Rapid Propagation of Ca2+ Waves and Electrical Signals in the Liverwort Marchantia polymorpha.

In response to both biotic and abiotic stresses, vascular plants transmit long-distance Ca2+ and electrical signals from localized stress sites to distant tissues through their vasculature. Various models have been proposed for the mechanisms underlying the long-distance signaling, primarily centered around the presence of vascular bundles. We here demonstrate that the non-vascular liverwort Marchantia polymorpha possesses a mechanism for propagating Ca2+ waves and electrical signals in response to wounding. The propagation velocity of these signals was approximately 1-2 mm s-1, equivalent to that observed in vascular plants. Both Ca2+ waves and electrical signals were inhibited by La3+ as well as tetraethylammonium chloride, suggesting the crucial importance of both Ca2+ channel(s) and K+ channel(s) in wound-induced membrane depolarization as well as the subsequent long-distance signal propagation. Simultaneous recordings of Ca2+ and electrical signals indicated a tight coupling between the dynamics of these two signaling modalities. Furthermore, molecular genetic studies revealed that a GLUTAMATE RECEPTOR-LIKE (GLR) channel plays a central role in the propagation of both Ca2+ waves and electrical signals. Conversely, none of the three two-pore channels were implicated in either signal propagation. These findings shed light on the evolutionary conservation of rapid long-distance Ca2+ wave and electrical signal propagation involving GLRs in land plants, even in the absence of vascular tissue.

Effects on low threshold mechanoreceptors in whisker hair follicles by 5-HT, Cd2+, tetraethylammonium, 4-aminopyridine, and Ba2.

Low threshold mechanoreceptors (LTMRs) are important for environmental exploration, social interaction, and tactile discrimination. Whisker hair follicles are mechanical sensory organs in non-primate mammals that are functionally equivalent to human fingertips. Several functional types of LTMRs have been identified in rodent whisker hair follicles, including rapidly adapting (RA), slow adapting type 1 (SA1), and slowly adapting type 2 (SA2) LTMRs. Properties of these LTMRs have not been fully characterized. In the present study, we have used pressure-clamped single-fiber recording technique to record impulses of RA, SA1, and SA2 LTMRs in mouse whisker hair follicles, and tested effects of 5-HT, Cd2+, tetraethylammonium (TEA), 4-aminopyridine (4-AP), and Ba2+ on the LTMR impulses. We show that 5-HT at 2 mM suppresses SA1 impulses but has no effects on RA and SA2 impulses. Cd2+ at 100 µM suppresses both SA1 and SA2 impulses but has no effects on RA impulses. TEA at 10 mM has no effects on RA and SA1 impulses but increased SA2 impulses. However, TEA at 1 mM and 200 µM decreases SA2 impulses. 4-AP at 1 mM suppresses both SA1 and SA2 impulses but has no effects on RA impulses. Ba2+ at 5 mM increases both RA and SA1 impulses but suppresses SA2 impulses. Collectively, RA, SA1, and SA2 LTMRs show distinct pharmacological properties, suggesting that these LTMRs may use different mechanisms to tune their mechanical signaling.

Inactivation of Native K Channels.

We have experimented with isolated cardiomyocytes of mollusks Helix. During the whole-cell patch-clamp recordings of K+ currents a considerable decrease in amplitude was observed upon repeated voltage steps at 0.96 Hz. For these experiments, ventricular cells were depolarized to identical + 20 mV from a holding potential of - 50 mV. The observed spontaneous inhibition of outward currents persisted in the presence of 4-aminopyridine, tetraethylammonium chloride or E-4031, the selective class III antiarrhythmic agent that blocks HERG channels. Similar tendency was retained when components of currents sensitive to either 4-AP or TEA were mathematically subtracted. Waveforms of currents sensitive to 1 and 10 micromolar concentration of E-4031 were distinct comprising prevailingly those activated during up to 200 ms pulses. The outward current activated by a voltage ramp at 60 mV x s-1 rate revealed an inward rectification around + 20 mV. This feature closely resembles those of the mammalian cardiac delayed rectifier IKr.

Effects of tetraethylammonium-sensitive K+ channel blockade on cholinergic and thermal sweating in endurance-trained and untrained men.

NEW FINDINGS: What is the central question of this study? Does inhibition of K+ channels modulate the exercise-training-induced augmentation in cholinergic and thermal sweating? What is the main finding and its importance? Iontophoretic administration of tetraethylammonium, a K+ channel blocker, blunted sweating induced by a low dose (0.001%) of the cholinergic agent pilocarpine, but not heat-induced sweating. However, no differences in the cholinergic sweating were observed between young endurance-trained and untrained men. Thus, while K+ channels play a role in the regulation of eccrine sweating, they do not contribute to the increase in sweating commonly observed in endurance-trained adults. Our findings provide important new insights into the mechanisms underlying the regulation of sweating by endurance conditioning. ABSTRACT: We evaluated the hypothesis that the activation of K+ channels mediates the exercise-training-induced augmentation of cholinergic and thermal sweating. On separate days, 11 endurance-trained and 10 untrained men participated in two experimental protocols. Prior to each protocol, we administered 2% tetraethylammonium (TEA, K+ channels blocker) and saline (Control) at forearm skin sites on both arms via transdermal iontophoresis. In protocol 1, low (0.001%) and high (1%) doses of pilocarpine were administered at the TEA-treated and Control sites over a 60-min period. In protocol 2, participants were passively heated by immersing their lower limbs in hot water (43°C) until core (rectal) temperature (Tc ) increased by 0.8°C above resting levels. Administration of TEA attenuated cholinergic sweating (P = 0.001) during the initial 20 min after the treatment of low dose of pilocarpine only whilst the response was similar between the groups (P = 0.163). Cholinergic and thermal sweating were higher in the trained relative to the untrained men (all P ≤ 0.033). Thermal sweating reached ∼90% of the response at a Tc elevation of 0.8°C during the initial 20 min of passive heating, which corresponds to the period wherein TEA attenuated cholinergic sweating in protocol 1. However, sweating did not differ between the Control and TEA sites in either group (P = 0.704). We showed that activation of K+ channels does not appear to mediate the elevated sweating response induced by a low dose of pilocarpine in trained men. We also demonstrated that K+ channels do not contribute to sweating during heat stress in either group.

The role of potassium channels in the proliferation and migration of endometrial adenocarcinoma HEC1-A cells.

BACKGROUND: Endometrial cancer is the most common gynecological cancer in developed countries. Potassium channels, which have many types, are suggested to play a major role in cancer progression. However, their role in endometrial cancer has not been fully investigated. We aimed to demonstrate whether the ATP-sensitive potassium channel blocker glibenclamide, voltage-sensitive potassium channel blocker 4-aminopyridine, non-selective (voltage-sensitive and calcium-activated) potassium channels blocker tetraethylammonium and potassium chloride (KCl) have any effect on the proliferation and migration of HEC1-A cells. METHODS AND RESULTS: Proliferation and migration were evaluated by real-time cell analysis (xCELLigence system) and wound healing assays, respectively. Proliferation was reduced by glibenclamide (0.1 and 0.2 mM, P < 0.05 and P < 0.01, respectively), 4-aminopyridine (10 and 20 mM, P < 0.001) and tetraethylammonium (10 and 20 mM, P < 0.01 and P < 0.001, respectively). However, KCl did not change the proliferation. Migration was reduced by glibenclamide (0.01, 0.1 and 0.2 mM, P < 0.001, P < 0.001 and P < 0.01, respectively) and 4-aminopyridine (10 and 20 mM, P < 0.05 and P < 0.01, respectively). Tetraethylammonium did not change migration. However, KCl reduced it (10, 25 and 50 mM, P < 0.05, P < 0.01 and P < 0.01, respectively). Both proliferation and migration were reduced by glibenclamide and 4-aminopyridine. However, tetraethylammonium only reduced proliferation and KCl only reduced migration. CONCLUSIONS: Potassium channels have an important role in HEC1-A cell proliferation and migration and potassium channel blockers needs to be further investigated for their therapeutic effect in endometrial cancer.

Tetraethylammonium chloride reduces anaesthetic-induced neurotoxicity in Caenorhabditis elegans and mice.

BACKGROUND: If anaesthetics cause permanent cognitive deficits in some children, the implications are enormous, but the molecular causes of anaesthetic-induced neurotoxicity, and consequently possible therapies, are still debated. Anaesthetic exposure early in development can be neurotoxic in the invertebrate Caenorhabditis elegans causing endoplasmic reticulum (ER) stress and defects in chemotaxis during adulthood. We screened this model organism for compounds that alleviated neurotoxicity, and then tested these candidates for efficacy in mice. METHODS: We screened compounds for alleviation of ER stress induction by isoflurane in C. elegans assayed by induction of a green fluorescent protein (GFP) reporter. Drugs that inhibited ER stress were screened for reduction of the anaesthetic-induced chemotaxis defect. Compounds that alleviated both aspects of neurotoxicity were then blindly tested for the ability to inhibit induction of caspase-3 by isoflurane in P7 mice. RESULTS: Isoflurane increased ER stress indicated by increased GFP reporter fluorescence (240% increase, P<0.001). Nine compounds reduced induction of ER stress by isoflurane by 90-95% (P<0.001 in all cases). Of these compounds, tetraethylammonium chloride and trehalose also alleviated the isoflurane-induced defect in chemotaxis (trehalose by 44%, P=0.001; tetraethylammonium chloride by 23%, P<0.001). In mouse brain, tetraethylammonium chloride reduced isoflurane-induced caspase staining in the anterior cortical (-54%, P=0.007) and hippocampal regions (-46%, P=0.002). DISCUSSION: Tetraethylammonium chloride alleviated isoflurane-induced neurotoxicity in two widely divergent species, raising the likelihood that it may have therapeutic value. In C. elegans, ER stress predicts isoflurane-induced neurotoxicity, but is not its cause.

The Amyloid Precursor Protein C99 Fragment Modulates Voltage-Gated Potassium Channels.

BACKGROUND/AIMS: The Amyloid Precursor Protein (APP) is involved in the regulation of multiple cellular functions via protein-protein interactions and has been most studied with respect to Alzheimer's disease (AD). Abnormal processing of the single transmembrane-spanning C99 fragment of APP contributes to the formation of amyloid plaques, which are causally related to AD. Pathological C99 accumulation is thought to associate with early cognitive defects in AD. Here, unexpectedly, sequence analysis revealed that C99 exhibits 24% sequence identity with the KCNE1 voltage-gated potassium (Kv) channel ß subunit, comparable to the identity between KCNE1 and KCNE2-5 (21-30%). This suggested the possibility of C99 regulating Kv channels. METHODS: We quantified the effects of C99 on Kv channel function, using electrophysiological analysis of subunits expressed in Xenopus laevis oocytes, biochemical and immunofluorescence techniques. RESULTS: C99 isoform-selectively inhibited (by 30-80%) activity of a range of Kv channels. Among the KCNQ (Kv7) family, C99 isoform-selectively inhibited, shifted the voltage dependence and/or slowed activation of KCNQ2, KCNQ3, KCNQ2/3 and KCNQ5, with no effects on KCNQ1, KCNQ1-KCNE1 or KCNQ4. C99/APP co-localized with KCNQ2 and KCNQ3 in adult rat sciatic nerve nodes of Ranvier. Both C99 and full-length APP co-immunoprecipitated with KCNQ2 in vitro, yet unlike C99, APP only weakly affected KCNQ2/3 activity. Finally, C99 altered the effects on KCNQ2/3 function of inhibitors tetraethylammounium and XE991, but not openers retigabine and ICA27243. CONCLUSION: Our findings raise the possibility of C99 accumulation early in AD altering cellular excitability by modulating Kv channel activity.

Blocking effect of ferritin on the ryanodine receptor-isoform 2.

Iron, an essential element for most living organism, participates in a wide variety of physiological processes. Disturbance in iron homeostasis has been associated with numerous pathologies, particularly in the heart and brain, which are the most susceptible organs. Under iron-overload conditions, the generation of reactive oxygen species leads to impairment in Ca2+ signaling, fundamentally implicated in cardiac and neuronal physiology. Since iron excess is accompanied by increased expression of iron-storage protein, ferritin, we examined whether ferritin has an effect on the ryanodine receptor - isoform 2 (RYR2), which is one of the major components of Ca2+ signaling. Using the method of planar lipid membranes, we show that ferritin induced an abrupt, permanent blockage of the RYR2 channel. The ferritin effect was strongly voltage dependent and competitively antagonized by cytosolic TEA+, an impermeant RYR2 blocker. Our results collectively indicate that monomeric ferritin highly likely blocks the RYR2 channel by a direct electrostatic interaction within the wider region of the channel permeation pathway.

Antispasmodic Effect of Asperidine B, a Pyrrolidine Derivative, through Inhibition of L-Type Ca2+ Channel in Rat Ileal Smooth Muscle.

Antispasmodic agents are used for modulating gastrointestinal motility. Several compounds isolated from terrestrial plants have antispasmodic properties. This study aimed to explore the inhibitory effect of the pyrrolidine derivative, asperidine B, isolated from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178, on spasmodic activity. Isolated rat ileum was set up in an organ bath. The contractile responses of asperidine B (0.3 to 30 µM) on potassium chloride and acetylcholine-induced contractions were recorded. To investigate its antispasmodic mechanism, CaCl2, acetylcholine, Nω-nitro-l-arginine methyl ester (l-NAME), nifedipine, methylene blue and tetraethylammonium chloride (TEA) were tested in the absence or in the presence of asperidine B. Cumulative concentrations of asperidine B reduced the ileal contraction by ~37%. The calcium chloride and acetylcholine-induced ileal contraction was suppressed by asperidine B. The effects of asperidine B combined with nifedipine, atropine or TEA were similar to those treated with nifedipine, atropine or TEA, respectively. In contrast, in the presence of l-NAME and methylene blue, the antispasmodic effect of asperidine B was unaltered. These results suggest that the antispasmodic property of asperidine B is probably due to the blockage of the L-type Ca2+ channel and is associated with K+ channels and muscarinic receptor, possibly by affecting non-selective cation channels and/or releasing intracellular calcium.

T-type Ca2+ channel activity increases in rat hippocampal CA1 region during kindling epileptogenesis.

Epileptogenesis is a dynamical process that involves synaptic plasticity changes such as synaptic reorganization of excitatory and inhibitory systems and axonal sprouting in the hippocampus, which is one of the most studied epileptogenic regions in the brain. However, the early events that trigger these changes are not understood well. We investigated short-term and long-term synaptic plasticity parameters and T-type Ca2+ channel activity changes in the early phase of a rat kindling model. Chronic pentylenetetrazole (PTZ) application was used in order to induce the kindling process in rats. The recordings were obtained from hippocampal slices in the CA1 region at 25th day of PTZ application. Tetraethylammonium was used in order to induce long-term potentiation and T-type Ca2+ channel activity was assessed in the presence of mibefradil. We found that tetraethylammonium-induced long-term potentiation was not prevented by mibefradil in the kindling group in contrast to control group. We also found an increase in paired-pulse ratios in the PTZ-applied group. Our findings indicate an increase in the "T-type Ca2+ channel component of LTP" in the kindling group, which may be an early mechanism in epileptogenesis.

The beneficial effect of clove essential oil and its major component, eugenol, on erectile function in diabetic rats.

Diabetic men are at a higher risk of erectile dysfunction (ED). A tropical plant, clove (Syn. Eugenia caryophyllata, Caryophyllus aromaticus L., Syzygium aromaticum (L.) Merr. & L.M. Perry) from the Myrtaceae family has displayed aphrodisiac activity. The present research aimed to investigate the impacts of clove essential oil (CEO) and the ingredient of CEO, eugenol (E) on ED in diabetic rats. We divided Sprague-Dawley rats into control and diabetic groups. Erectile function was evaluated before and after CEO and E intracavernosal injection. CEO- and E-induced relaxation responses were investigated in isolated corpus cavernosum (CC) using various inhibitors. The intracavernous administration of CEO and E restored erectile responses in diabetic rats. CEO and E induced remarkable relaxation in all groups. CEO- and E-induced relaxation responses were partially inhibited after pre-contraction with KCl. Tetraethylammonium and glibenclamide inhibited the relaxation response to CEO. Glibenclamide inhibited maximum relaxation to E. The inhibitors of nitric oxide synthase (NOS), soluble guanylyl cyclase and nifedipine did not change CEO- and E-induced relaxation responses. The current results suggest that CEO and the major compound of the essential oil, E improved diabetes-induced ED in rats, and CEO caused CC relaxation via K+ channels independently NO signalling pathway.

Contribution of KCNQ and TREK Channels to the Resting Membrane Potential in Sympathetic Neurons at Physiological Temperature.

The ionic mechanisms controlling the resting membrane potential (RMP) in superior cervical ganglion (SCG) neurons have been widely studied and the M-current (IM, KCNQ) is one of the key players. Recently, with the discovery of the presence of functional TREK-2 (TWIK-related K+ channel 2) channels in SCG neurons, another potential main contributor for setting the value of the resting membrane potential has appeared. In the present work, we quantified the contribution of TREK-2 channels to the resting membrane potential at physiological temperature and studied its role in excitability using patch-clamp techniques. In the process we have discovered that TREK-2 channels are sensitive to the classic M-current blockers linopirdine and XE991 (IC50 = 0.310 ± 0.06 µM and 0.044 ± 0.013 µM, respectively). An increase from room temperature (23 °C) to physiological temperature (37 °C) enhanced both IM and TREK-2 currents. Likewise, inhibition of IM by tetraethylammonium (TEA) and TREK-2 current by XE991 depolarized the RMP at room and physiological temperatures. Temperature rise also enhanced adaptation in SCG neurons which was reduced due to TREK-2 and IM inhibition by XE991 application. In summary, TREK-2 and M currents contribute to the resting membrane potential and excitability at room and physiological temperature in the primary culture of mouse SCG neurons.

TRPM8 Channel Activation Reduces the Spontaneous Contractions in Human Distal Colon.

The transient receptor potential-melastatin 8 (TRPM8) is a non-selective Ca2+-permeable channel, activated by cold, membrane depolarization, and different cooling compounds. TRPM8 expression has been found in gut mucosal, submucosal, and muscular nerve endings. Although TRPM8 plays a role in pathological conditions, being involved in visceral pain and inflammation, the physiological functions in the digestive system remain unclear as yet. The aims of the present study were: (i) to verify the TRPM8 expression in human distal colon; (ii) to examine the effects of TRPM8 activation on colonic contractility; (iii) to characterize the mechanism of action. Reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting were used to analyze TRPM8 expression. The responses of human colon circular strips to different TRPM8 agonists [1-[Dialkyl-phosphinoyl]-alkane (DAPA) 2-5, 1-[Diisopropyl-phosphinoyl]-alkane (DIPA) 1-7, DIPA 1-8, DIPA 1-9, DIPA 1-10, and DIPA 1-12) were recorded using a vertical organ bath. The biomolecular analysis revealed gene and protein expression of TRPM8 in both mucosal and smooth muscle layers. All the agonists tested, except-DIPA 1-12, produced a concentration-dependent decrease in spontaneous contraction amplitude. The effect was significantly antagonized by 5-benzyloxytryptamine, a TRPM8 antagonist. The DIPA 1-8 agonist resulted in the most efficacious and potent activation among the tested molecules. The DIPA 1-8 effects were not affected by tetrodotoxin, a neural blocker, but they were significantly reduced by tetraethylammonium chloride, a non-selective blocker of K+ channels. Moreover, iberiotoxin, a blocker of the large-conductance Ca2+-dependent K+-channels, but not apamin, a blocker of small-conductance Ca2+-dependent K+ channels, significantly reduced the inhibitory DIPA 1-8 actions. The results of the present study demonstrated that TRPM8 receptors are also expressed in human distal colon in healthy conditions and that ligand-dependent TRPM8 activation is able to reduce the colonic spontaneous motility, probably by the opening of the large-conductance Ca2+-dependent K+-channels.

Endothelium-Independent Vasodilatory Effects of Isodillapiolglycol Isolated from Ostericum citriodorum.

Ostericum citriodorum is a plant with a native range in China used in herbal medicine for treating angina pectoris. In this study, we investigated the vasodilatory effects of isodillapiolglycol (IDG), which is one of the main ingredients isolated from O. citriodorum ethyl acetate extract, in Sprague-Dawley rat aortic rings, and measured intracellular Ca2+ ([Ca2+]in) using a molecular fluo-3/AM probe. The results show that IDG dose-dependently relaxed endothelium-intact or -denuded aortic rings pre-contracted with noradrenaline (NE) or potassium chloride (KCl), and inhibited CaCl2-induced contraction in high K+ depolarized aortic rings. Tetraethyl ammonium chloride (a Ca2+-activated K+ channel blocker) or verapamil (an L-type Ca2+ channel blocker) significantly reduced the relaxation of IDG in aortic rings pre-contracted with NE. In vascular smooth muscle cells, IDG inhibited the increase in [Ca2+]in stimulated by KCl in Krebs solution; likewise, IDG also attenuated the increase in [Ca2+]in induced by NE or subsequent supplementation of CaCl2. These findings demonstrate that IDG relaxes aortic rings in an endothelium-independent manner by reducing [Ca2+]in, likely through inhibition of the receptor-gated Ca2+ channel and the voltage-dependent Ca2+ channel, and through opening of the Ca2+-activated K+ channel.

Vasodilatory Effect of Phellinus linteus Extract in Rat Mesenteric Arteries.

Phellinus linteus is a well-known medicinal mushroom that is widely used in Asian countries. In several experimental models, Phellinus linteus extracts were reported to have various biological effects, including anti-inflammatory, anti-cancer, hepatoprotective, anti-diabetic, neuroprotective, and anti-angiogenic activity. In the present study, several bioactive compounds, including palmitic acid ethyl ester and linoleic acid, were identified in Phellinus linteus. The intermediate-conductance calcium-activated potassium channel (IKCa) plays an important role in the regulation of the vascular smooth muscle cells' (VSMCs) contraction and relaxation. The activation of the IKCa channel causes the hyperpolarization and relaxation of VSMCs. To examine whether Phellinus linteus extract causes vasodilation in the mesenteric arteries of rats, we measured the isometric tension using a wire myograph. After the arteries were pre-contracted with U46619 (a thromboxane analogue, 1 µM), Phellinus linteus extract was administered. The Phellinus linteus extract induced vasodilation in a dose-dependent manner, which was independent of the endothelium. To further investigate the mechanism, we used the non-selective K+ channel blocker tetraethylammonium (TEA). TEA significantly abolished Phellinus linteus extract-induced vasodilation. Thus, we tested three different types of K+ channel blockers: iberiotoxin (BKca channel blocker), apamin (SKca channel blocker), and charybdotoxin (IKca channel blocker). Charybdotoxin significantly inhibited Phellinus linteus extract-induced relaxation, while there was no effect from apamin and iberiotoxin. Membrane potential was measured using the voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC4(3)) in the primary isolated vascular smooth muscle cells (VSMCs). We found that the Phellinus linteus extract induced hyperpolarization of VSMCs, which is associated with a reduced phosphorylation level of 20 KDa myosin light chain (MLC20).

4,4'-Diisothiocyanatostilbene-2,2'-disulfonate modulates voltage-gated K+ current and influences cell cycle arrest in androgen sensitive and insensitive human prostate cancer cell lines.

The stilbene derivative, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), an anion channel blocker is used in the present study to evaluate its modulatory effect on voltage-gated K+ current (IK) in human prostate cancer cell lines (LNCaP and PC-3). Voltage-gated K+ (KV) channels in the plasma membrane are critically involved in the proliferation of tumor cells. Therefore, KV channels are considered as a novel potential target for cancer treatment. The results of the present study show that the external perfusion of DIDS activates IK in a concentration-dependent manner, although the known K+ channel blocker TEA failed to block the DIDS activated IK in PC-3 cells. Whereas, in LNCaP cells, the higher concentration of DIDS blocked IK, though this effect was not completely recovered after washout. The difference in function of DIDS might be due to the expression of different Kv channel isoforms in LNCaP and PC-3 cells. Further, the anticancer studies show that treatment of DIDS significantly induced G2/M phase cell cycle arrest and induced moderate and low level of cell death in LNCaP and PC-3 cells respectively. This finding reveals that DIDS modulates IK and exerts cell cycle arrest and cell death in LNCaP and PC-3 cells.

Separate and combined effects of KCa and KATP channel blockade with NOS inhibition on cutaneous vasodilation and sweating in older men during heat stress.

Our objective in this study was to examine the separate and combined effects of potassium (K+) channels and nitric oxide synthase (NOS) on cutaneous vasodilation and sweating in older men during rest and exercise in the heat. In 13 habitually active men (61 ± 4 yr), cutaneous vascular conductance and local sweat rate were assessed at six dorsal forearm skin sites continuously perfused with either 1) lactated Ringer (control), 2) 10 mM NG-nitro-l-arginine methyl ester (l-NAME, NOS inhibitor), 3) 50 mM tetraethylammonium (TEA; Ca2+-activated K+ channel blocker), 4) 5 mM glybenclamide (GLY; ATP-sensitive K+ channel blocker), 5) 50 mM TEA + 10 mM l-NAME, and 6) 5 mM GLY + 10 mM l-NAME via microdialysis. Participants rested in non-heat stress (25°C) and heat stress (35°C) conditions for ∼60 min each, followed by 50 min of moderate-intensity cycling (∼55% V̇o2peak) and 30 min of recovery in the heat. During rest and exercise in the heat, l-NAME, TEA + l-NAME, and GLY + l-NAME attenuated CVC relative to control (all P ≤ 0.05), although l-NAME was not different from TEA + l-NAME or GLY + l-NAME (all P > 0.05). TEA attenuated CVC during rest, whereas GLY attenuated CVC during exercise (both P ≤ 0.05). Additionally, whereas neither l-NAME nor TEA altered sweating throughout the protocol (all P > 0.05), combined TEA + l-NAME attenuated sweating during exercise in the heat (P ≤ 0.05). We conclude that in habitually active older men blockade of KCa and KATP channels attenuates cutaneous vasodilation during rest and exercise in the heat, respectively, and these effects are NOS dependent. Furthermore, combined NOS inhibition and KCa channel blockade attenuates sweating during exercise in the heat.

Relevant effects of Taohong Siwu decoction on isolated rat aortic ring dependent on endothelium nitric oxide-cGMP pathway.

Using rat thoracic aortic rings to test the relaxing effects of the 95% ethanol extract and aqueous extract of Taohong Siwu decoction (THSW) on endothelium intact or endothelium removed aortic rings. Results showed that the 95% ethanol extract (0.1, 1, 10, 100, 1000 mg•L-1) and aqueous extract (0.1, 1, 10, 100, 1000 mg•L-1) of THSW were able to relax the intact endothelium aortic rings pre-contracted by 10-6 mol•L-1 PE. 10-4 mol•L-1 L-NAME and 10-5 mol•L-1 methylene blue both were able to inhibit the relaxation other than indomethacin. For the endothelium removed aortic rings, potassium channel blocker 3×10-3mol•L-1 tetraethylammonium chloride and 10-5 mol•L-1 glibenclamide had no effect on the relaxation effects caused by the 95% ethanol extract and aqueous extract of THSW. It could be concluded that the 95% ethanol extract and aqueous extract of THSW relax blood vessel by endothelium-dependent way.

Generation and Role of Oscillatory Contractions in Mouse Airway Smooth Muscle.

BACKGROUND/AIMS: Tetraethylammonium chloride (TEA) induces oscillatory contractions in mouse airway smooth muscle (ASM); however, the generation and maintenance of oscillatory contractions and their role in ASM are unclear. METHODS: In this study, oscillations of ASM contraction and intracellular Ca2+ were measured using force measuring and Ca2+ imaging technique, respectively. TEA, nifedipine, niflumic acid, acetylcholine chloride, lithium chloride, KB-R7943, ouabain, 2-Aminoethoxydiphenyl borate, thapsigargin, tetrodotoxin, and ryanodine were used to assess the mechanism of oscillatory contractions. RESULTS: TEA induced depolarization, resulting in activation of L-type voltage-dependent Ca2+ channels (LVDCCs) and voltage-dependent Na+ (VNa) channels. The former mediated Ca2+ influx to trigger a contraction and the latter mediated Na+ entry to enhance the contraction via activating LVDCCs. Meanwhile, increased Ca2+-activated Cl- channels, inducing depolarization that resulted in contraction through LVDCCs. In addition, the contraction was enhanced by intracellular Ca2+ release from Ca2+ stores mediated by inositol (1,4,5)-trisphosphate receptors (IP3Rs). These pathways together produce the contractile phase of the oscillatory contractions. Furthermore, the increased Ca2+ activated the Na+-Ca2+ exchanger (NCX), which transferred Ca2+ out of and Na+ into the cells. The former induced relaxation and the latter activated Na+/K+-ATPase that induced hypopolarization to inactivate LVDCCs causing further relaxation. This can also explain the relaxant phase of the oscillatory contractions. Moreover, the depolarization induced by VNa channels and NCX might be greater than the hypopolarization caused by Na+/K+-ATPase alone, inducing LVDCC activation and resulting in further contraction. CONCLUSIONS: These data indicate that the TEA-induced oscillatory contractions were cooperatively produced by LVDCCs, VNa channels, Ca2+-activated Cl- channels, NCX, Na+/K+ ATPase, IP3Rs-mediated Ca2+ release, and extracellular Ca2+.

The Insensitivity of TASK-3 K2P Channels to External Tetraethylammonium (TEA) Partially Depends on the Cap Structure.

Two-pore domain K⁺ channels (K2P) display a characteristic extracellular cap structure formed by two M1-P1 linkers, the functional role of which is poorly understood. It has been proposed that the presence of the cap explains the insensitivity of K2P channels to several K⁺ channel blockers including tetraethylammonium (TEA). We have explored this hypothesis using mutagenesis and functional analysis, followed by molecular simulations. Our results show that the deletion of the cap structure of TASK-3 (TWIK-related acid-sensitive K⁺ channel) generates a TEA-sensitive channel with an IC50 of 11.8 ± 0.4 mM. The enhanced sensitivity to TEA displayed by the cap-less channel is also explained by the presence of an extra tyrosine residue at position 99. These results were corroborated by molecular simulation analysis, which shows an increased stability in the binding of TEA to the cap-less channel when a ring of four tyrosine is present at the external entrance of the permeation pathway. Consistently, Y99A or Y205A single-residue mutants generated in a cap-less channel backbone resulted in TASK-3 channels with low affinity to external TEA.