Big Conductance Calcium-activated Potassium Channel Research Articles

The Roles of Potassium Channels and Nitric oxide in the Modulation of Apelin induced-relaxation in Isolated Diabetic Rat Aorta

Diabetes is associated with endothelial dysfunction, which impairs blood vesselscapacity to maintain vascular tone. Apelin is an adipocyte-produced relaxing factor that has endothelium-dependent and nitric oxide (NO)-mediated vasorelaxant effects. The current study investigated how streptozotocin (STZ)-induced type one diabetes modulates the mechanisms involved in aortic vascular response to apelin, focusing on the role of potassium channels and endothelial derived relaxing factors (EDRF). In this study, precontracted rat thoracic aortic segments were pre-incubated with the NO inhibitor, cyclooxygenase (COX) inhibitor and potassium channels blockers including: non-selective calcium-activated potassium channel, big conductance calcium-activated potassium channels (BKca), intermediate conductance calcium activated potassium channels (IKca), delayed inward rectifier potassium channels (Kir), adenosine triphosphates-sensitive potassium channels (KATP) and voltage sensitive potassium channels (Kv) blockers, then cumulative concentrations of apelin were applied to each group in both non-diabetic and diabetic conditions. The statistical analysis between diabetic and non-diabetic groups revealed that endothelial impairment induced by diabetes in rat thoracic aorta remarkably attenuated the vascular responses to apelin. An important new finding in this study was that almost all potassium channels blockers noticeably P<0.001 increased apelin efficacy with relatively no changes in the peptide potency. However, in diabetic aortic segments, the non-selective Kca, BKca blockers, NO inhibitor and COX inhibitor reversed vascular responses to apelin, but Kir, KATP and Kv blockers significantly reduced vascular responses to apelin in comparison to control rats. It is worth noting that diabetes did not only alter the peptide potency in these experimental groups but also significantly increased the maximum responses when Kca and BKca blockers preincubated. In conclusion, our findings shed light on the mechanisms behind diabetes-induced aortic artery hypo-reactivity to apelin which involve the inhibition of endothelial NO synthase activities and decreased contribution of Kca, BKca, IKca, Kv, Kir and KATP channels.

BK ablation attenuates osteoblast bone formation via integrin pathway

Impaired bone formation is one of the major causes of low bone mass and skeletal fragility that occurs in osteoporosis. However, the mechanisms underlying the defects in bone formation are not well understood. Here, we report that big conductance calcium-activated potassium channels (BKs) are required for bone formation and osteoblast function both in vivo and in vitro. By 15 weeks of age, BK knockout (BKO) mice exhibited a decline in bone mineral density and trabecular bone volume of the tibiae and lumbar vertebrae, which were associated with impaired bone formation and osteoblast activity. Mechanistically, BK ablation in bone and bone marrow mesenchymal stem cells (BMSCs) of BKO mice inhibited integrin signaling. Furthermore, the binding of α subunit of BK with integrin β1 protein in osteoblasts was confirmed, and FAK-ERK1/2 signaling was proved to be involved by genetic modification of KCNMA1 (which encodes the α subunit of BK) in ROS17/2.8 osteoblast cells. These findings indicated that BK regulates bone formation by promoting osteoblast differentiation via integrin pathway, which provided novel insight into ion transporter crosstalk with the extracellular matrix in osteoblast regulation and revealed a new potential strategy for intervention in correcting bone formation defects.

Functional Coupling of Cav2.3 and BK Potassium Channels Regulates Action Potential Repolarization and Short-Term Plasticity in the Mouse Hippocampus.

Voltage-gated ion channels are essential for signal generation and propagation in neurons and other excitable cells. The high-voltage activated calcium-channel Cav2.3 is expressed throughout the central and peripheral nervous system, and within CA1 hippocampal pyramidal neurons it is localized throughout the somato-dendritic region and dendritic spines. Cav2.3 has been shown to provide calcium for other calcium-dependent potassium channels including small-conductance calcium-activated potassium channels (SK), but big-conductance calcium-activated potassium channels (BK) have been thought to be activated by calcium from all known voltage-gated calcium channels, except Cav2.3. Here we show for the first time that CA1 pyramidal cells which lack Cav2.3 show altered action potential (AP) waveforms, which can be traced back to reduced SK- and BK-channel function. This change in AP waveform leads to strengthened synaptic transmission between CA1 and the subiculum, resulting in increased short-term plasticity. Our results demonstrate that Cav2.3 impacts cellular excitability through functional interaction with BK channels, impacting communication between hippocampal subregions.

The inhibitory effect of BKCa channels induced by autoantibodies against angiotensin II type 1 receptor is independent of AT1R.

Autoantibodies against angiotensin II Type 1 receptor (AT1-AA) are routinely detected in the serum of preeclampsia patients, which results in an increase in vascular tone and an elevation in intracellular calcium concentration of rat vascular smooth muscle (VSM). The big conductance calcium-activated potassium channels (BKCa channels) account for the dominant outward currents in VSMCs, contributing to membrane hyperpolarization and vasodilation. In the present study, we investigated the effect of AT1-AA on BKCa channels. A preeclampsia model was established by passively immunizing healthy pregnant BALB/c mice with AT1-AA extracted from hybridoma culture supernatant. Blood pressure, serum AT1-AA levels, and urinary protein were measured in the immunized mice. BKCa channel expression was detected using qRT-PCR and immunohistochemical technique. The patch-clamp technique was used to record the single currents of BKCa channels in the HEK293T cells that had been transfected. AT1-AA immunized mice exhibited elevated AT1-AA and urinary protein levels compared with mice of the vehicle group. Systolic blood pressure was also increased in the immunized group. BKCa channel β1-subunit expression was reduced in the mesenteric arteries of immunized mice. AT1-AA could inhibit the BKCa currents and the inhibitory effects were not completely reversed following the application of valsartan, an inhibitor of AT1 receptor. In conclusion, AT1-AA could decrease BKCa expression and inhibit BKCa activity independent of AT1R. These inhibitory effects are likely to be contributory factors in the promotion of increased vascular tone caused by AT1-AA in preeclampsia.

Abstract P508: Inhibition of Big-conductance Calcium-activated Potassium Channels in Vascular Myocytes is a Key Novel Mechanism for Tacrolimus-induced Hypertension

Background and Purpose: Tacrolimus (TAC, also called FK506), a common immunosuppressive drug, causes hypertension. In this study, we tested a novel hypothesis that TAC inhibits big-conductance Ca 2+ -activated K + (BK) channels in artery smooth muscle cells (SMCs), leading to hypertension. Experimental Approach: Noninvasive and invasive blood pressure measurements were used to determine hypertension; organ bath technique and live videomicroscopy to assess vasoconstriction; fluorescence imaging and confocal microscope to detect whole-cell and local intracellular Ca 2+ concentration; patch-clamp techniques to record channel activity; Western blot analysis and real-time RT-PCR to evaluate channel expression; and gene knockout mice to determine its functions. Key Results: Intraperitoneal injection of TAC once a day for a week causes hypertension with a peak at 4 weeks. Application of norepinephrine (NE) induces a larger vasoconstriction in arteries and increase in [Ca 2+ ] i in artery SMCs from mice treated with TAC for 4 weeks. Whole-cell BK channel activity is significantly decreased in SMCs from TAC-treated mice, whereas Ca 2+ spark activity is increased. The voltage sensitivity, Ca 2+ sensitivity and open time of single BK channels are all decreased in TAC-administrated mouse cells. BK channel β1, but not α, subunit protein and mRNA expression levels are significantly decreased in arteries from TAC-treated mice. Chronic administration of TAC fails to further increase blood pressure in BK channel β1 knockout mice with hypertension. Conclusion and Implications: Our findings provide novel evidence that TAC inhibits BK channel β1 subunit expression and functions in artery SMCs, thereby leading to enhanced vasoconstriction and hypertension.

Tirofiban induces vasorelaxation of the coronary artery via an endothelium-dependent NO-cGMP signaling by activating the PI3K/Akt/eNOS pathway

Tirofiban, a glycoprotein IIb/IIIa inhibitor, is an antiplatelet drug extensively used in patients with acute coronary syndrome (ACS) and exerts an therapeutic effect on no-reflow phenomenon during percutaneous coronary intervention (PCI). Previous studies elucidated the vasodilation caused by tirofiban in the peripheral artery. However, whether tirofiban exerts a vasodilator effect on the coronary artery is unclear. Our present study found that tirofiban induced endothelium-dependent vasodilation in a concentration- and time-dependent manner in the isolated rat coronary artery pre-constricted by 5-hydroxytryptamine (5-HT). Further study showed that incubation of human umbilical venous endothelial cells (HUVECs) with tirofiban increased NO production, which was ascribed to the increased eNOS phosphorylation. This was confirmed by the loss of the vasorelaxant effect of tirofiban in the presence of l-NAME (eNOS inhibitor) and L-NMMA (NOS inhibitor) but not SMT (iNOS inhibitor) on isolated rat coronary arteries. The vasorelaxation was also blocked by the PI3K inhibitors, wortmannin and LY294002, as well as the Akt inhibitor SH-5, indicating the role of PI3K and Akt in tirofiban-mediated vasodilation. Moreover, further study showed that soluble guanylyl cyclase (sGC) inhibitor ODQ, or blockers of potassium channel (big-conductance calcium-activated potassium channel) blocked tirofiban-induced vasodilation of the coronary artery. These findings suggest that tirofiban induces vasorelaxation via an endothelium-dependent NO-cGMP signaling through the activation of the Akt/eNOS/sGC pathway.

Effects of cisplatin on potassium currents in CT26 cells.

Cisplatin, a platinum-based drug, is an important weapon against many types of cancer. It is well-known that cisplatin induces apoptosis. Potassium channel plays very important role in several signaling pathways. To investigate the possibility that potassium channels also have a role in the cellular response to cisplatin, we examined the effect of cisplatin on the activity of potassium channels on CT26 cell, the colon carcinoma cell line. The cells were cultured in DMEM, supplemented with 10< heat-inactivated fetal bovine serum. At mid-log phase, cultures were harvested, washed twice in phosphate-buffered saline, and resuspended in culture medium before use. Cells were voltage-clamped using the whole-cell patch clamp technique. Membrane current data were collected and amplified. Differences between two groups were assessed by paired t-test and one sample t-test to compare the relative values. One-way ANOVA was used for all experiment with more than two groups. Potassium currents were detected in CT26 cells and the currents were reduced by the application of tetraethylammonium (TEA) chloride, iberiotoxin, a big conductance calcium-activated potassium channel blocker and barium. The potassium currents were enhanced to 192< by the application of cisplatin (0.5 mM). Moreover, the increase of potassium currents by cisplatin was further inhibited by the application of TEA confirming the action of cisplatin on potassium channels. In addition, relative current induced by cisplatin in CT26 cells was bit larger than in normal IEC-6 cells. Potassium currents were detected in CT26 cells and the currents were reduced by the application of tetraethylammonium (TEA) chloride, iberiotoxin, a big conductance calcium-activated potassium channel blocker and barium. The potassium currents were enhanced to 192< by the application of cisplatin (0.5 mM). Moreover, the increase of potassium currents by cisplatin was further inhibited by the application of TEA confirming the action of cisplatin on potassium channels. In addition, relative current induced by cisplatin in CT26 cells was bit larger than in normal IEC-6 cells.

A Combination of Intravenous Genistein Plus Mg2+ Enhances Antihypertensive Effects in SHR by Endothelial Protection and BKCa Channel Inhibition.

The effects of combining genistein (GST) plus magnesium (Mg) upon the development of hypertension were examined in 28 twelve-week-old male spontaneous hypertension rats (SHRs). Four experimental groups were tested: SHR (0.9% NaCl and DMSO), SHR + GST (0.9% NaCl and GST 5mg/kg/day), SHR + Mg (Mg(2+) 0.75 mmol/kg/day and DMSO), and SHR + GST + Mg (Mg(2+) 0.75 mmol/kg/day and GST 5mg/kg/day). A group of normotensive genetic control, Wistar-Kyoto (WKY) rats were also included for comparison. Drugs were administrated intravenously daily for 30 days. Systolic blood pressure (SBP) and heart rate were measured by tail-cuff plethysmography every five days. Vascular tone of mesenteric arteries was examined by an isometric force transducer. Big-conductance calcium-activated potassium channel (BKCa) currents were detected by whole-cell patch-clamp techniques. SBP in SHRs was significantly elevated vs. that in WKY rats. GST or Mg lowered SBP of SHRs. Their combination enhanced antihypertensive effects, as indicated by significantly lowered SBP and shorter onset times. GST or Mg individually improved endothelial dysfunction of SHRs. However, again their combination enhanced endothelial protection, nearly restoring maximal relaxation responses to those seen in WKY. BKCa currents in SHRs were increased compared with WKY rats. GST, Mg, and their combination restored BKCa currents to those of WKY rats. The combination of GST and Mg produces antihypertensive effects and improvement of endothelial dysfunction, which are substantially greater than that when either is used individually. These results suggest a novel and feasible protocol for the prevention and treatment of hypertension and related cardio- and cerebro-vascular diseases.

Fenamates block gap junction coupling and potentiate BKCa channels in guinea pig arteriolar cells

We determined the actions of the fenamates, flufenamic acid (FFA) and niflumic acid (NFA), on gap junction-mediated intercellular coupling between vascular smooth muscle cells (VSMC) in situ of acutely isolated arteriole segments from the three vascular beds: the spiral modiolar artery (SMA), anterior inferior cerebellar artery (AICA) and mesenteric artery (MA), and on non-junctional membrane channels in dispersed VSMCs. Conventional whole-cell recording methods were used. FFA reversibly suppressed the input conductance (Ginput) or increased the input resistance (Rinput) in a concentration dependent manner, with slightly different IC50s for the SMA, AICA and MA segments (26, 33 and 56μM respectively, P>0.05). Complete electrical isolation of the recorded VSMC was normally reached at ≥300μM. NFA had a similar effect on gap junction among VSMCs with an IC50 of 40, 48 and 62μM in SMA, AICA and MA segments, respectively. In dispersed VSMCs, FFA and NFA increased outward rectifier K+-current mediated by the big conductance calcium-activated potassium channel (BKCa) in a concentration-dependent manner, with a similar EC50 of ∼300μM for both FFA and NFA in the three vessels. Iberiotoxin, a selective blocker of the BKCa, suppressed the enhancement of the BKCa by FFA and NFA. The KV blocker 4-AP had no effect on the fenamates-induced K+-current enhancement. We conclude that FFA and NFA blocked the vascular gap junction mediated electrical couplings uniformly in arterioles of the three vascular beds, and complete electrical isolation of the recorded VSMC is obtained at ≧300μM; FFA and NFA also activate BKCa channels in the arteriolar smooth muscle cells in addition to their known inhibitory effects on chloride channels.

Function and Protein Expression of Potassium Channels in Mesenteric Resistance Arteries Isolated from 2K-1C Hypertensive Rats

The present study aimed to evaluate the K+ channels activation in vascular relaxation induced by the nitric oxide (NO) donors ruthenium-derived complex (Terpy) and sodium nitroprusside (SNP), as well as its protein expression, on mesenteric resistance arteries (MRA) isolated from renal hypertensive rats (2K-1C) and sham-operated rats (Sham). The NO donors Terpy and SNP induced relaxation with similar efficacy in isolated MRA from both 2K-1C and Sham rats, although SNP was more potent than Terpy. The maximum relaxation induced by Terpy was decreased when the voltagegated potassium channels were blocked in MRA from Sham, but not in 2K-1C rat arteries. The blockade of ATP-sensitive (KATP), big and small conductance Ca2+-activated (SKCa) or inward rectifier (KIR) potassium channels decreased the maximum relaxation induced by Terpy in MRA from Sham and 2K-1C rats. However, the maximum relaxation induced by SNP was inhibited in Sham but not in 2K-1C rats when the big conductance calcium-activated potassium channel was blocked. However, it remained the same when the other potassium channels were blocked. The protein expression of the SKCa and KATP were not altered in 2K-1C hypertensive rat MRA whereas the expression of KV and BKCa were augmented in MRA from 2K-1C rats. Therefore, the potassium channels play different role on the relaxation induced by SNP and Terpy. The activation of different potassium channels and the protein expression of potassium channels may be differently modulated in arteries from 2K-1C hypertensive rats when compared to normotensive rats.

Relaxant effect of all-trans-retinoic acid via NO-sGC-cGMP pathway and calcium-activated potassium channels in rat mesenteric artery

Intraperitoneal injection of all-trans-retinoic acid (ATRA) results in a reduction of blood pressure in spontaneously hypertensive rats. However, the mechanisms involved in this effect are not clear. We hypothesized that ATRA may relax resistance arteries. In this study, we found that ATRA relaxed phenylephrine-preconstricted mesenteric arterial rings, which were abrogated by the removal of the endothelium. Pretreatment of endothelium-intact arterial rings with an inhibitor of endothelial nitric oxide (NO) synthase, N(G)-nitro-l-arginine methyl ester (l-NAME), or soluble guanylyl cyclase, 1H-[1,2,4]-oxadiazole-[4,3-α]-quinoxaline-1-one, reduced the vasorelaxant effect of ATRA. Incubation of mesenteric arterial rings with ATRA increased the production of NO and cGMP, which were blocked by N(G)-nitro-l-arginine methyl ester. The vasorelaxant effect of ATRA was markedly attenuated in the presence of an inhibitor of big conductance calcium-activated potassium channels (charybdotoxin), but not with an inhibitor of voltage-dependent potassium channel (4-aminopyridine) or ATP-sensitive potassium channel (glibenclamide). Activation of retinoic acid receptors (RARs) with CH55 or retinoic X receptors (RXRs) with LGD1069 induced the vasorelaxation of phenylephrine-preconstricted mesenteric arterial rings. The RAR (BMS493) and RXR (UVI3003) antagonists blocked the ATRA-induced vasorelaxation. The vasorelaxant effect ATRA is physiologically relevant because the intravenous infusion of ATRA decreased blood pressure in normotensive rats. We conclude that ATRA relaxes resistance vessels via both RARs and RXRs receptors that are mediated by the endothelium-dependent NO-cGMP pathway, which may participate in the control of blood pressure.

Naturally occurring monoepoxides of eicosapentaenoic acid and docosahexaenoic acid are bioactive antihyperalgesic lipids

Beneficial physiological effects of long-chain n-3 polyunsaturated fatty acids are widely accepted but the mechanism(s) by which these fatty acids act remains unclear. Herein, we report the presence, distribution, and regulation of the levels of n-3 epoxy-fatty acids by soluble epoxide hydrolase (sEH) and a direct antinociceptive role of n-3 epoxy-fatty acids, specifically those originating from docosahexaenoic acid (DHA). The monoepoxides of the C18:1 to C22:6 fatty acids in both the n-6 and n-3 series were prepared and the individual regioisomers purified. The kinetic constants of the hydrolysis of the pure regioisomers by sEH were measured. Surprisingly, the best substrates are the mid-chain DHA epoxides. We also demonstrate that the DHA epoxides are present in considerable amounts in the rat central nervous system. Furthermore, using an animal model of pain associated with inflammation, we show that DHA epoxides, but neither the parent fatty acid nor the corresponding diols, selectively modulate nociceptive pathophysiology. Our findings support an important function of epoxy-fatty acids in the n-3 series in modulating nociceptive signaling. Consequently, the DHA and eicosapentaenoic acid epoxides may be responsible for some of the beneficial effects associated with dietary n-3 fatty acid intake.

Endothelium-Derived Nitric Oxide Inhibits the Relaxation of the Porcine Coronary Artery to Natriuretic Peptides by Desensitizing Big Conductance Calcium-Activated Potassium Channels of Vascular Smooth Muscle

The present experiments investigated whether endothelium-derived mediators modulate the effect of natriuretic peptides in porcine coronary arteries. Rings with and without endothelium were suspended in organ chambers for isometric tension recording. Concentration-relaxation curves to C-type natriuretic peptide (CNP) and atrial natriuretic peptide (ANP) were obtained during contractions to endothelin-1. Removal of the endothelium potentiated relaxations to both CNP and ANP. N(omega)-nitro-L-arginine methyl ester potentiated relaxations to natriuretic peptides only in arteries with endothelium. Sodium nitroprusside (SNP) inhibited the response to the natriuretic peptides only in the absence of the endothelium. In rings with endothelium, 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) and 4H-8-bromo-1,2,4-oxadiazolo[3,4-d]benz[b][1,4]oxazin-1-one (NS2028) potentiated CNP-mediated relaxations. Iberiotoxin (IBTX) reduced the response only in rings without endothelium. Glybenclamide inhibited the relaxations in both the presence and absence of endothelium. CNP-induced relaxations were reduced by 8-bromoguanosine 3',5'-cGMP (8-bromo-cGMP) to the same extent in rings with and without endothelium. There was no significant difference between the increased cGMP content caused by CNP in porcine coronary arteries with or without endothelium. In patch-clamp studies in porcine coronary arterial smooth muscle cells, the natriuretic peptide-mediated enhancement of the IBTX-sensitive big conductance calcium-activated potassium channel (BK(Ca)) amplitude was reversed by SNP and 8-bromo-cGMP. These findings demonstrate that, in the porcine coronary artery, the opening of BK(Ca) and ATP-dependent potassium channels of the vascular smooth muscle contributes to CNP-mediated relaxations. Endothelium-derived and exogenous NO inhibit the direct relaxing effect of natriuretic peptides by desensitizing the response of the BK(Ca)s of the vascular smooth muscle to the generation of cGMP.

BK channels in Purkinje cell plasma membranes are concentrated in plasmerosomes at sites of hypolemmal cisternae

Calcium-activated potassium channels have been shown to be critically involved in neuronal function but an elucidation of their detailed roles awaits identification of the subcellular domains and microdomains where they are located. This study was undertaken to unravel the precise subcellular distribution of the big-conductance calcium-activated potassium channels (called BK, KCa1.1 or Slo1) in the somato-dendritic compartment of cerebellar Purkinje cells by means of postembedding immunogold cytochemistry and SDS-digested freeze-fracture replica labeling (SDS-FRL). We found BK channels to be unevenly distributed over the Purkinje cell plasma membrane and localized to specific subcellular domains. At distal dendritic compartments, BK channels were scattered over the plasma membrane of shafts and spines, but absent from postsynaptic densities. At the soma and proximal dendrites, BK channels formed two distinct pools. One pool was scattered over the plasma membrane, the other pool was clustered in plasma membrane domains overlying subsurface membrane cisterns, also called hypolemmal cisternae. These subcompartments of the endoplasmic reticulum likely represent calciosomes that unload and refill Ca2+ independently. Purkinje cell subsurface cisterns are enriched in inositol 1,4,5-triphosphate receptors that mediate the effects of several neurotransmitters, hormones and growth factors by releasing Ca2+ into the cytosol, generating local Ca2+ sparks. Such increases in cytosolic [Ca2+] may be sufficient for BK channel activation. Clustered BK channels in the plasma membrane may thus participate in building a functional unit (plasmerosome) with the underlying calciosome that contributes significantly to local signaling in Purkinje cells.

Some structural changes on triazolyl-benzotriazoles and triazolyl-benzimidazolones as potential potassium channel activators. III

This paper reports the synthesis and pharmacological evaluation of some compounds, obtained by structural modifications of 1,2,3-triazolyl-benzotriazoles and 1,2,3-triazolyl-benzimidazolones, which had shown activity as potential activators of the big-conductance calcium-activated potassium channels (BK Ca). Changes have concerned the introduction of a hinderer substituent in the 5-position of the benzimidazolone ( 4a, b) and benzotriazole ( 5a, b) rings, opening of the benzimidazolone ring ( 7) and substitution of the 1,2,3-triazole ring with a 2-hydroxyphenyl ring ( 10). Furthermore a series of 3-aryl-benzotriazin-4-one derivatives ( 13a– e) has been studied, which appears as a modification and/or combination of the benzimidazolone and benzotriazole rings. Only compound 10 shows interesting activity, while the other structural modifications either do not increase (compounds 4 and 5) or reduce (compounds 7 and 13) the pharmacological activity. However, these results provide useful information about structure–activity relationships.

Triazolyl–benzimidazolones and triazolyl–benzotriazoles: new potential potassium channel activators. II

This paper reports the synthesis and pharmacological evaluation of a series of 5-substituted-triazolyl–benzotriazoles ( 2a–f) and the corresponding series of 5-substituted-triazolyl–benzimidazolones ( 6a–f), as potential activators of the big-conductance calcium-activated potassium channels (BK Ca). The synthesis and structure demonstration of the stock compounds of the two series have been described in our previous works, as well as the common starting compounds 4-carboxamido-5-(4-substituted-2-amino-anilino)-1,2,3-triazoles ( 1a–f). The triazolyl–benzotriazoles were obtained by diazotization, while the triazolyl–benzimidazolones were obtained by thermal intramolecular cyclization of ethoxycarbonylamino derivatives or directly with phosgene. Benzimidazolone compounds generally showed little effect whilst the compounds with a benzotriazole ring showed full efficacy, with vasorelaxing properties and potency parameters a little lower than that of the reference compound NS 1619. These effects were significantly reduced by an increased membrane depolarization. This depolarization-sensitive response is in agreement with the pharmacodynamic hypothesis of activation of potassium channels.