Intermediate Conductance Calcium-activated Potassium Research Articles

M2 muscarinic receptors negatively modulate cell migration in human glioblastoma cells

Glioblastoma (GB) is a very aggressive human brain tumor. The high growth potential and invasiveness make this tumor surgically and pharmacologically untreatable. Our previous work demonstrated that the activation of the M2 muscarinic acetylcholine receptors (M2 mAChRs) inhibited cell proliferation and survival in GB cell lines and in the cancer stem cells derived from human biopsies. The aim of the present study was to investigate the ability of M2 mAChR to modulate cell migration in two different GB cell lines: U87 and U251. By wound healing assay and single cell migration analysis performed by time-lapse microscopy, we demonstrated the ability of M2 mAChRs to negatively modulate cell migration in U251 but not in the U87 cell line. In order to explain the different effects observed in the two cell lines we have evaluated the possible involvement of the intermediate conductance calcium-activated potassium (IKCa) channel. IKCa channel is present in the GB cells, and it has been demonstrated to modulate cell migration. Using the perforated patch-clamp technique we have found that selective activation of M2 mAChR significantly reduced functional density of the IKCa current in U251 but not in U87 cells. To understand whether the M2 mAChR mediated reduction of ion channel density in the U251 cell line was relevant for the cell migration impairment, we tested the effects of TRAM-34, a selective inhibitor of the IKCa channel, in wound healing assay. We found that it was able to markedly reduce U251 cell migration and significantly decrease the number of invadopodia-like structure formations. These results suggest that only in U251 cells the reduced cell migration M2 mAChR-mediated might involve, at least in part, the IKCa channel.

Functional cooperation between IKCa and TRPC1 channels regulates serum-induced vascular smooth muscle cell proliferation via mediating Ca2+ influx and ERK1/2 activation.

The increased proliferation of vascular smooth muscle cells (VSMCs) contributes to the pathogenesis of vascular diseases. The intermediate conductance calcium-activated potassium (IKCa ) channel plays a critical role in VSMC proliferation by raising the intracellular calcium concentration ([Ca2+ ]i ), but the underlying mechanism is still not unclear. Here we investigated the cooperation between IKCa and transient receptor potential canonical 1 (TRPC1) channels in mediating extracellular Ca2+ entry, which in turn activates downstream Ca2+ signalling in the regulation of VSMC proliferation using serum-induced cell proliferation model. Serum-induced cell proliferation was accompanied with up-regulation of IKCa expression and an increase in [Ca2+ ]i . Serum-induced cell proliferation and increase in [Ca2+ ]i were suppressed by IKCa inhibition with TRAM-34 or IKCa knockdown. Serum-induced cell proliferation was strongly reduced by the removal of extracellular Ca2+ with EGTA or intracellular Ca2+ with BAPTA-AM and, additionally, by TRPC1 knockdown. Moreover, the increase in [Ca2+ ]i induced by serum or by IKCa activation with 1-EBIO was attenuated by TRPC1 knockdown. Finally, serum induced ERK1/2 activation, which was attenuated by treatment with TRAM-34 or BAPTA-AM, as well as TRPC1 knockdown. Consistently, serum-induced cell proliferation was suppressed by ERK1/2 inhibition with PD98059. Taken together, these results suggest that the IKCa and TRPC1 channels cooperate in mediating Ca2+ influx that activates the ERK1/2 pathway to promote cell proliferation, thus providing new mechanistic insights into VSMC proliferation.

LY294002 Inhibits Intermediate Conductance Calcium-Activated Potassium (KCa3.1) Current in Human Glioblastoma Cells.

Glioblastomas (GBs) are among the most common tumors with high malignancy and invasiveness of the central nervous system. Several alterations in protein kinase and ion channel activity are involved to maintain the malignancy. Among them, phosphatidylinositol 3-kinase (PI3K) activity and intermediate conductance calcium-activated potassium (KCa3.1) current are involved in several aspects of GB biology. By using the electrophysiological approach and noise analysis, we observed that KCa3.1 channel activity is LY294002-sensitive and Wortmannin-resistant in accordance with the involvement of PI3K class IIβ (PI3KC2β). This modulation was observed also during the endogenous activation of KCa3.1 current with histamine. The principal action of PI3KC2β regulation was the reduction of open probability in intracellular free calcium saturating concentration. An explanation based on the “three-gate” model of the KCa3.1 channel by PI3KC2β was proposed. Based on the roles of KCa3.1 and PI3KC2β in GB biology, a therapeutic implication was suggested to prevent chemo- and radioresistance mechanisms.

Physiological role of K+ channels in irisin-induced vasodilation in rat thoracic aorta

Irisin, an exercise-induced myokine, has been shown to have a peripheral vasodilator effect. However, little is known about the mechanisms underlying its effects. In this study, it was aimed to investigate the vasoactive effects of irisin on rat thoracic aorta, and the hypothesis that voltage-gated potassium (KV) channels, ATP-sensitive potassium (KATP) channels, small-conductance calcium-activated potassium (SKCa) channels, large-conductance calcium-activated potassium (BKCa) channels, intermediate-conductance calcium-activated potassium (IKCa) channels, inward rectifier potassium (Kir) channels, and two-pore domain potassium (K2P) channels may have roles in these effects. Isometric contraction-relaxation responses of isolated thoracic aorta rings were measured with an organ bath model. The steady contraction was induced with both 10−5 M phenylephrine and 45 mM KCl, and then the concentration-dependent responses of irisin (10-9-10-6 M) were examined. Irisin exerted the vasorelaxant effects in both endothelium-intact and -denuded aortic rings at concentrations of 10-8, 10-7, and 10-6 M (p < 0.001). Besides, KV channel blocker 4-aminopyridine, KATP channel blocker glibenclamide, SKCa channel blocker apamin, BKCa channel blockers tetraethylammonium and iberiotoxin, IKCa channel blocker TRAM-34, and Kir channel blocker barium chloride incubations significantly inhibited the irisin-induced relaxation responses. However, incubation of K2P TASK-1 channel blocker anandamide did not cause a significant decrease in the relaxation responses of irisin. In conclusion, the first physiological findings were obtained regarding the functional relaxing effects of irisin in rat thoracic aorta. Furthermore, this study is the first to report that irisin-induced relaxation responses are associated with the activity of KV, KATP, SKCa, BKCa, IKCa, and Kir channels.

Hippocampal Vascular Dysfunction and Impaired Memory in a Neuroendocrine Model of Stress and Hypertension

The hippocampus is a deep brain structure critically involved in learning and memory that is highly susceptible to hypoxic/ischemic injury. Hypertension and psychological stress are major risk factors of dementia; however, their effects on the vasculature of the cognition-centric hippocampus remain unclear. This study investigated hippocampal vascular function and memory in a novel model of neuroendocrine-derived stress and hypertension. This model is induced via vector mediated brain-derived neurotrophic factor (BDNF) overexpression in the paraventricular nucleus of the hypothalamus (PVN) that causes chronic stimulation of the major hypothalamic stress pathways (e.g. hypothalamic-pituitary-adrenal axis), leading to significant long-term elevation of blood pressure. We hypothesized that function of the arterioles supplying the hippocampus and memory would be impaired in this BDNF-PVN model of chronic stress and hypertension. Potential sex differences in the susceptibility of the hippocampus to dysfunction were also investigated. Male and female Sprague Dawley rats received bilateral PVN injections of AAV2 vectors expressing BDNF or GFP for control (n=6-8/group) at 8 weeks of age. Four weeks later, an object recognition task was used to quantify the percent of time spent investigating a novel object as a measure of hippocampal-dependent long-term memory function. Hippocampal arterioles (HAs) from the same rats were then studied isolated and pressurized in an arteriograph chamber. Vasodilator responses of HAs to mediators of functional hyperemia were measured, including small- and intermediate-conductance calcium-activated potassium (SK/IK) channel activation via NS309, and activation of inward rectifier potassium (KIR) channels by increasing extracellular K+ from 3-15mM. Memory function was significantly impaired by PVN-BDNF treatment (two-way ANOVA, Tukey's post hoc test, F1,22=16.53; p=0.001), but sex had no effect (F1,22=1.16; p=0.29). Object recognition was lower in both male and female PVN-BDNF rats that spent 49±3% and 52±6% of the time with the novel object, compared to control male and female rats that spent 66±3% and 72±4% of the time investigating the novel object. HAs from male and female PVN-BDNF rats dilated significantly less to NS309 than controls (F1,20=12.35; p=0.002), suggesting endothelial damage. Surprisingly, activation of KIR channels with 15mM K+ caused a 3-5% vasoconstriction of HAs from both male and female PVN-BDNF rats that was in striking contrast to the 20-30% vasodilation that occurred in response to KIR channel activation in HAs from control rats (F1,19=24.90; p<0.0001). These data show that chronic neuroendocrine stress and hypertension caused hippocampal vascular dysfunction and impaired memory similarly in male and female rats. The blunted vasodilation of HAs from PVN-BDNF rats suggests functional hyperemia may be impaired in the hippocampus that could lead to neuronal dysfunction, memory loss and dementia.

New Insights on KCa3.1 Channel Modulation.

The human intermediate conductance calcium-activated potassium channel, KCa3.1, is involved in several pathophysiological conditions playing a critical role in cell secretory machinery and calcium signalling. The recent cryo-EM analysis provides new insights for understanding the modulation by both endogenous and pharmacological agents. A typical feature of this channel is the low open probability in saturating calcium concentrations and its modulation by potassium channel openers (KCOs), such as benzo imidazolone 1-EBIO, without changing calcium-dependent activation. In this paper, we proposed a model of KCOs action in the modulation of channel activity. The KCa3.1 channel has a very rich pharmacological profile with several classes of molecules that selectively interact with different binding sites of the channel. Among them, benzo imidazolones can be openers (positive modulators such as 1-EBIO, DC-EBIO) or blockers (negative modulators such as NS1619). Through computation modelling techniques, we identified the 1,4-benzothiazin-3-one as a promising scaffold to develop new KCa3.1 channel modulators. Further studies are needed to explore the potential use of 1-4 benzothiazine- 3-one in KCa3.1 modulation and its pharmacological application.

Activation of the AMP-related kinase (AMPK) induces renal vasodilatation and downregulates Nox-derived reactive oxygen species (ROS) generation

AMP-activated protein kinase (AMPK) is a cellular energy sensor activated during energy stress to stimulate ATP production pathways and restore homeostasis. AMPK is widely expressed in the kidney and involved in mitochondrial protection and biogenesis upon acute renal ischemia, AMPK activity being blunted in metabolic disease-associated kidney disease. Since little is known about AMPK in the regulation of renal blood flow, the present study aimed to assess the role of AMPK in renal vascular function. Functional responses to the selective AMPK activator A769662 were assessed in intrarenal small arteries isolated from the kidney of renal tumour patients and Wistar rats and mounted in microvascular myographs to perform simultaneous measurements of intracellular calcium [Ca2+]i and tension. Superoxide (O2.-) and hydrogen peroxide (H2O2) production were measured by chemiluminescence and fluorescence and protein expression by Western blot. Activation of AMPK with A769662 increased AMPKα phosphorylation at Thr-172 and induced potent relaxations compared to AICAR in isolated human and rat intrarenal arteries, through both endothelium-dependent mechanisms involving nitric oxide (NO) and intermediate-conductance calcium-activated potassium (IKCa) channels, as well as activation of ATP-sensitive (KATP) channels and sarcoplasmic reticulum Ca2+-ATPase (SERCA) in vascular smooth muscle (VSM). Furthermore, AMPK activator reduced NADPH oxidase 4 (Nox4) and Nox2-derived reactive oxygen species (ROS) production. These results demonstrate that A769662 has potent vasodilator and antioxidant effects in intrarenal arteries. The benefits of AMPK activation in rat kidney are reproduced in human arteries and therefore vascular AMPK activation might be a therapeutic target in the treatment of metabolic disease-associated kidney injury.

Modulation of Cardiovascular Function in Primary Hypertension in Rat by SKA-31, an Activator of KCa2.x and KCa3.1 Channels.

The aim of this study was to investigate the hemodynamic effects of SKA-31, an activator of the small (KCa2.x) and intermediate (KCa3.1) conductance calcium-activated potassium channels, and to evaluate its influence on endothelium-derived hyperpolarization (EDH)-KCa2.3/KCa3.1 type relaxation in isolated endothelium-intact small mesenteric arteries (sMAs) from spontaneously hypertensive rats (SHRs). Functional in vivo and in vitro experiments were performed on SHRs or their normotensive controls, Wistar-Kyoto rats (WKY). SKA-31 (1, 3 and 10 mg/kg) caused a brief decrease in blood pressure and bradycardia in both SHR and WKY rats. In phenylephrine-pre-constricted sMAs of SHRs, SKA-31 (0.01–10 µM)-mediated relaxation was reduced and SKA-31 potentiated acetylcholine-evoked endothelium-dependent relaxation. Endothelium denudation and inhibition of nitric oxide synthase (eNOS) and cyclooxygenase (COX) by the respective inhibitors l-NAME or indomethacin, attenuated SKA-31-mediated vasorelaxation. The inhibition of KCa3.1, KCa2.3, KIR and Na+/K+-ATPase by TRAM-34, UCL1684, Ba2+ and ouabain, respectively, reduced the potency and efficacy of the EDH-response evoked by SKA-31. The mRNA expression of eNOS, prostacyclin synthase, KCa2.3, KCa3.1 and KIR were decreased, while Na+/K+-ATPase expression was increased. Collectively, SKA-31 promoted hypotension and vasodilatation, potentiated agonist-stimulated vasodilation, and maintained KCa2.3/KCa3.1-EDH-response in sMAs of SHR with downstream signaling that involved KIR and Na+/K+-ATPase channels. In view of the importance of the dysfunction of endothelium-mediated vasodilatation in the mechanism of hypertension, application of activators of KCa2.3/KCa3.1 channels such as SKA-31 seem to be a promising avenue in pharmacotherapy of hypertension.

KCNN4 promotes invasion and metastasis through the MAPK/ERK pathway in hepatocellular carcinoma

Hepatocellular Carcinoma (HCC) is one of the most common malignancies in the world, and is well-known for its bad prognosis. Potassium calcium-activated channel subfamily N member 4 (KCNN4) is a...

Memory impairment in spontaneously hypertensive rats is associated with hippocampal hypoperfusion and hippocampal vascular dysfunction

We investigated the effect of chronic hypertension on hippocampal arterioles (HippAs) and hippocampal perfusion as underlying mechanisms of memory impairment, and how large artery stiffness relates to HippA remodeling. Using male spontaneously hypertensive rats (SHR) and normotensive Wistar rats (n = 12/group), long-term (LTM) and spatial memory were tested using object recognition and spontaneous alternation tasks. Hippocampal blood flow was measured via hydrogen clearance basally and during hypercapnia. Reactivity of isolated and pressurized HippAs to pressure and pharmacological activators and inhibitors was investigated. To determine large artery stiffness, distensibility and elastin content were measured in thoracic aorta. SHR had impaired LTM and spatial memory associated with decreased basal blood flow (68 ± 12 mL/100 g/min) vs. Wistar (111 ± 28 mL/100 g/min, p < 0.01) that increased during hypercapnia similarly between groups. Compared to Wistar, HippAs from SHR had increased tone at 60 mmHg (58 ± 9% vs. 37 ± 7%, p < 0.01), and decreased reactivity to small- and intermediate-conductance calcium-activated potassium (SK/IK) channel activation. HippAs in both groups were unaffected by NOS inhibition. Decreased elastin content correlated with increased stiffness in aorta of SHR that was associated with increased stiffness and hypertrophic remodeling of HippAs. Hippocampal vascular dysfunction during hypertension could potentiate memory deficits and may provide a therapeutic target to limit vascular cognitive impairment.

Preferred Formation of Heteromeric Channels between Coexpressed SK1 and IKCa Channel Subunits Provides a Unique Pharmacological Profile of Ca2+-Activated Potassium Channels

Three small conductance calcium-activated potassium channel (SK) subunits have been cloned and found to preferentially form heteromeric channels when expressed in a heterologous expression system. The original cloning of the gene encoding the intermediate conductance calcium-activated potassium channel (IKCa) was termed SK4 because of the high homology between channel subtypes. Recent immunovisualization suggests that IKCa is expressed in the same subcellular compartments of some neurons as SK channel subunits. Stochastic optical reconstruction microscopy super-resolution microscopy revealed that coexpressed IKCa and SK1 channel subunits were closely associated, a finding substantiated by measurement of fluorescence resonance energy transfer between coexpressed fluorophore-tagged subunits. Expression of homomeric SK1 channels produced current that displayed typical sensitivity to SK channel inhibitors, while expressed IKCa channel current was inhibited by known IKCa channel blockers. Expression of both SK1 and IKCa subunits gave a current that displayed no sensitivity to SK channel inhibitors and a decreased sensitivity to IKCa current inhibitors. Single channel recording indicated that coexpression of SK1 and IKCa subunits produced channels with properties intermediate between those observed for homomeric channels. These data indicate that SK1 and IKCa channel subunits preferentially combine to form heteromeric channels that display pharmacological and biophysical properties distinct from those seen with homomeric channels.

The potassium channel KCa3.1 promotes cell proliferation by activating SKP2 and metastasis through the EMT pathway in hepatocellular carcinoma.

The intermediate conductance calcium-activated potassium channel (KCa3.1) plays an important role in maintaining intracellular calcium homeostasis and is involved in the tumorigenesis of many human cancers. However, it is unknown whether KCa3.1 plays a role in the genesis of hepatocellular carcinoma (HCC), one of the most common malignant tumors worldwide with a very poor prognosis. In our study, we found that the expression of KCa3.1 was significantly elevated in poorly differentiated HCC tissues compared to adjacent noncancerous tissues. In vitro and in vivo experiments showed that KCa3.1 could promote cell proliferation, migration, and invasion of HCC. Mechanistically, KCa3.1 promoted cell cycle progression and migration and invasion of HCC cells by activating S-phase protein kinase 2 (SKP2) to trigger the degradation of p21 and p27 and targeting Reelin (RELN) to induce epithelial-mesenchymal transition (EMT), respectively. Taken together, our results demonstrate that KCa3.1 plays an important role in the genesis and progression of HCC, implying that it might be a promising therapeutic target in HCC.

The role of small and intermediate conductance calcium-activated potassium channels in endothelial-dependent hyperpolarization in physiology and arterial hypertension

The endothelium plays a crucial role in modulating vascular tone by synthesizing and releasing endothelium-derived relaxing factors, including nitric oxide (NO) and prostacyclin I2 (PGI2). Additionally, endothelium-dependent hyperpolarization (EDH) that is NO – and PGI2–independent participates in the relaxation of small-diameter blood vessels (&lt;300 μm). EDH response is initiated by agonists (e.g. acetylcholine, bradykinin) – or shear stress – induced increase of calcium ions level in the endothelium and involves opening of the endothelial small (KCa2.3) and intermediate conductance (KCa3.1) calcium-activated potassium channels. The efflux of potassium ions could elicit the hyperpolarization of the surrounding myocytes by the activation of the inward-rectifier potassium ion channel (KIR) and/or Na+/K+-ATPase. The reduced release and/or bioavailability of NO, which is characteristic for endothelial dysfunction and may result in arterial hypertension, stimulate the generation of EDH signals, as a compensatory mechanism to maintain the endothelial control of vasodilator tone. The contribution of EDH in endothelium-dependent relaxation varies between vascular beds, animal and experimental model. In arterial hypertension the reduced expression/activity of KCa3.1 and KCa2.3 results in impaired vasorelaxation. Currently, the use of modulatory compounds (activators and inhibitors) of KCa3.1 and KCa2.3 as the potential therapeutic targets in cardiovascular diseases is under intensive investigation. It has already been known that application of activators of KCa3.1 and KCa2.3 potassium channels such (as SKA-31) can improve the EDH-type responses, the endothelial function and decrease mean arterial blood pressure. This may suggest the usefulness of these compounds in the treatment of arterial hypertension.

Impaired Mesenteric Arterial Function of Male UC Davis Type 2 Diabetes Mellitus (UCD‐T2DM) Rats: Possible Involvement of Small Conductance Calcium‐activated Potassium Channels (SKca)

UC Davis type‐2 diabetes mellitus (UCD‐T2DM) model is a novel and validated model of type‐2 diabetes exhibiting etiology very similar to clinical T2DM seen in humans. This model is characterized by polygenic adult‐onset obesity and diabetes observed in both sexes along with development of insulin resistance, impaired glucose tolerance, and subsequent β‐cell decompensation. We have previously shown that vascular reactivity in mesenteric arteries (MA) of male UCD‐T2DM is altered. Endothelium‐dependent hyperpolarizing factors (EDHF) is an important regulator of vascular tone, especially in small vessels such as MA. The objective of this study was to investigate the contribution of small or intermediate conductance calcium‐activated potassium channels (SKca &amp; IKca) in modulating vascular function of MA in this model. Endothelium‐dependent vasodilation (EDV) to acetylcholine (ACh, 10−8 to 10−5 M) in pre‐contracted MA with phenylephrine (PE, 2μM) was measured before and after pretreatment with indomethacin (Indo, 10μm), a cyclooxygenase inhibitor, followed by addition of L‐NAME (200uM), a nitric oxide synthase inhibitor, and then Apamin (1μm), a selective inhibitor of SKca; and TRAM‐34 (1μm), a selective inhibitor of IKca. We demonstrated that the sensitivity to ACh‐induced relaxation was significantly reduced in MA from male UCD‐T2DM rats compared to controls. Addition of L‐NAME reduced Indo‐insensitive vasorelaxation in both control and diabetic animals. However, when compared with that of control, the effect of L‐NAME was much greater in MA of UCD‐T2DM, indicating a reduced contribution of EDHFtype relaxation or an enhanced role of NO in this disease model. The remaining EDV to ACh which is referred to as the L‐NAME/Indo‐insensitive component, or EDHF‐type relaxation was subsequently blocked by inhibiting intermediate (but not small) conductance calcium‐activated potassium channels in MA of diabetic model. In control animals, however, incubation of MA with apamin (a SKca inhibitor) led to a further reduction of EDV. The subsequent addition of TRAM‐34 eventually blunted the EDHF‐type relaxation in this group. These data, for the first time, show that the contribution of EDHF in EDV is reduced in the MA of UCD‐T2DM group, and the down regulation of SKca may be involved. Clearly, further studies will be needed to document the underlying mechanism(s) of altered vascular function in male UCD‐T2DM rats.Support or Funding InformationSupported by NIH/NHLBIThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Detection of salivary protein biomarkers of saliva secretion disorder in a primary Sjögren syndrome murine model

Saliva secretion disorder is one of the most common symptoms in primary Sjögren syndrome (pSS). Salivary biomarkers related to saliva secretion disorder were identified in a pSS murine model, NOD/ShiLtJ mouse, using differential proteomic analysis. Candidate biomarkers were screened with Kyoto Encyclopedia of Genes and Genomes pathway analysis and validated in saliva and salivary glands by western blotting and immunohistochemistry (IHC). Biological functions were detected using ingenuity pathway analysis (IPA). We identified 1101 salivary proteins from NOD/ShiLtJ mice and BALB/c mice (control). Catenin alpha-3 (CTNNA3), tyrosine-protein phosphatase non-receptor type 6 (PTPN6), Ras-related C3 botulinum toxin substrate (RAC2), and intermediate conductance calcium-activated potassium channel protein 4 (KCNN4) were screened as candidate biomarkers from 225 significantly dysregulated salivary proteins. These proteins participated in adherens junction or saliva secretion pathway and may be related to saliva secretion disorder. Proteomic analysis revealed that CTNNA3, PTPN6, and RAC2 were dysregulated in saliva and salivary glands and showed satisfactory sensitivity and specificity in receiver-operating characteristic curve; KCNN4 showed no statistical difference. IHC and IPA indicated that CTNNA3 may regulate acinar cell morphology, while PTPN6 and RAC2 promoted lymphocyte adhesion in salivary glands. Thus, CTNNA3, PTPN6, and RAC2 may be related to saliva secretion disorder in pSS.

Mechanisms of l-alpha-lysophosphatidylinositol-induced relaxation in human pulmonary arteries

Aimsl-Alpha-lysophosphatidylinositol (LPI) is an endogenous agonist of G protein-coupled receptor 55 (GPR55) which relaxes mesenteric arteries on activation. The aim of the present study was to determine the influence and underlying mechanisms of LPI-induced relaxation in human pulmonary arteries (hPAs). Main methodsFunctional studies were performed in isolated hPAs using organ bath technique. The expression of GPR55 in hPAs and bronchioles was determined by real-time qPCR, Western blot analysis, and immunohistochemistry. Key findingsLPI induced a concentration-dependent vasorelaxation in endothelium-intact hPAs. This effect was attenuated by the GPR55 antagonist CID16020046, the peroxisome proliferator-activated receptor-γ (PPARγ) antagonist GW9662, the putative endothelial cannabinoid receptor (CBe) antagonist O-1918 and the inhibitor of nitric oxide (NO) synthase (L-NAME). In addition, vasorelaxation was also attenuated by the presence of a high KCl concentration, selective blockers of small (KCa2.3; UCL1684), intermediate (KCa3.1; TRAM-34) and large conductance (KCa1.1; iberiotoxin) calcium-activated potassium channels and by endothelium denudation. However, vasorelaxation was not attenuated by the cannabinoid CB1 receptor antagonist AM251 or by the cyclooxygenase inhibitor indomethacin. SignificanceThe study showed that the LPI-induced vasorelaxation was endothelium-dependent and mediated by GPR55, PPARγ and CBe receptors, occurred in a NO- and calcium-activated potassium channel-dependent manner in isolated hPAs. LPI seems to possess positive, hypotensive properties in pulmonary vascular bed.

The influence of DOCA-salt hypertension and chronic administration of the FAAH inhibitor URB597 on KCa2.3/KCa3.1-EDH-type relaxation in rat small mesenteric arteries

The aim of this study was to examine the influence of deoxycorticosterone acetate-salt (DOCA-salt) hypertension and chronic treatment with the fatty acid amide hydrolase inhibitor, URB597, on small and intermediate conductance calcium-activated potassium channels and endothelium-dependent hyperpolarization (KCa2.3/KCa3.1-EDH) in rat small mesenteric arteries (sMAs).The EDH-type response was investigated, in endothelium-intact sMAs using a wire myograph, by examining acetylcholine-evoked vasorelaxation in the presence of Nω-nitro-L-arginine methyl ester and indomethacin (inhibitors of nitric oxide synthase and cyclooxygenase, respectively). In normo- and hypertension the efficacy of EDH-type relaxation was similar and inhibition of KCa2.3 and KCa3.1 by UCL1684 and TRAM-34, respectively, given alone or in combination, attenuated EDH-mediated vasorelaxation. KCa3.1 expression and NS309 (KCa2.3/KCa3.1 activator)-induced relaxation was reduced in sMAs of DOCA-salt rats. Endothelium denudation and incubation with UCL1684 and TRAM-34 attenuated the maximal NS309-evoked vasorelaxation in both groups. URB597 had no effect in functional studies, but increased the expression of KCa3.1 in the sMAs.KCa2.3/KCa3.1-EDH-mediated relaxation was maintained in the sMAs of DOCA-salt rats despite endothelial dysfunction and down-regulation of KCa3.1. Furthermore, KCa3.1 played a key role in the EDH-type dilator response of sMAs in normo- and hypertension. The hypotensive effect of URB597 is independent of KCa2.3/KCa3.1-EDH-type relaxation.

KCa3.1 channels modulate the processing of noxious chemical stimuli in mice

Intermediate conductance calcium-activated potassium channels (KCa3.1) have been recently implicated in pain processing. However, the functional role and localization of KCa3.1 in the nociceptive system are largely unknown. We here characterized the behavior of mice lacking KCa3.1 (KCa3.1−/−) in various pain models and analyzed the expression pattern of KCa3.1 in dorsal root ganglia (DRG) and the spinal cord. KCa3.1−/− mice demonstrated normal behavioral responses in models of acute nociceptive, persistent inflammatory, and persistent neuropathic pain. However, their behavioral responses to noxious chemical stimuli such as formalin and capsaicin were increased. Accordingly, formalin-induced nociceptive behavior was increased in wild-type mice after administration of the KCa3.1 inhibitor TRAM-34. In situ hybridization experiments detected KCa3.1 in most DRG satellite glial cells, in a minority of DRG neurons, and in ependymal cells lining the central canal of the spinal cord. Together, our data point to a specific inhibitory role of KCa3.1 for the processing of noxious chemical stimuli.

IK channel activation increases tumor growth and induces differential behavioral responses in two breast epithelial cell lines.

Many potassium channel families are over-expressed in cancer, but their mechanistic role in disease progression is poorly understood. Potassium channels modulate membrane potential (Vmem) and thereby influence calcium ion dynamics and other voltage-sensitive signaling mechanisms, potentially acting as transcriptional regulators. This study investigated the differential response to over-expression and activation of a cancer-associated potassium channel, the intermediate conductance calcium-activated potassium channel (IK), on aggressive behaviors in mammary epithelial and breast cancer cell lines. IK was over-expressed in the highly metastatic breast cancer cell line MDA-MB-231 and the spontaneously immortalized breast epithelial cell line MCF-10A, and the effect on cancer-associated behaviors was assessed. IK over-expression increased primary tumor growth and metastasis of MDA-MB-231 in orthotopic xenografts, demonstrating for the first time in any cancer type that increased IK is sufficient to promote cancer aggression. The primary tumors had similar vascularization as determined by CD31 staining and similar histological characteristics. Interestingly, despite the increased in vivo growth and metastasis, neither IK over-expression nor activation with agonist had a significant effect on MDA-MB-231 proliferation, invasion, or migration in vitro. In contrast, IK decreased MCF-10A proliferation and invasion through Matrigel but had no effect on migration in a scratch-wound assay. We conclude that IK activity is sufficient to promote cell aggression in vivo. Our data provide novel evidence supporting IK and downstream signaling networks as potential targets for cancer therapies.

KCa3.1 as an Effective Target for Inhibition of Growth and Progression of Intrahepatic Cholangiocarcinoma.

Background: Intrahepatic cholangiocarcinoma (ICC) is a high malignant tumor arising from the bile ducts in the liver with a poor prognosis. As current molecular targeted therapies and systemic chemotherapies had limited success in ICC, novel therapeutic targets are needed. In this study, we attempted to investigate the expression and the role of the intermediate conductance calcium-activated potassium channel (KCa3.1) in ICC.Methods: The expression levels of KCa3.1 channel were measured in 81 resected ICC tumor specimens and the clinicopathological significance of these levels were determined. KCa3.1 channel inhibitor and siRNA were used to study the role of KCa3.1 in proliferation, migration, and invasion of ICC cell lines. The effect of KCa3.1 channel blockade on tumor growth in vivo was also studied using xenograft model in nude mice.Results: The protein expression of KCa3.1 channel was upregulated in ICC tissues and was correlated with age, lymph node metastasis and TNM stage. And high KCa3.1 expression indicated a worse prognosis in ICC patients. Blocking KCa3.1 channel with a specific inhibitor TRAM-34 reduced the proliferation and invasion of ICC cells. Knockdown of KCa3.1 could achieve the same effects through decreasing NF-κB activation. Further in vivo studies demonstrated that KCa3.1 channel blockade suppressed ICC tumor growth.Conclusions: Our observations suggested KCa3.1 might be a promising novel therapeutic target in intrahepatic cholangiocarcinoma.