaS-induced relaxation. This inhibitor of the Na+/K+ pump increased the pancreatic vasoconstriction induced by L-NAME or by the co-administration of L-NAME and indomethacin. Ouabain did not modify either the L-NAME or the L-NAME/indomethacin resistant part of the ADPbetaS vasodilatation. 4. The K(ATP) inhibitor tolbutamide (185 microM) did not significantly modify basal pancreatic flow rate and ADPbetaS-induced relaxation. This inhibitor which did not change L-NAME-induced vasoconstriction, significantly diminished the L-NAME resistant part of ADPbetaS-induced vasodilatation. Tolbutamide intensified the vasoconstriction induced by the co-administration of L-NAME and indomethacin. In contrast, the L-NAME/indomethacin resistant part of ADPbetaS vasodilatation was not changed by the closure of K(ATP). 5. The SK(Ca) inhibitor apamin (0.1 microM) did not significantly change pancreatic vascular resistance whatever the experimental conditions (in the absence or in presence of L-NAME or L-NAME/indomethacin). In the presence of L-NAME, the closure of SK(Ca) channels changed the one minute vasodilator effect of ADPbetaS into a potent vasoconstriction and thereafter modified only the beginning of the second part of the L-NAME-resistant part of the ADPbetaS-induced vasodilatation. In contrast, the L-NAME/indomethacin resistant part of ADPbetaS-induced relaxation remained unchanged in the presence of apamin. 6. Charybdotoxin (0.2 microM), an inhibitor of BK(Ca), increased pancreatic vascular resistance in the presence of L-NAME/indomethacin. In the presence of L-NAME, the closure of BK(Ca) channels reversed the one minute vasodilator effect of ADPbetaS into a potent vasoconstriction and drastically diminished the sustained vasodilatation. In contrast the L-NAME/indomethacin resistant part of ADPbetaS-induced relaxation was not modified by the presence of charybdotoxin. Under L-NAME/indomethacin/charybdotoxin/apamin infusions, ADPbetaS evoked a drastic and transient vasoconstriction reaching a maximum at the second minute, which was followed by a sustained increase in the flow rate throughout the ADPbetaS infusion. The maximal vasodilator effect of ADPbetaS observed was not modified by the addition of apamin. 7. The results suggest that the L-NAME-resistant relaxation induced by ADPbetaS in the pancreatic vascular bed involves activation of BK(Ca), K(ATP) and to a lesser extent of SK(Ca) channels, but the L-NAME/indomethacin resistant part of ADPbetaS-induced relaxation is insensitive to the closure of K(ATP), SK(Ca) and BK(Ca) channels.

1. The cellular mechanism(s) of action of endothelium-derived vasodilator substances in the rabbit middle cerebral artery (RMCA) were investigated. Specifically, the subtypes of potassium channels involved in the effects of endothelium-derived relaxing factors (EDRFs) in acetylcholine (ACh)-induced endothelium-dependent vasorelaxation in this vessel were systematically compared. 2. In the endothelium-intact RMCA precontracted with histamine (3 microM), ACh induced a concentration-dependent vasorelaxation, which was sensitive to indomethacin (10 microM) or N(G)-nitro-L-arginine (L-NOARG; 100 microM); pD2 values 8.36 vs 7.40 and 6.38, P < 0.01 for both, n = 6 and abolished by a combination of both agents. ACh caused relaxation in the presence of high K+ PSS (40 mM KCl), which was not affected by indomethacin, but abolished by L-NOARG and a combination of indomethacin and L-NOARG. 3. In the presence of indomethacin, relaxation to ACh in the endothelium-intact RMCA precontracted with histamine was unaffected by either glibenclamide (10 microM), an ATP-sensitive K+ channel (K[ATP]) blocker, 4-aminopyridine (4-AP, 1 mM) or dendrotoxin (DTX, 0.1 microM), delayed rectifier K channel (Kv) blockers. However, relaxation responses to ACh were significantly inhibited by either LY83583 (10 microM) and 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 10 microM), guanylyl cyclase inhibitors, or charybdotoxin (CTX; 0.1 microM), iberiotoxin (ITX, 0.1 microM) and apamin (APA, 0.1 microM), large conductance Ca2+-activated K+ channels (BK[Ca]) blocker and small conductance Ca2+-activated K+ channel (SK[Ca]) blocker, respectively. 4. In the presence of L-NOARG, relaxation to ACh was unaffected by glibenclamide or the cytochrome P450 mono-oxygenase inhibitor, clotrimazole (1 microM), but was significantly inhibited by either 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536, 10 microM) and 2',3'-dideoxyadenosine (2',3'-DDA, 30 microM), adenylyl cyclase inhibitors, or 4-AP, DTX, CTX, ITX and APA. 5. In the endothelium-denuded RMCA precontracted with histamine, authentic NO-induced relaxation was unaffected by glibenclamide, 4-AP and DTX, but significantly reduced by ODQ, ITX and APA. Authentic prostaglandin I2 (PGI2)-induced relaxation was unaffected by glibenclamide, but significantly reduced by 2',3'-DDA, 4-AP, DTX, ITX and APA. Forskolin-induced relaxation was significantly inhibited by high K+, CTX and 4-AP. 6. These results indicate that: (1) in the RMCA the EDRFs released by ACh are NO and a prostanoid (presumably PGI2), and there is no evidence for the release of a non-NO/PGI2 endothelium-derived hyperpolarizing factor (EDHF), (2) K(Ca) channels are involved in NO-mediated relaxation of the RMCA but both K(Ca) and Kv channels are involved in PGI2-mediated relaxation.

Small airway and vessels play a critical role in chronic airway and pulmonary vascular diseases, but their pharmacology has not been well characterised. We have studied airway and vascular responses in rat lung slices and separately in vitro using myography. In lung slices, under basal conditions, acetylcholine contracted airways, but had no vascular effect. The thromboxane mimetic, U46619 contracted both vessels and airways. In the presence of U46619, acetylcholine dilated vessels, but further contracted airways, an effect that was blocked by the nitric oxide synthase inhibitor L-NG-nitro-L-arginine or apamin plus charybdotoxin, which inhibit endothelial-derived hyperpolarising factor. Airway responses in lung slices were unaffected by L-NGnitro-L-arginine methyl ester, indomethacin or apamin plus charybdotoxin. By contrast, apamin plus charybdotoxin contracted bronchi studied in isolation. Our observations are the first to identify mechanisms of endothelium dependent dilations in precision cut lung slices and the potential for transverse hormonal communication between airways and vessels.

1. Penile small arteries (effective internal lumen diameter of 300 600 microm) were isolated from the horse corpus cavernosum and mounted in microvascular myographs in order to investigate the mechanisms underlying the endothelium-dependent relaxations to acetylcholine (ACh) and bradykinin (BK). 2. In arteries preconstricted with the thromboxane analogue U46619 (3-30 nM), ACh and BK elicited concentration-dependent relaxations, pD2 and maximal responses being 7.71+/-0.09 and 91+/-1 % (n=23), and 8.80+/-0.07 and 89+/-2% (n=24) for ACh and BK, respectively. These relaxations were abolished by mechanical endothelial cell removal, attenuated by the nitric oxide (NO) synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NOARG, 100 microM) and unchanged by indomethacin (3 microM). However, raising extracellular K+ to concentrations of 20-30 mM significantly inhibited the ACh and BK relaxant responses to 63+/-4% (P<0.01, n=7) and to 59+/-4% (P<0.01, n=6), respectively. ACh- and BK-elicited relaxations were abolished in arteries preconstricted with K+ in the presence of 100 microM L-NOARG. 3. In contrast to the inhibitor of ATP-sensitive K channels, the blockers of Ca2+-activated K+ (K(Ca)) channels, charybdotoxin (30 nM) and apamin (0.3 microM), each induced slight but significant rightward shifts of the relaxations to ACh and BK without affecting the maximal responses. Combination of charybdotoxin and apamin did not cause further inhibition of the relaxations compared to either toxin alone. In the presence of L-NOARG (100 microM), combined application of the two toxins resulted in the most effective inhibition of the relaxations to both ACh and BK. Thus, pD2 and maximal responses for ACh and BK were 7.65+/-0.08 and 98+/-1%, and 9.17+/-0.09 and 100+/-0%, respectively, in controls, and 5.87+/-0.09 (P<0.05, n=6) and 38+/-11% (P<0.05, n=6), and 8.09+/-0.14 (P<0.01, n=6) and 98+/-1% (n=6), respectively, after combined application of charybdotoxin plus apamin and L-NOARG. 4. The selective inhibitor of guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 microM) did not alter the maximal responses to either ACh or BK, but slightly decreased the sensitivity to both agonists, deltapD2 being 0.25+/-0.07 (P<0.05, n=6) and 0.62+/-0.12 (P< 0.01, n=6) for ACh and BK, respectively. Combined application of ODQ and charybdotoxin plus apamin produced further inhibition of the sensitivity to both ACh (deltapD2=1.39+/-0.09, P<0.01, n=6) and BK (1.29+/-0.11, P<0.01, n=6), compared to either ODQ or charybdotoxin plus apamin alone. 5. Exogenous nitric oxide (NO) present in acidified solutions of sodium nitrite (NaNO2) and S-nitrosocysteine (SNC) both concentration-dependently relaxed penile resistance arteries, pD2 and maximal responses being 4.84+/-0.06 and 82+/-3% (n=12), and 6.72+/-0.07 and 85+/-4% (n=19), respectively. Charybdotoxin displaced to the right the dose-relaxation curves for both NO (deltapD2 0.38+/-0.06, P<0.01, n=6) and SNC (deltapD2 0.50+/-0.10, P<0.01, n=5), whereas apamin only reduced sensitivity (deltapD2=0.35+/-0.12, P<0.05, n=5) and maximum response (65+/-9%, P<0.05, n=6) to SNC. ODQ shifted to the right the dose-relaxation curves to both NO and SNC. The relaxant responses to either NO or SNC were not further inhibited by a combination of ODQ and charybdotoxin or ODQ and charybdotoxin plus apamin, respectively, compared to either blocker alone. 6. In the presence of 3 microM phentolamine, 5 microM ouabain contracted penile resistance arteries by 50+/-6% (n=17) of K-PSS, but did not significantly change the relaxant responses to either ACh, BK or NO. However, in the presence of L-NOARG ouabain reduced the ACh- and BK-elicited relaxation from 94+/-3% to 16+/-5% (P<0.0001, n=6), and from 98+/-2% to 13+/-3% (P<0.0001, n=5), respectively. Combined application of ODQ and ouabain inhibited the relaxations to NO from 92+/-2% to 26+/-3% (P<0.0001, n=6). 7. The present results demonstrate that the endothelium-dependent relaxations of penile small arteries involve the release of NO and a non-NO non-prostanoid factor(s) which probably hyperpolarize(s) smooth muscle by two different mechanisms: an increased charybdotoxin and apamin-sensitive K+ conductance and an activation of the Na+-K+ATPase. These two mechanisms appear to be independent of guanylate cyclase stimulation, although NO itself can also activate charybdotoxin-sensitive K+ channels and the Na+-K+ pump through both cyclic GMP-dependent and independent mechanisms, respectively.

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.

The inducible endothelium-dependent hyperpolarizing factor (iEDHF) pathway is activated as a compensatory response to adverse changes in the body. It causes vasorelaxation and maintains circulatory homeostasis in the organs. Small to moderate quantities of ethanol enhance vascular relaxation. However, its mechanism and the involvement of the iEDHF pathway in this process are unknown. Therefore, we studied iEDHF-mediated, acetylcholine-induced, endothelium-dependent relaxation in the superior mesenteric arteries (SMAs) of rats chronically fed ethanol. Rats were administered a standard diet (S-Control group), Lieber's control diet (L-Control group), or Lieber's ethanol diet (EtOH group). SMA relaxation was assessed by isometric tension measurements. Arachidonate 15-lipoxygenase (ALOX15) and soluble epoxide hydrolase (sEH) were determined by immunoblot. Acetylcholine-induced, endothelium-dependent relaxation was significantly greater in the EtOH than the control groups. These differences persisted after PGI2 and NO blockade. Thus, the increase in acetylcholine-induced relaxation was EDHF-mediated. In the EtOH group, however, it was prevented by iEDHF inhibitors. ALOX15 and sEH protein expression levels were higher in the EtOH than the L-Control group. The increase in acetylcholine-induced relaxation by chronic ethanol consumption was mediated by the iEDHF pathway. This mechanism may compensate for the blood pressure elevation induced by ethanol. This study suggests that iEDHF is induced during proper drinking and may help prevent the onset of cardiovascular conditions.

Short chain fatty acids, including propionate, are generated in the caecum and large intestine, and when absorbed may elicit localised increases in intestinal blood flow. We sought to assess the mechanism by which propionate caused vasorelaxation. Propionate-mediated relaxation of noradrenaline-preconstricted rat mesenteric small arteries (RMSAs, i.d. 200-300 microm) was studied using small vessel myography. Propionate (1-30 mM) produced a concentration-dependent relaxation. Relaxation induced by 10 mM propionate (the approximate EC50) was almost abolished by endothelial denudation, although a marked relaxation to a very high concentration of propionate (50 mM) persisted in the absence of the endothelium. In endothelium-intact RMSAs, relaxation to 10 mM propionate was almost abolished by elevating [K+]o to 25 mM, but was unaffected by 100 microM N(omega)-nitro-L-arginine methyl ester (L-NAME) (68 +/- 4 vs. 66 +/- 3% in controls, n = 35), or by 1 microM indomethacin (60 +/- 4 vs. 61 +/- 7 % in controls, n = 15). In the presence of L-NAME, relaxation to 10 mM propionate was significantly and markedly (i.e. > 50 %) inhibited by 50 microM Ba2+ and by the combination of 100 nM charybdotoxin and 100 nM apamin. A similar effect on propionate-mediated relaxation was also exerted by 100 microM ouabain, and by the combination of 50 microM barium with ouabain. Relaxation was also significantly and markedly inhibited by pre-treatment of RMSAs with 100 nM thapsigargin or 10 microM cyclopiazonic acid (CPA). The results demonstrate that 10 mM propionate relaxes RMSAs via endothelium-derived hyperpolarising factor (EDHF). The observation that relaxation by propionate is inhibited by thapsigargin and CPA suggests that this action of propionate involves the release of endothelial cell Ca2+ stores.

Luteolin (3',4',5,7-tetrahydroxyflavone) is a flavone with a yellow crystalline appearance present in numerous plants such as broccoli, green chili, and carrot. Luteolin is considered to be an endocrine disruptor with potent estrogen agonist activity and potent progesterone antagonist activity. Luteolin has effects on smooth muscle. Luteolin relaxed guinea pig trachea smooth muscle as it inhibited both phosphodiesterase and reduced intracellular Ca2+. Luteolin also caused vasorelaxation in rat thoracic aorta smooth muscle by inhibiting intracellular Ca2+ release, inhibition of sarcolemmal Ca2+ channels, and activation of K+ channels. Luteolin or its glycosides from artichoke extracts may have an ameliorating effect on irritable bowel syndrome. The purpose of this study was to determine if luteolin had an effect on gallbladder motility.

We have investigated the involvement of potassium channels in the NO-induced relaxation of small ET-1 precontracted arteries from placentas of normal pregnancies in the presence of the potassium channel modulating agents charybdotoxin, 4-AP, glibenclamide, TEA and the blocker of soluble guanylyl cyclase, ODQ, respectively. We have studied the effect of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) in vessels precontracted by different concentrations of potassium and we have also investigated the presence of BK(Ca) channels in placental arteries by immunohistochemistry and immunoblotting. Our results show that charybdotoxin, an inhibitor of large- and intermediate-conductance Ca(2+)-activated potassium channels, inhibits relaxation in placental arteries. In presence of both charybdotoxin and ODQ, the inhibition of relaxation was significantly stronger, which indicates that NO-induced relaxation of human placental arteries is partly mediated through cGMP, and partly through a direct effect on potassium channels of the BK(Ca) type. The NO-donor SNAP preferentially relaxes contractions induced by 75 mM K(+) as compared to 100 mM K(+). This effect profile is a unique feature of drugs acting by K(+) channel opening. The immunohistochemistry shows that BK(Ca) channels are located both in smooth muscle and in endothelium in placental arteries.

The present study investigated whether calcium-activated K+ channels are involved in acetylcholine-evoked nitric oxide (NO) release and relaxation.