Alcohol withdrawal is associated with a downregulation of large-conductance Ca2+-activated K+ channels in rat inferior colliculus neurons

Rationale

Large conductance calcium-activated potassium (BK_Ca or K_Ca1.1) channels are well-known molecular targets for the action of alcohol and therefore may play an important role in the pathogenesis of alcohol withdrawal syndrome.

Objectives

We evaluate the modifications of total outward K⁺ currents and protein expression of BK_Ca channels α-subunit in inferior colliculus (IC) neurons obtained from controls and rats subjected to alcohol withdrawal associated with enhanced susceptibility to seizures.

Methods

Outward K⁺ currents and BK_Ca channel proteins were measured using the whole cell configuration of patch clamp techniques and Western blot analysis, respectively.

Results

Total outward K⁺ current density was significantly reduced in IC neurons at 24 and 48 h during the alcohol withdrawal period when the susceptibility to seizures was maximal and absent, respectively. The iberiotoxin-sensitive (BK_Ca) current density and conductance also were significantly reduced at 24 h following alcohol withdrawal. Consistent with functional data, the levels of protein expression of α-subunit associated with BK_Ca channels also was significantly reduced in IC neurons at 24 and 48 h following alcohol withdrawal.

Conclusions

The downregulation of BK_Ca channels outlasts the finite period of elevated susceptibility to alcohol withdrawal seizures. These findings indicate that BK_Ca channels, per se, may not be fundamentally important for the generation of alcohol withdrawal seizures.     

K(Ca) 3.1 channels maintain endothelium-dependent vasodilatation in isolated perfused kidneys of spontaneously hypertensive rats after chronic inhibition of NOS

BACKGROUND AND PURPOSE

The purpose of the study was to investigate renal endothelium-dependent vasodilatation in a model of severe hypertension associated with kidney injury.

EXPERIMENTAL APPROACH

Changes in perfusion pressure were measured in isolated, perfused kidneys taken from 18-week-old Wistar–Kyoto rat (WKY), spontaneously hypertensive rats (SHR) and SHR treated for 2 weeks with N^ω-nitro-L-arginine methyl ester in the drinking water (L-NAME-treated SHR, 6 mg·kg⁻¹·day⁻¹).

KEY RESULTS

Acetylcholine caused similar dose-dependent renal dilatation in the three groups. In vitro administration of indomethacin did not alter the vasodilatation, while the addition of N^w-nitro-L-arginine (L-NA) produced a differential inhibition of the vasodilatation, (inhibition in WKY > SHR > L-NAME-treated SHR). Further addition of ODQ, an inhibitor of soluble guanylyl cyclase, abolished the responses to sodium nitroprusside but did not affect the vasodilatation to acetylcholine. However, the addition of TRAM-34 (or charybdotoxin) inhibitors of Ca²⁺-activated K⁺ channels of intermediate conductance (K_Ca3.1), blocked the vasodilatation to acetylcholine, while apamin, an inhibitor of Ca²⁺-activated K⁺ channels of small conductance (K_Ca2.3), was ineffective. Dilatation induced by an opener of K_Ca3.1/K_Ca2.3 channels, NS-309, was also blocked by TRAM-34, but not by apamin. The magnitude and duration of NS-309-induced vasodilatation and the renal expression of mRNA for K_Ca3.1, but not K_Ca2.3, channels followed the same ranking order (WKY < SHR < L-NAME-treated SHR).

CONCLUSIONS AND IMPLICATIONS

In SHR kidneys, an EDHF-mediated response, involving activation of K_Ca3.1 channels, contributed to the mechanism of endothelium-dependent vasodilatation. In kidneys from L-NAME-treated SHR, up-regulation of this pathway fully compensated for the decrease in NO availability.     

Targeting BK (big potassium) channels in epilepsy

Introduction: Epilepsies are disorders of neuronal excitability characterized by spontaneous and recurrent seizures. Ion channels are critical for regulating neuronal excitability and, therefore, can contribute significantly to epilepsy pathophysiology. In particular, large conductance, Ca²⁺-activated K⁺ (BK_Ca) channels play an important role in seizure etiology. These channels are activated by both membrane depolarization and increased intracellular Ca²⁺. This unique coupling of Ca²⁺ signaling to membrane depolarization is important in controlling neuronal hyperexcitability, as outward K⁺ current through BK_Ca channels hyperpolarizes neurons.

Areas covered: BK_Ca channel structure–function and the role of these channels in epilepsy pathophysiology.

Expert opinion: Loss-of-function BK_Ca channel mutations contribute to neuronal hyperexcitability that can lead to temporal lobe epilepsy, tonic–clonic seizures and alcohol withdrawal seizures. Similarly, BK_Ca channel blockade can trigger seizures and status epilepticus. Paradoxically, some mutations in BK_Ca channel subunit can give rise to channel gain-of-function that leads to development of idiopathic epilepsy (primarily absence epilepsy). Seizures themselves also enhance BK_Ca channel currents associated with neuronal hyperexcitability, and blocking BK_Ca channels suppresses generalized tonic–clonic seizures. Thus, both loss-of-function and gain-of-function BK_Ca channels might serve as molecular targets for drugs to suppress certain seizure phenotypes including temporal lobe seizures and absence seizures, respectively.     

Large- and small-conductance Ca2+-activated K+ channels: their role in the nicotinic receptor-mediated catecholamine secretion in bovine adrenal medulla

In cultured bovine adrenal chromaffln cells, charybdotoxin and iberiotoxin (inhibitors of the large-conductance Ca²⁺-activated K⁺ channel) as well as apamin (an inhibitor of the small-conductance Ca²⁺-activated K⁺ channel), at 1–100 nM, suppressed carbachol-induced ⁸⁶Rb⁺ efflux, augmented carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels and catecholamine secretion and had no effect on carbachol-induced ²²Na⁺ influx via nicotinic receptors, a prerequisite for Ca²⁺ channel activation by carbachol. ⁴⁵Ca²⁺ influx caused by high K⁺ (a direct activation of voltage-dependent Ca²⁺ channels) was also enhanced by these K⁺ channel inhibitors, with the concentration-response curves being similar to those for carbachol-induced ⁴⁵Ca²⁺ influx. Dendrotoxin and mast cell degranulating peptide (inhibitors of voltage-dependent K⁺ channels), on the other hand, did not alter carbachol-induced ⁸⁶Rb⁺ efflux or ⁴⁵Ca²⁺ influx.These results suggest that the stimulation of nicotinic receptors eventually opens large- and small-conductance Ca²⁺-activated K⁺ channels, and that the blockade of these Ca²⁺-activated K⁺ channels results in gating of voltage-dependent Ca²⁺ channels and thereby augments catecholamine secretion from bovine adrenal chromaffln cells.     

Gambierol Blocks a K+ Current Fraction without Affecting Catecholamine Release in Rat Fetal Adrenomedullary Cultured Chromaffin Cells

Gambierol inhibits voltage-gated K⁺ (K_V) channels in various excitable and non-excitable cells. The purpose of this work was to study the effects of gambierol on single rat fetal (F19–F20) adrenomedullary cultured chromaffin cells. These excitable cells have different types of K_V channels and release catecholamines. Perforated whole-cell voltage-clamp recordings revealed that gambierol (100 nM) blocked only a fraction of the total outward K⁺ current and slowed the kinetics of K⁺ current activation. The use of selective channel blockers disclosed that gambierol did not affect calcium-activated K⁺ (K_Ca) and ATP-sensitive K⁺ (K_ATP) channels. The gambierol concentration necessary to inhibit 50% of the K⁺ current-component sensitive to the polyether (IC₅₀) was 5.8 nM. Simultaneous whole-cell current-clamp and single-cell amperometry recordings revealed that gambierol did not modify the membrane potential following 11s depolarizing current-steps, in both quiescent and active cells displaying repetitive firing of action potentials, and it did not increase the number of exocytotic catecholamine release events, with respect to controls. The subsequent addition of apamin and iberiotoxin, which selectively block the K_Ca channels, both depolarized the membrane and enhanced by 2.7 and 3.5-fold the exocytotic event frequency in quiescent and active cells, respectively. These results highlight the important modulatory role played by K_Ca channels in the control of exocytosis from fetal (F19–F20) adrenomedullary chromaffin cells.     

Inhibitory effects of the antiepileptic drug ethosuximide on G protein-activated inwardly rectifying K+ channels

Antiepileptic drugs protect against seizures by modulating neuronal excitability. Ethosuximide is selectively used for the treatment of absence epilepsy, and has also been shown to have the potential for treating several other neuropsychiatric disorders in addition to several antiepileptic drugs. Although ethosuximide inhibits T-type Ca²⁺, noninactivating Na⁺, and Ca²⁺-activated K⁺ channels, the molecular mechanisms underlying the effects of ethosuximide have not yet been sufficiently clarified. G protein-activated inwardly rectifying K⁺ channels (GIRK, or Kir3) play an important role in regulating neuronal excitability, heart rate and platelet aggregation. In the present study, the effects of various antiepileptic drugs on GIRK channels were examined first by using the Xenopus oocyte expression assay. Ethosuximide at clinically relevant concentrations inhibited GIRK channels expressed in Xenopus oocytes. The inhibition was concentration-dependent, but voltage-independent, and time-independent during each voltage pulse. However, the other antiepileptic drugs tested: phenytoin, valproic acid, carbamazepine, phenobarbital, gabapentin, topiramate and zonisamide, had no significant effects on GIRK channels even at toxic concentrations. In contrast, Kir1.1 and Kir2.1 channels were insensitive to all of the drugs tested. Ethosuximide also attenuated ethanol-induced GIRK currents. These inhibitory effects of ethosuximide were not observed when ethosuximide was applied intracellularly. In granule cells of cerebellar slices, ethosuximide inhibited GTPγS-activated GIRK currents. Moreover, ADP- and epinephrine-induced platelet aggregation was inhibited by ethosuximide, but not by charybdotoxin, a platelet Ca²⁺-activated K⁺ channel blocker. These results suggest that the inhibitory effects of ethosuximide on GIRK channels may affect some of brain, heart and platelet functions.     

Pharmacological properties of Ca2+-activated K+ currents of ramified murine brain macrophages

Using the whole-cell configuration of the patch clamp technique, calcium-activated potassium currents (I_K,Ca) were investigated in ramified murine brain macrophages. In order to induce I_K,Ca the intracellular concentration of nominal free Ca²⁺ was adjusted to 1μM. The Ca²⁺-activated K⁺ current of brain macrophages did not show any voltage dependence at test potentials between –120 and +30mV. A tenfold change in extracellular K⁺ concentration shifted the reversal potential of I_K,Ca by 51mV. The bee venom toxin apamin applied at concentrations of up to 1μM did not affect I_K,Ca. Ca²⁺-activated K⁺ currents of ramified brain macrophages were highly sensitive to extracellularly applied charybdotoxin (CTX). The half-maximal effective concentration of CTX was calculated to be 4.3nM. In contrast to CTX, the scorpion toxin kaliotoxin did not inhibit I_K,Ca at concentrations between 1 and 50nM. Tetraethylammonium (TEA) blocked 8.0% of I_K,Ca at a concentration of 1mM, whereas 31.4% of current was blocked by 10mM TEA. Several inorganic polyvalent cations were tested at a concentration of 2mM for their ability to block I_K,Ca. La³⁺ reduced I_K,Ca by 72.8%, whereas Cd²⁺ decreased I_K,Ca by 17.4%; in contrast, Ni²⁺ did not have any effect on I_K,Ca. Ba²⁺ applied at a concentration of 1mM reduced I_K,Ca voltage-dependently at hyperpolarizing potentials. Received: 17 January / Accepted: 5 May 1997     

The K+ channels KCa3.1 and Kv1.3 as novel targets for asthma therapy

Asthma affects 10% of the UK population and is an important cause of morbidity and mortality at all ages. Current treatments are either ineffective or carry unacceptable side effects for a number of patients; in consequence, development of new approaches to therapy are important. Ion channels are emerging as attractive therapeutic targets in a variety of non-excitable cells. Ion channels conducting K⁺ modulate the activity of several structural and inflammatory cells which play important roles in the pathophysiology of asthma. Two channels of particular interest are the voltage-gated K⁺ channel K_v1.3 and the intermediate conductance Ca²⁺-activated K⁺ channel K_Ca3.1 (also known as IK_Ca1 or SK4). K_v1.3 is expressed in IFNγ-producing T cells while K_Ca3.1 is expressed in T cells, mast cells, macrophages, airway smooth muscle cells, fibroblasts and epithelial cells. Both channels play important roles in cell activation, migration, and proliferation through the regulation of membrane potential and calcium signalling. We hypothesize that K_Ca3.1- and/or K_v1.3-dependent cell processes are one of the common denominators in asthma pathophysiology. If true, these channels might serve as novel targets for the treatment of asthma. Emerging evidence lends support to this hypothesis. Further validation through the study of the role that these channels play in normal and asthmatic airway cell (patho)physiology and in vivo models will provide further justification for the assessment of small molecule blockers of K_v1.3 and K_Ca3.1 in the treatment of asthma.     

Intermediate-conductance calcium-activated potassium channels participate in neurovascular coupling

BACKGROUND AND PURPOSE

Controlling vascular tone involves K⁺ efflux through endothelial cell small- and intermediate-conductance calcium-activated potassium channels (K_Ca2.3 and K_Ca3.1, respectively). We investigated the expression of these channels in astrocytes and the possibility that, by a similar mechanism, they might contribute to neurovascular coupling.

EXPERIMENTAL APPROACH

Transgenic mice expressing enhanced green fluorescent protein (eGFP) in astrocytes were used to assess K_Ca2.3 and K_Ca3.1 expression by immunohistochemistry and RT-PCR. K_Ca currents in eGFP-positive astrocytes were determined in situ using whole-cell patch clamp electrophysiology. The contribution of K_Ca3.1 to neurovascular coupling was investigated in pharmacological experiments using electrical field stimulation (EFS) to evoke parenchymal arteriole dilatation in FVB/NJ mouse brain slices and whisker stimulation to evoke changes in cerebral blood flow in vivo, measured by laser Doppler flowmetry.

KEY RESULTS

K_Ca3.1 immunoreactivity was restricted to astrocyte processes and endfeet and RT-PCR confirmed astrocytic K_Ca2.3 and K_Ca3.1 mRNA expression. With 200 nM [Ca²⁺]_i, the K_Ca2.1-2.3/K_Ca3.1 opener NS309 increased whole-cell currents. CyPPA, a K_Ca2.2/K_Ca2.3 opener, was without effect. With 1 µM [Ca²⁺]_i, the K_Ca3.1 inhibitor TRAM-34 reduced currents whereas apamin (K_Ca2.1-2.3 blocker) had no effect. CyPPA also inhibited currents evoked by NS309 in HEK293 cells expressing K_Ca3.1. EFS-evoked Fluo-4 fluorescence confirmed astrocyte endfoot recruitment into neurovascular coupling. TRAM-34 inhibited EFS-evoked arteriolar dilatation by 50% whereas charybdotoxin, a blocker of K_Ca3.1 and the large-conductance K_Ca channel, K_Ca1.1, inhibited dilatation by 82%. TRAM-34 reduced the cortical hyperaemic response to whisker stimulation by 40%.

CONCLUSION AND IMPLICATIONS

Astrocytes express functional K_Ca3.1 channels, and these contribute to neurovascular coupling.

LINKED ARTICLES

This article is part of a themed issue on Vascular Endothelium in Health and Disease. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.164.issue-3     

EFFECTS OF BKCa CHANNELS ON THE REDUCTION OF CYTOSOLIC Ca2+ IN cGMP-INDUCED RELAXATION OF GUINEAPIG TRACHEA

1. In order to examine the mechanisms of cGMP-induced relaxation in airway smooth muscle, the effects of atrial natriuretic peptide (ANP) and 8-brom cGMP on muscle tone were studied by measuring isometric tension, while the effects on cytosolic Ca²⁺ concentrations were studied by measuring the spectra of fura-2 loaded in guinea-pig tracheal strips. 2. Atrial natriuretic peptide and 8-brom cGMP caused a concentration-dependent inhibition of spontaneous tone in the guinea-pig trachea. The relaxant effects of these agents on spontaneous tone were markedly suppressed in the presence of iberiotoxin (IbTX), a selective inhibitor of large-conductance Ca²⁺-activated K⁺ (BKca) channels. Iberiotoxin (30 nmol/L) markedly affected the maximal effect induced by ANP and 8-brom cGMP and augmented EC₇₀ values for ANP and EC₅₀values for 8-brom cGMP approximately 27- and 17-fold, respectively. The inhibitory effects of IbTX on relaxation induced by these agents were diminished in the presence of 1 μmol/L nifedipine, an antagonist of voltage-operated Ca²⁺channels (VOCC). 3. The inhibitory action of ANP and 8-brom cGMP on spontaneous tone was not affected by the presence of 10 μmol/L glibenclamide, an inhibitor of ATP-sensitive K⁺ channels, and 100 nmol/L apamin, an inhibitor of small-conductance Ca²+-activated K⁺ channels. When these agents were applied to tissues precontracted by high (40mmol/L) K⁺, the relaxant effects of these agents markedly diminished. 4. The extracellular Ca²⁺-dependent contraction was inhibited in the presence of 0.3 μmoI/L ANP or 0.1 mmol/L 8-brom cGMP. Concentration—response curves to extracellular Ca²⁺ (0.03—2.4 mmol/L) were markedly diminished by exposure to these agents. The maximal effect induced by extracellular Ca²⁺ was affected by these agents. 5. Atrial natriuretic peptide caused an inhibition of spontaneous tone accompanied by a reduction in the intracellular Ca²⁺ concentration. In the presence of IbTX, the elimination of both muscle tone and cytosolic Ca²⁺ by ANP was suppressed. 6. We conclude that ANP and 8-brom cGMP activate BKca channels and that the inhibition of Ca²⁺ influx through VOCC, mediated by BKca channel activation, may be involved in cGMP-dependent bronchodilation.     

Microglial SK3 and SK4 Currents and Activation State are Modulated by the Neuroprotective Drug, Riluzole

Microglia monitor the CNS for ‘danger’ signals after acute injury, such as stroke and trauma, and then undergo complex activation processes. Classical activation of microglia can produce neurotoxic levels of glutamate and immune mediators (e.g., pro-inflammatory cytokines, reactive oxygen and nitrogen species), while alternative activation up-regulates anti-inflammatory molecules and is thought to resolve inflammation and protect the brain. Thus, pharmacological strategies to decrease classical- and/or promote alternative activation are of interest. Here, we assessed actions of the neuroprotective drug, riluzole, on two Ca²⁺-activated K⁺ channels in microglia — SK3 (KCa2.3, KCNN3) and SK4 (KCa3.1, KCNN4) — and on classical versus alternative microglial activation. Riluzole is used to treat amyotrophic lateral sclerosis, and is in clinical trials for several other CNS disorders, where it has been presumed to target neurons and reduce glutamate-mediated toxicity. We show that simply elevating intracellular Ca²⁺ to micromolar levels in whole-cell recordings does not activate SK channels in a cell line derived from primary rat microglia (MLS-9). In intact cells, riluzole raised cytoplasmic Ca²⁺, but it was marginal (~200 nM) and transient (2 min). Surprisingly then, in whole cell recordings, riluzole rapidly activated SK3 and SK4 channels for as long as it was present, and did not require elevated intracellular Ca²⁺. We then used primary rat microglia to analyze expression of several activation markers and inflammatory mediators. Riluzole decreased classical LPS-induced activation, and increased some aspects of IL-4-induced alternative activation. These actions on microglia suggest an additional mechanism underlying the neuroprotective actions of riluzole.     

Activation of large‐conductance Ca2+‐activated K+ channels by pinacidil in human umbilical vascular endothelial cells

The effect of pinacidil, an opener of ATP‐sensitive K⁺ (K_ATP) channels, on large‐conductance Ca²⁺‐activated K+ (BK_Ca) channels was investigated in cultured endothelial cells of human umbilical veins. In whole cell configuration, pinacidil (30 μM) increased the amplitude of K⁺ outward currents (I_K). Charybdotoxin (100 nM), but not glibenclamide (10 μM), suppressed pinacidil‐induced increase in I_K. Neither carbonyl cyanide m‐chlorophenyl hydrazone (CCCP; 10 μM), an inhibitor of mitochondrial Ca²⁺‐uniporter, nor cyclosporin A (200 nM), an inhibitor of the mitochondrial permeability transition pore, affected pinacidil‐induced increase in I_K. In inside‐out patch configuration, bath application of pinacidil (30 μM) did not change single channel conductance but increased the activity of BK_Ca channels. Pinacidil (30 μM) shifted the activation curve of BK_Ca channels to less positive membrane potential by approximately 15 mV. Pinacidil stimulated the activity of these channels in a concentration‐dependent manner. The EC₅₀ value for pinacidil‐induced channel activity was 20 μM. After BK_Ca channels had been enhanced by Evans blue (100 μM), subsequent application of pinacidil (100 μM) did not further increase the channel activity. These results clearly indicate that in addition to the activation of K_ATP channels, pinacidil can also stimulate BK_Ca channels in endothelial cells. These effects could contribute to the regulation of vascular tone if similar results were found in endothelial cells in vivo. Drug Dev. Res. 48:6–16, 1999. © 1999 Wiley‐Liss, Inc.     

Involvement of sex hormonal regulation of K+ channels in electrophysiological and contractile functions of muscle tissues

Sex hormones, such as testosterone, progesterone, and 17β-estradiol, control various physiological functions. This review focuses on the sex hormonal regulation of K⁺ channels and the effects of such regulation on electrophysiological and contractile functions of muscles. In the cardiac tissue, testosterone and progesterone shorten action potential, and estrogen lengthens QT interval, a marker of increased risk of ventricular tachyarrhythmias. We have shown that testosterone and progesterone in physiological concentration activate KCNQ1 channels via membrane-delimited sex hormone receptor/eNOS pathways to shorten the action potential duration. Mitochondrial K⁺ channels are also involved in the protection of cardiac muscle. Testosterone and 17β-estradiol directly activate mitochondrial inner membrane K⁺ channels (Ca²⁺ activated K⁺ channel (K_Ca channel) and ATP-sensitive K⁺ channel (K_ATP channel)) that are involved in ischemic preconditioning and cardiac protection. During pregnancy, uterine blood flow increases to support fetal growth and development. It has been reported that 17β-estradiol directly activates large-conductance Ca²⁺-activated K⁺ channel (BK_Ca channel) attenuating arterial contraction. Furthermore, 17β-estradiol increases expression of BK_Ca channel β1 subunit which enhances BK_Ca channel activity by DNA demethylation. These findings are useful for understanding the mechanisms of sex or generation-dependent differences in the physiological and pathological functions of muscles, and the mechanisms of drug actions.     

ROLE OF CALCIUM-ACTIVATED POTASSIUM CHANNELS IN THE RELAXATION OF TRACHEAL SMOOTH MUSCLES BY FORSKOLIN

1. The role of calcium-activated potassium (K_Ca) channels in bronchodilation produced by a direct adenylyl cyclase activator, forskolin, was investigated. The involvement of intracellular cyclic AMP (cAMP) in the process was also examined. 2. The isometric tension records from guinea-pig tracheal smooth muscles indicated that application of charybdotoxin (ChTX), a selective inhibitor of large conductance K_Ca channels, led to a suppression of the relaxant effect of forskolin in the precontracted tissue by carbachol (CCh). However, the inhibitory action by ChTX had a much greater effect on the relaxation caused by isoproterenol than by forskolin. 3. In contrast to the effect of ChTX, glybenclamide, a cromakalim-sensitive K⁺ channel inhibitor and apamin, a small conductance K_Ca channel blocker, had no effects on the bronchodilation produced by forskolin. 4. The effects of forskolin and nifedipine on tone produced by high K⁺ was compared. Concentration-inhibition curves in guinea-pig trachealis precontracted by 20 mmol/L K⁺ solution were similar for forskolin and nifedipine. Conversely, relaxation by forskolin was significantly diminished when tissues were contracted with 40 mmol/ L K⁺ solution, whereas nifedipine relaxations were unaffected. 5. A single channel record from a cell-attached patch in a porcine tracheal myocyte demonstrated that forskolin stimulates reversibly K_Ca channels without affecting the unitary amplitude. 6. The results are consistent with forskolin-induced relaxation occurring at least in part through the opening of ChTX-sensitive K_Ca channels, by means of a cAMP-dependent channel modulation. The lesser effect of ChTX on forskolin compared with isoproterenol-induced relaxations suggests a tighter coupling between relaxation and channel opening by β-adrenergic receptor stimulation, and is consistent with cAMP-independent mechanisms of receptor-channel coupling.     

Losartan Normalizes Endothelium-Derived Hyperpolarizing Factor–Mediated Relaxation by Activating Ca2+-Activated K+ Channels in Mesenteric Artery From Type 2 Diabetic GK Rat

Ca²⁺-activated K⁺ (K _Ca ) channels are important for endothelium-derived hyperpolarizing factor (EDHF) signaling. Since treatment with angiotensin II receptor blockers (ARBs) improves vasculopathies in type 2 diabetic patients, we asked whether the EDHF-type relaxation and its associated K _Ca channels [small (SK _Ca )–, intermediate (IK _Ca )–, and large (BK _Ca )–conductance channels] are abnormal in mesenteric arteries isolated from Goto-Kakizaki (GK) rats at the chronic stage of type 2 diabetes (34 – 38 weeks) and whether an ARBs (losartan, 25 mg · kg^?1 · day^?1 for 2 weeks) might correct these abnormalities. Although the acetylcholine chloride–induced EDHF-type relaxation in mesenteric arteries from GK rats was reduced versus the Wistar controls, it was significantly restored by losartan treatment. The SK _Ca-blocker apamin or the IK _Ca-blocker 1-[(2-chlorophenyl)diphenylmethyl]-1 H-pyrazole (TRAM-34) inhibited such relaxations in the losartan-treated or -untreated Wistar groups and in the losartan-treated GK group, but not in the losartan-untreated GK group. The BK _Ca-blocker iberiotoxin had a significant inhibitory effect in only one of these groups, the losartan-treated GK. The relaxations induced by the SK _Ca /IK _Ca acti-vator NS309 and the BK _Ca activator NS1619, which were impaired in GK rats, were normalized by losartan treatment. We conclude that losartan improves EDHF-type relaxation in GK rats at least partly by normalizing SK _Ca /IK _Ca activities and increasing BK _Ca activity.     

Antinociceptive action of myricitrin: involvement of the K+ and Ca2+ channels

The present study was designed to investigate the mechanisms involved in the antinociception afforded by myricitrin in chemical models of nociception in mice. Myricitrin given by intrathecal (i.t.) or intracerebroventricular (i.c.v.) route produced dose-related antinociception when evaluated against acetic acid-induced visceral pain in mice. In addition, the intraperitoneal administration of myricitrin caused significant inhibition of biting behaviour induced by i.t. injection of glutamate, substance P, capsaicin, interleukin 1 β (IL-1β) and tumor necrosis factor-α (TNF-α). The antinociception caused by myricitrin in the acetic acid test was fully prevented by i.t. pre-treatment with pertussis toxin, a Gi/o protein inactivator, and by i.c.v. injection of calcium chloride (CaCl₂). In addition, the i.t. pre-treatment of mice with apamin, a blocker of small (or low)-conductance calcium-gated K⁺ channels and tetraethylammonium, a blocker of voltage-gated K⁺ channels significantly reversed the antinociception induced by myricitrin. The charybdotoxin, a blocker of large (or fast)-conductance calcium-gated K⁺ channels and glibenclamide, a blocker of the ATP-gated K⁺ channels had no effect on myricitrin-induced antinociception. Calcium uptake analysis revealed that myricitrin inhibited ⁴⁵Ca²⁺ influx under a K⁺-induced depolarization condition. However, calcium movement was modified in a non-depolarizing condition only when the highest concentration of myricitrin was used. In summary, our findings indicate that myricitrin produces consistent antinociception in chemical models of nociception in mice. These results clearly demonstrate an involvement of the Gi/o protein dependent mechanism on antinociception caused by myricitrin. The opening of voltage- and small-conductance calcium-gated K⁺ channels and the reduction of calcium influx led to the antinociceptive of myricitrin.     

NS11021, a novel opener of large-conductance Ca2+-activated K+ channels,enhances erectile responses in rats

Background and purpose:

Large-conductance Ca²⁺-activated K⁺ channels (BK_Ca), located on the arterial and corporal smooth muscle, are potential targets for treatment of erectile dysfunction (ED). This study investigated whether NS11021 (1-(3,5-Bis-trifluoromethyl-phenyl)-3-[4-bromo-2-(1H-tetrazol-5-yl)-phenyl]-thiourea), a novel opener of BK_Ca channels, relaxes erectile tissue in vitro and enhances erectile responses in intact rats. The effects were compared with sildenafil, an inhibitor of phosphodiesterase type 5.

Experimental approach:

Patch clamp was used to record whole cell current in rat isolated corpus cavernosum smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs). Isometric tension was measured in intracavernous arterial rings and corpus cavernosum strips isolated from rats and men, and simultaneous measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and tension were performed in intracavernous arteries. Erectile response was measured in anaesthetized rats.

Key results:

In patch clamp recordings, NS11021 increased currents sensitive to the selective BK_Ca channel blocker, iberiotoxin (IbTX) in SMCs, but did not modulate K⁺ current in HUVECs. NS11021 reduced [Ca²⁺]_i and tension in penile arteries. IbTX inhibited the vasorelaxation induced by NS11021 and sildenafil in human erectile tissue. NS11021 and sildenafil but not vehicle increased erectile responses in anaesthetized rats, an effect which was abolished after pretreatment with tetraethylammonium.

Conclusions and implications:

NS11021 leads to relaxation of both intracavernous arteries and corpus cavernosum strips primarily through opening of BK_Ca channels. It is also effective in facilitating erectile responses in anaesthetized rats. These results suggest a potential for use of BK_Ca openers in the treatment of ED.     

Functional evidence for different roles of GABAA and GABAB receptors in modulating mouse gastric tone

The aims of the present study were to investigate, using mouse whole stomach in vitro, the effects of γ-aminobutyric acid (GABA) and GABA receptor agonists on the spontaneous gastric tone, to examine the subtypes of GABA receptors involved in the responses and to determine the possible site(s) of action.GABA induced gastric relaxation, which was antagonized by the GABA_A-receptor antagonist, bicuculline, potentiated by phaclofen, GABA_B-receptor antagonist, but not affected by 1,2,5,6-Tetrahydropyridin-4-yl methylphosphinic acid hydrate (TPMPA), GABA_C-receptor antagonist. Muscimol, GABA_A-receptor agonist, mimicked GABA effects inducing relaxation, which was significantly reduced by bicuculline, Nω-nitro-l-arginine methyl ester (l-NAME), inhibitor of NO synthase or apamin, inhibitor of small conductance Ca²⁺-dependent K⁺ channels, which blocks the purinergic transmission in this preparation. It was abolished by tetrodotoxin (TTX) or l-NAME plus apamin. Baclofen, a specific GABA_B-receptor agonist, induced an increase in the gastric tone, which was antagonized by phaclofen and abolished by TTX or atropine. Bicuculline, but not phaclofen or TPMPA, per se induced an increase in gastric tone, which was prevented by l-NAME. In conclusion, our results suggest that GABA is involved in the regulation of mouse gastric tone, through modulation of intrinsic neurons. Activation of GABA_A-receptors mediates relaxation through neural release of NO and neurotransmitters, activating Ca²⁺-dependent K⁺ channels, likely purines, while activation of GABA_B-receptors leads to contraction through acetylcholine release.