Imaging of the calcium activated potassium channel 3.1 (KCa 3.1) in vivo using a senicapoc-derived positron emission tomography tracer.

The calcium-activated potassium channel 3.1 (KCa 3.1) is overexpressed in many tumor entities and has predictive power concerning disease progression and outcome. Imaging of the KCa 3.1 channel in vivo using a radiotracer for positron emission tomography (PET) could therefore establish a potentially powerful diagnostic tool. Senicapoc shows high affinity and excellent selectivity toward the KCa 3.1 channel. We have successfully pursued the synthesis of the 18 F-labeled derivative [18 F]3 of senicapoc using the prosthetic group approach with 1-azido-2-[18 F]fluoroethane ([18 F]6) in a "click" reaction. The biological activity of the new PET tracer was evaluated in vitro and in vivo. Inhibition of the KCa 3.1 channel by 3 was demonstrated by patch clamp experiments and the binding pose was analyzed by docking studies. In mouse and human serum, [18 F]3 was stable for at least one half-life of [18 F]fluorine. Biodistribution experiments in wild-type mice were promising, showing rapid and predominantly renal excretion. An in vivo study using A549-based tumor-bearing mice was performed. The tumor signal could be delineated and image analysis showed a tumor-to-muscle ratio of 1.47 ± 0.24. The approach using 1-azido-2-[18 F]fluoroethane seems to be a good general strategy to achieve triarylacetamide-based fluorinated PET tracers for imaging of the KCa 3.1 channel in vivo.

Protein kinase Cγ in cerebellar Purkinje cells regulates Ca2+-activated large-conductance K+ channels and motor coordination.

The cerebellum, the site where protein kinase C (PKC) was first discovered, contains the highest amount of PKC in the central nervous system, with PKCγ being the major isoform. Systemic PKCγ-knockout (KO) mice showed impaired motor coordination and deficient pruning of surplus climbing fibers (CFs) from developing cerebellar Purkinje cells (PCs). However, the physiological significance of PKCγ in the mature cerebellum and the cause of motor incoordination remain unknown. Using adeno-associated virus vectors targeting PCs, we showed that impaired motor coordination was restored by re-expression of PKCγ in mature PKCγ-KO mouse PCs in a kinase activity-dependent manner, while normal motor coordination in mature Prkcgfl/fl mice was impaired by the Cre-dependent removal of PKCγ from PCs. Notably, the rescue or removal of PKCγ from mature PKCγ-KO or Prkcgfl/fl mice, respectively, did not affect the CF innervation profile of PCs, suggesting the presence of a mechanism distinct from multiple CF innervation of PCs for the motor defects in PKCγ-deficient mice. We found marked potentiation of Ca2+-activated large-conductance K+ (BK) channel currents in PKCγ-deficient mice, as compared to wild-type mice, which decreased the membrane resistance, resulting in attenuation of the electrical signal during the propagation and significant alterations of the complex spike waveform. These changes in PKCγ-deficient mice were restored by the rescue of PKCγ or pharmacological suppression of BK channels. Our results suggest that PKCγ is a critical regulator that negatively modulates BK currents in PCs, which significantly influences PC output from the cerebellar cortex and, eventually, motor coordination.

Role for calcium-activated potassium channels (BK) in migration control of human hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Its high metastasis rate is significantly correlated with poor patient prognosis. Elucidating the molecular mechanism underlying HCC metastasis is essential for HCC treatment. Owing to their high conductance, large-conductance calcium-activated potassium channels (BK channels) play a critical role in the control of membrane potential and have repeatedly been proposed as potential targets for cancer therapy. Emerging evidence suggests that BK channels are involved in the progression of cancer malignancies. The present study investigated the role of BK channels in mediating the hypoxia-stimulated migration of HCC cells both in vitro and in vivo in the absence and presence of various BK channels modulators. We found that BK channels were functionally expressed on the membranes of the SMMC-7721 and Huh7 HCC cell lines. Furthermore, blockage or activation of BK channels on the surface of HCC cells correspondingly inhibited or promoted HCC cell proliferation, migration and invasion in hypoxia conditions, with altered expression and distribution of cell-cell adhesion molecule E-cadherin and typical marker of mesenchymal cells, Vimentin, but not N-cadherin. Hypoxia conditions did not alter BK channels expression but increased its open probability. Moreover, BK channels blocker IbTX significantly inhibited HCC cell remote colonization in HCC cell xenografted mice. In conclusion, the results of this study suggest that blocking BK channels offers an attractive strategy for treating HCC.

Activation of Neuronal Nicotinic Receptors Inhibits Acetylcholine Release in the Neuromuscular Junction by Increasing Ca2+ Flux through Cav1 Channels.

Cholinergic neurotransmission is a key signal pathway in the peripheral nervous system and in several branches of the central nervous system. Despite the fact that it has been studied extensively for a long period of time, some aspects of its regulation still have not yet been established. One is the relationship between the nicotine-induced autoregulation of acetylcholine (ACh) release with changes in the concentration of presynaptic calcium levels. The mouse neuromuscular junction of m. Levator Auris Longus was chosen as the model of the cholinergic synapse. ACh release was assessed by electrophysiological methods. Changes in calcium transients were recorded using a calcium-sensitive dye. Nicotine hydrogen tartrate salt application (10 µM) decreased the amount of evoked ACh release, while the calcium transient increased in the motor nerve terminal. Both of these effects of nicotine were abolished by the neuronal ACh receptor antagonist dihydro-beta-erythroidine and Cav1 blockers, verapamil, and nitrendipine. These data allow us to suggest that neuronal nicotinic ACh receptor activation decreases the number of ACh quanta released by boosting calcium influx through Cav1 channels.

Phylogenetic and developmental analyses indicate complex functions of calcium-activated potassium channels in zebrafish embryonic development.

BACKGROUND: Calcium-activated potassium channels (KCa) are a specific type of potassium channel activated by intracellular calcium concentration changes. This group of potassium channels plays fundamental roles ranging from regulating neuronal excitability to immune cell activation. Many human diseases such as schizophrenia, hypertension, epilepsy, and cancers have been linked to mutations in this group of potassium channels. Although the KCa channels have been extensively studied electrophysiologically and pharmacologically, their spatiotemporal gene expression during embryogenesis remains mostly unknown. RESULTS: Using zebrafish as a model, we identified and renamed 14 KCa genes. We further performed phylogenetic and syntenic analyses on vertebrate KCa genes. Our data revealed that the number of KCa genes in zebrafish was increased, most likely due to teleost-specific whole-genome duplication. Moreover, we examined zebrafish KCa gene expression during early embryogenesis. The duplicated ohnologous genes show distinct and overlapped gene expression. Furthermore, we found that zebrafish KCa genes are expressed in various tissues and organs (somites, fins, olfactory regions, eye, kidney, and so on) and neuronal tissues, suggesting that they may play important roles during zebrafish embryogenesis. CONCLUSIONS: Our phylogenetic and developmental analyses shed light on the potential functions of the KCa genes during embryogenesis related to congenital diseases and human channelopathies.

Opening of Intermediate Conductance Ca2+-Activated K+ Channels in C2C12 Skeletal Muscle Cells Increases the Myotube Diameter via the Akt/Mammalian Target of Rapamycin Pathway.

The activation of potassium channels and the ensuing hyperpolarization in skeletal myoblasts are essential for myogenic differentiation. However, the effects of K+ channel opening in myoblasts on skeletal muscle mass are unclear. Our previous study revealed that pharmacological activation of intermediate conductance Ca2+-activated K+ channels (IKCa channels) increases myotube formation. In this study, we investigated the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO), a Ca2+-activated K+ channel opener, on the mass of skeletal muscle. Application of DCEBIO to C2C12 cells during myogenesis increased the diameter of C2C12 myotubes in a concentration-dependent manner. This DCEBIO-induced hypertrophy was abolished by gene silencing of IKCa channels. However, it was resistant to 1 µM but sensitive to 10 µM TRAM-34, a specific IKCa channel blocker. Furthermore, DCEBIO reduced the mitochondrial membrane potential by opening IKCa channels. Therefore, DCEBIO should increase myotube mass by opening of IKCa channels distributed in mitochondria. Pharmacological studies revealed that mitochondrial reactive oxygen species (mitoROS), Akt, and mammalian target of rapamycin (mTOR) are involved in DCEBIO-induced myotube hypertrophy. An additional study demonstrated that DCEBIO-induced muscle hypertrophic effects are only observed when applied in the early stage of myogenic differentiation. In an in vitro myotube inflammatory atrophy experiment, DCEBIO attenuated the reduction of myotube diameter induced by endotoxin. Thus, we concluded that DCEBIO increases muscle mass by activating the IKCa channel/mitoROS/Akt/mTOR pathway. Our study suggests the potential of DCEBIO in the treatment of muscle wasting diseases. SIGNIFICANCE STATEMENT: Our study shows that 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO), a small molecule opener of Ca2+-activated K+ channel, increased muscle diameter via the mitochondrial reactive oxygen species/Akt/mammalian target of rapamycin pathway. And DCEBIO overwhelms C2C12 myotube atrophy induced by endotoxin challenge. Our report should inform novel role of K+ channel in muscle development and novel usage of K+ channel opener such as for the treatment of muscle wasting diseases.

Calcium-activated potassium channel family in coronary artery bypass grafts.

OBJECTIVES: We examined the expression, distribution, and contribution to vasodilatation of the calcium-activated potassium (KCa) channel family in the commonly used coronary artery bypass graft internal thoracic artery (ITA) and saphenous vein (SV) to understand the role of large conductance KCa (BKCa), intermediate-conductance KCa (IKCa), and small-conductance KCa (SKCa) channel subtypes in graft dilating properties determined by endothelium-smooth muscle interaction that is essential to the postoperative performance of the graft. METHODS: Real-time polymerase chain reaction and western blot were employed to detect the messenger RNA and protein level of KCa channel subtypes. Distribution of KCa channel subtypes was examined by immunohistochemistry. KCa subtype-mediated vasorelaxation was studied using wire myography. RESULTS: Both ITA and SV express all KCa channel subtypes with each subtype distributed in both endothelium and smooth muscle. ITA and SV do not differ in the overall expression level of each KCa channel subtype, corresponding to comparable relaxant responses to respective subtype activators. In ITA, BKCa is more abundantly expressed in smooth muscle than in endothelium, whereas SKCa exhibits more abundance in the endothelium. In comparison, SV shows even distribution of KCa channel subtypes in the 2 layers. The BKCa subtype in the KCa family plays a significant role in vasodilatation of ITA, whereas its contribution in SV is quite limited. CONCLUSIONS: KCa family is abundantly expressed in ITA and SV. There are differences between these 2 grafts in the abundance of KCa channel subtypes in the endothelium and the smooth muscle. The significance of the BKCa subtype in vasodilatation of ITA may suggest the potential of development of BKCa modulators for the prevention and treatment of ITA spasm during/after coronary artery bypass graft surgery.

KCa channels are major contributors to ATP-induced cutaneous vasodilation in healthy older adults.

OBJECTIVE: To examine the contributions of calcium-activated K+ (KCa) channels and nitric oxide synthase (NOS) to adenosine triphosphate (ATP)-induced cutaneous vasodilation in healthy older adults. METHODS: In eleven older adults (69 ± 2 years, 5 females), cutaneous vascular conductance, normalized to maximum vasodilation (%CVCmax) was assessed at four dorsal forearm skin sites that were continuously perfused with either 1) lactated Ringer solution (Control), 2) 50 mM tetraethylammonium (TEA, KCa channel blocker), 3) 10 mM Nω-nitro-L-arginine (L-NNA, NOS inhibitor), or 4) combined 50 mM TEA +10 mM L-NNA, via microdialysis. Local skin temperature was fixed at 33 °C at all sites with local heaters throughout the protocol while the cutaneous vasodilator response was assessed during coadministration of ATP (0.03, 0.3, 3, 30, 300 mM; 20 min per dose), followed by 50 mM sodium nitroprusside and local skin heating to 43 °C to achieve maximum vasodilation (20-30 min). RESULTS: Blockade of KCa channels blunted %CVCmax relative to Control from 0.3 to 300 mM ATP (All P < 0.05). A similar response was observed for the combined KCa channel blockade and NOS inhibition site from 3 to 300 mM ATP (All P < 0.05). Conversely, NOS inhibition alone did not influence %CVCmax across all ATP doses (All P > 0.05). CONCLUSION: In healthy older adults, KCa channels play an important role in modulating ATP-induced cutaneous vasodilation, while the NOS contribution to this response is negligible.

Signaling pathways targeting mitochondrial potassium channels.

In this review, we describe key signaling pathways regulating potassium channels present in the inner mitochondrial membrane. The signaling cascades covered here include phosphorylation, redox reactions, modulation by calcium ions and nucleotides. The following types of potassium channels have been identified in the inner mitochondrial membrane of various tissues: ATP-sensitive, Ca2+-activated, voltage-gated and two-pore domain potassium channels. The direct roles of these channels involve regulation of mitochondrial respiration, membrane potential and synthesis of reactive oxygen species (ROS). Changes in channel activity lead to diverse pro-life and pro-death responses in different cell types. Hence, characterizing the signaling pathways regulating mitochondrial potassium channels will facilitate understanding the physiological role of these proteins. Additionally, we describe in this paper certain regulatory mechanisms, which are unique to mitochondrial potassium channels.

Case Report on: Very Early Afterdepolarizations in HiPSC-Cardiomyocytes-An Artifact by Big Conductance Calcium Activated Potassium Current (Ibk,Ca).

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent an unlimited source of human CMs that could be a standard tool in drug research. However, there is concern whether hiPSC-CMs express all cardiac ion channels at physiological level and whether they might express non-cardiac ion channels. In a control hiPSC line, we found large, "noisy" outward K+ currents, when we measured outward potassium currents in isolated hiPSC-CMs. Currents were sensitive to iberiotoxin, the selective blocker of big conductance Ca2+-activated K+ current (IBK,Ca). Seven of 16 individual differentiation batches showed a strong initial repolarization in the action potentials (AP) recorded from engineered heart tissue (EHT) followed by very early afterdepolarizations, sometimes even with consecutive oscillations. Iberiotoxin stopped oscillations and normalized AP shape, but had no effect in other EHTs without oscillations or in human left ventricular tissue (LV). Expression levels of the alpha-subunit (KCa1.1) of the BKCa correlated with the presence of oscillations in hiPSC-CMs and was not detectable in LV. Taken together, individual batches of hiPSC-CMs can express sarcolemmal ion channels that are otherwise not found in the human heart, resulting in oscillating afterdepolarizations in the AP. HiPSC-CMs should be screened for expression of non-cardiac ion channels before being applied to drug research.

[Ca2+-activated K+ channels as cancer therapeutic targets].

Similar to calcium (Ca2+) and chloride (Cl-) ion channels/transporters, potassium (K+) channels have been recognized as a crucial cancer treatment target. Recent studies have provided convincing evidences of positive correlation between elevated expression levels of Ca2+-activated K+ (KCa) channels and cancer proliferation, metastasis, and poor patient prognosis. In cancer cells, KCa1.1 and KCa3.1 KCa channels are co-localized with Ca2+-permeable Orai/TRP channels to provide a positive-feedback loop for Ca2+ entry. They are responsible for the promotion of cell growth and metastasis in the different types of cancer, and are therefore potential therapeutic targets and biomarkers for cancer. We determined the epigenetic and post-transcriptional dysregulation of KCa3.1 by class I histone deacetylase inhibitors in breast and prostate cancer cells. We further determined the transcriptional repression and protein degradation of KCa1.1 by vitamin D receptor agonists and androgen receptor antagonists, which are expected as potential therapeutic drugs for triple-negative breast cancer. The anti-inflammatory cytokine, interleukin-10 (IL-10) is an immunosuppressive factor involved in tumorigenesis, and plays a crucial role in escape from tumor immune surveillance. We determined KCa3.1 activators are a possible therapeutic option to suppress the tumor-promoting activities of IL-10. These results may provide new insights into cancer treatment focused on Ca2+-activated K+ channels.

Participation of PKG and PKA-related pathways in the IFN-γ induced modulation of the BKCa channel activity in human cardiac fibroblasts.

This study was devoted to elucidating the interferon (IFN)-γ-induced signaling pathway and the interaction between protein kinase G (PKG) and protein kinase A (PKA) through large-conductance Ca(2+)-activated K(+) channels in human cardiac fibroblasts. The IK currents were recorded using a whole-cell patch clamp method. A large depolarization (+50 mV) and a high Ca2+ concentration (pCa 6.0) were used in the internal pipette solution to activate only the KCa channels. Iberiotoxin (Ibtx), which selectively inhibits BKCa channels at a concentration of 100 nmol/l, caused a significant reduction of basal IK. Adding IFN-γ in the presence of Ibtx had no effect on IK. Application of the IFN-γ caused a significant reduction in total K+ current amplitude, recorded with a 500 ms depolarizing pulse duration, to +50 mV from a holding potential of -80 mV. We tested the involvement of the sGC/cGMP/PKG signaling pathway by using specific PKG inhibitor KT 5823, potent sGC inhibitor NS 2028, and specific sGC agonist BAY 41-8543. The obtained data confirmed that only sGC participated in the IFN-γ-mediated BKCa channel modulation, which was mediated further by PKA. This study represents first evidence about the participation of the IFN-γ in the mechanisms responsible for BKCa modulation in HCFs. We also believe that this process occurs via negative crosstalk between the PKG- and PKA-associated pathways.

Aldosterone impairs coronary adenosine-mediated vasodilation via reduced functional expression of Ca2+-activated K+ channels.

Elevated plasma aldosterone (Aldo) levels are associated with greater risk of cardiac ischemic events and cardiovascular mortality. Adenosine-mediated coronary vasodilation is a critical cardioprotective mechanism during ischemia; however, whether this response is impaired by increased Aldo is unclear. We hypothesized that chronic Aldo impairs coronary adenosine-mediated vasodilation via downregulation of vascular K+ channels. Male C57BL/6J mice were treated with vehicle (Con) or subpressor Aldo for 4 wk. Coronary artery function, assessed by wire myography, revealed Aldo-induced reductions in vasodilation to adenosine and the endothelium-dependent vasodilator acetylcholine but not to the nitric oxide donor sodium nitroprusside. Coronary vasoconstriction to endothelin-1 and the thromboxane A2 mimetic U-46619 was unchanged by Aldo. Additional mechanistic studies revealed impaired adenosine A2A, not A2B, receptor-dependent vasodilation by Aldo with a tendency for Aldo-induced reduction of coronary A2A gene expression. Adenylate cyclase inhibition attenuated coronary adenosine dilation but did not eliminate group differences, and adenosine-stimulated vascular cAMP production was similar between Con and Aldo mice. Similarly, blockade of inward rectifier K+ channels reduced but did not eliminate group differences in adenosine dilation whereas group differences were eliminated by blockade of Ca2+-activated K+ (KCa) channels that blunted and abrogated adenosine and A2A-dependent dilation, respectively. Gene expression of several coronary KCa channels was reduced by Aldo. Together, these data demonstrate Aldo-induced impairment of adenosine-mediated coronary vasodilation involving blunted A2A-KCa-dependent vasodilation, independent of blood pressure, providing important insights into the link between plasma Aldo and cardiac mortality and rationale for aldosterone antagonist use to preserve coronary microvascular function.NEW & NOTEWORTHY Increased plasma aldosterone levels are associated with worsened cardiac outcomes in diverse patient groups by unclear mechanisms. We identified that, in male mice, elevated aldosterone impairs coronary adenosine-mediated vasodilation, an important cardioprotective mechanism. This aldosterone-induced impairment involves reduced adenosine A2A, not A2B, receptor-dependent vasodilation associated with downregulation of coronary KCa channels and does not involve altered adenylate cyclase/cAMP signaling. Importantly, this effect of aldosterone occurred independent of changes in coronary vasoconstrictor responsiveness and blood pressure.

Glucose-mediated inhibition of calcium-activated potassium channels limits α-cell calcium influx and glucagon secretion.

Pancreatic α-cells exhibit oscillations in cytosolic Ca2+ (Ca2+c), which control pulsatile glucagon (GCG) secretion. However, the mechanisms that modulate α-cell Ca2+c oscillations have not been elucidated. As ß-cell Ca2+c oscillations are regulated in part by Ca2+-activated K+ (Kslow) currents, this work investigated the role of Kslow in α-cell Ca2+ handling and GCG secretion. α-Cells displayed Kslow currents that were dependent on Ca2+ influx through L- and P/Q-type voltage-dependent Ca2+ channels (VDCCs) as well as Ca2+ released from endoplasmic reticulum stores. α-Cell Kslow was decreased by small-conductance Ca2+-activated K+ (SK) channel inhibitors apamin and UCL 1684, large-conductance Ca2+-activated K+ (BK) channel inhibitor iberiotoxin (IbTx), and intermediate-conductance Ca2+-activated K+ (IK) channel inhibitor TRAM 34. Moreover, partial inhibition of α-cell Kslow with apamin depolarized membrane potential ( Vm) (3.8 ± 0.7 mV) and reduced action potential (AP) amplitude (10.4 ± 1.9 mV). Although apamin transiently increased Ca2+ influx into α-cells at low glucose (42.9 ± 10.6%), sustained SK (38.5 ± 10.4%) or BK channel inhibition (31.0 ± 11.7%) decreased α-cell Ca2+ influx. Total α-cell Ca2+c was similarly reduced (28.3 ± 11.1%) following prolonged treatment with high glucose, but it was not decreased further by SK or BK channel inhibition. Consistent with reduced α-cell Ca2+c following prolonged Kslow inhibition, apamin decreased GCG secretion from mouse (20.4 ± 4.2%) and human (27.7 ± 13.1%) islets at low glucose. These data demonstrate that Kslow activation provides a hyperpolarizing influence on α-cell Vm that sustains Ca2+ entry during hypoglycemic conditions, presumably by preventing voltage-dependent inactivation of P/Q-type VDCCs. Thus, when α-cell Ca2+c is elevated during secretagogue stimulation, Kslow activation helps to preserve GCG secretion.

[A Cellular Pharmacological Approach to the Development of Drugs to Treat Muscle Wasting].

　Skeletal muscle atrophy reduces quality of life and increases mortality. However, there are few available drugs for the treatment of muscle atrophy. Recently, cell signaling pathways involved in skeletal muscle atrophy or hypertrophy have been determined. To develop drugs for skeletal muscle atrophy, we have studied compounds which modulate pathways of myogenic differentiation, a pivotal step for the maintenance of skeletal muscle mass. First, we examined a K+ channel opener on myogenic differentiation, since hyperpolarization is a trigger for skeletal muscle differentiation. 5,6-Dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO), an opener of the small/intermediate conductance Ca2+ activated K+ (SKCa/IKCa) channels, increases myogenic differentiation in C2C12 mouse skeletal myoblasts. This effect was inhibited by TRAM-34, an IKCa channel blocker. This suggests that K+ channels in skeletal muscle stem cells are potential targets for an anti-muscle atrophy drug. Next, we searched for drugs which prevent sepsis-induced muscle atrophy. Lipopolysaccharide (LPS), an inducer of sepsis, attenuates myogenic differentiation in C2C12 myoblasts. LPS also increases the protein expression of myostatin and activates NFκB during differentiation. The TLR4 signal inhibitor TAK-242, and an anti-TNFα neutralizing antibody, reduce these inflammatory responses. Our data suggest that LPS inhibits myogenic differentiation via the NFκB/TNFα pathway. This pathway may be involved in the development of muscle wasting caused by sepsis.

Clock-Generated Temporal Codes Determine Synaptic Plasticity to Control Sleep.

Neurons use two main schemes to encode information: rate coding (frequency of firing) and temporal coding (timing or pattern of firing). While the importance of rate coding is well established, it remains controversial whether temporal codes alone are sufficient for controlling behavior. Moreover, the molecular mechanisms underlying the generation of specific temporal codes are enigmatic. Here, we show in Drosophila clock neurons that distinct temporal spike patterns, dissociated from changes in firing rate, encode time-dependent arousal and regulate sleep. From a large-scale genetic screen, we identify the molecular pathways mediating the circadian-dependent changes in ionic flux and spike morphology that rhythmically modulate spike timing. Remarkably, the daytime spiking pattern alone is sufficient to drive plasticity in downstream arousal neurons, leading to increased firing of these cells. These findings demonstrate a causal role for temporal coding in behavior and define a form of synaptic plasticity triggered solely by temporal spike patterns.

Relaxant effect of Lippia origanoides essential oil in guinea-pig trachea smooth muscle involves potassium channels and soluble guanylyl cyclase.

ETHNOPHARMACOLOGICAL RELEVANCE: Lippia origanoides H.B.K. is an aromatic species used in folk medicine to treat respiratory diseases, including asthma. AIM OF THE STUDY: The aim of this work was to evaluate the relaxing potential and mechanism of action of the L. origanoides (LOO) essential oil in isolated guinea-pig trachea. MATERIALS AND METHODS: Leaves from L. origanoides were collected at experimental fields under organic cultivation, at the Forest Garden of Universidade Estadual de Feira de Santana. Essential oil was extracted by hydrodistillation, analyzed by GC/FID and GC/MS and the volatile constituents were identified. Spasmolytic activity and relaxant mechanism of LOO were assayed in isolated guinea-pig trachea contracted with histamine, carbachol or hyperpolarizing KCl. RESULTS: Chemical analysis revealed the presence of carvacrol (53.89%) as major constituent. LOO relaxed isolated guinea-pig trachea pre-contracted with KCl 60 mM [EC50 = 30.02 µg/mL], histamine 1 µM [EC50 = 9.28 µg/mL] or carbachol 1 µM [EC50 = 51.80 µg/mL]. The pre-incubation of glibenclamide, CsCl, propranolol, indomethacin, hexamethonium, aminophylline or L-NAME in histamine-induced contractions did not alter significantly the relaxant effect of LOO. However, the presence of 4-aminopyridine, tetraethylammonium or methylene blue reduced LOO effect, while the presence of dexamethasone or atropine potentialized the LOO relaxant effect. LOO pre-incubation inhibited carbachol-evoked contractions, with this effect potentialized in the presence of sodium nitroprusside and blocked in the presence of ODQ. CONCLUSIONS: The relaxant mechanism of LOO on the tracheal smooth muscle possibly involves stimulating of soluble guanylyl cyclase with consequent activation of the voltage-gated and Ca2+-activated K+ channels.

Small-conductance Ca2+-activated K+ channels: insights into their roles in cardiovascular disease.

Life-threatening malignant arrhythmias in pathophysiological conditions can increase the mortality and morbidity of patients with cardiovascular diseases. Cardiac electrical activity depends on the coordinated propagation of excitatory stimuli and the generation of action potentials in cardiomyocytes. Action potential formation results from the opening and closing of ion channels. Recent studies have indicated that small-conductance calcium-activated potassium (SK) channels play a critical role in cardiac repolarization in pathophysiological but not normal physiological conditions. The aim of this review is to describe the role of SK channels in healthy and diseased hearts, to suggest cardiovascular pathophysiologic targets for intervention, and to discuss studies of agents that target SK channels for the treatment of cardiovascular diseases.

Differential contributions of Ca2+ -activated K+ channels and Na+ /K+ -ATPases to the generation of the slow afterhyperpolarization in CA1 pyramidal cells.

In many types of CNS neurons, repetitive spiking produces a slow afterhyperpolarization (sAHP), providing sustained, intrinsically generated negative feedback to neuronal excitation. Changes in the sAHP have been implicated in learning behaviors, in cognitive decline in aging, and in epileptogenesis. Despite its importance in brain function, the mechanisms generating the sAHP are still controversial. Here we have addressed the roles of M-type K+ current (IM ), Ca2+ -gated K+ currents (ICa(K) 's) and Na+ /K+ -ATPases (NKAs) current to sAHP generation in adult rat CA1 pyramidal cells maintained at near-physiological temperature (35 °C). No evidence for IM contribution to the sAHP was found in these neurons. Both ICa(K) 's and NKA current contributed to sAHP generation, the latter being the predominant generator of the sAHP, particularly when evoked with short trains of spikes. Of the different NKA isoenzymes, α1 -NKA played the key role, endowing the sAHP a steep voltage-dependence. Thus normal and pathological changes in α1 -NKA expression or function may affect cognitive processes by modulating the inhibitory efficacy of the sAHP.