SKA-31, an activator of Ca2+-activated K+ channels, improves cardiovascular function in aging.

Aging represents an independent risk factor for the development of cardiovascular disease, and is associated with complex structural and functional alterations in the vasculature, such as endothelial dysfunction. Small- and intermediate-conductance, Ca2+-activated K+ channels (KCa2.3 and KCa3.1, respectively) are prominently expressed in the vascular endothelium, and pharmacological activators of these channels induce robust vasodilation upon acute exposure in isolated arteries and intact animals. However, the effects of prolonged in vivo administration of such compounds are unknown. In our study, we hypothesized that such treatment would ameliorate aging-related cardiovascular deficits. Aged (∼18 months) male Sprague Dawley rats were treated daily with either vehicle or the KCa channel activator SKA-31 (10 mg/kg, intraperitoneal injection; n = 6/group) for 8 weeks, followed by echocardiography, arterial pressure myography, immune cell and plasma cytokine characterization, and tissue histology. Our results show that SKA-31 administration improved endothelium-dependent vasodilation, reduced agonist-induced vascular contractility, and prevented the aging-associated declines in cardiac ejection fraction, stroke volume and fractional shortening, and further improved the expression of endothelial KCa channels and associated cell signalling components to levels similar to those observed in young male rats (∼5 months at end of study). SKA-31 administration did not promote pro-inflammatory changes in either T cell populations or plasma cytokines/chemokines, and we observed no overt tissue histopathology in heart, kidney, aorta, brain, liver and spleen. SKA-31 treatment in young rats had little to no effect on vascular reactivity, select protein expression, tissue histology, plasma cytokines/chemokines or immune cell properties. Collectively, these data demonstrate that administration of the KCa channel activator SKA-31 improved aging-related cardiovascular function, without adversely affecting the immune system or promoting tissue toxicity.

Decreased coronary arteriolar response to KCa channel opener after cardioplegic arrest in diabetic patients.

We have recently found that diabetes is associated with the inactivation of the calcium-activated potassium channels (KCa) in endothelial cells, which may contribute to endothelial dysfunction in diabetic patients at baseline. In the current study, we further investigated the effects of diabetes on coronary arteriolar responses to the small (SK) and intermediate (IK) KCa opener NS309 in diabetic and non-diabetic patients and correlated that data with the changes in the SK/IK protein expression/distribution in the setting of cardioplegic ischemia and reperfusion (CP) and cardiopulmonary bypass (CPB). Coronary arterioles from the harvested right atrial tissue samples from diabetic and non-diabetic patients (n = 8/group) undergoing cardiac surgery were dissected pre- and post-CP/CPB. The in vitro relaxation response of pre-contracted arterioles was examined in the presence of the selective SK/IK opener NS309 (10-9-10-5 M). The protein expression/localization of KCa channels in the harvested atrial tissue samples, coronary microvessels, and primary cultured human coronary endothelial cells were assayed by Western blotting and immunohistochemistry. The relaxation response to NS309 post-CP/CPB was significantly decreased in diabetic and non-diabetic groups compared to their pre-CP/CPB responses, respectively (P < 0.05). Furthermore, this decrease was greater in the diabetic group than that of the non-diabetic group (P < 0.05). There were no significant differences in the total protein expression/distribution of SK/IK in the human myocardium, coronary microvessels or coronary endothelial cells between diabetic and non-diabetic groups or between pre- and post-CP/CPB (P > 0.05). Our results suggest that diabetes further inactivates SK/IK channels of coronary microvasculature early after CP/CPB and cardiac surgery. The lack of diabetic changes in SK/IK protein abundances in the setting of CP/CPB suggests that the effect is post-translational. This alteration may contribute to post-operative endothelial dysfunction in the diabetic patients early after CP/CPB and cardiac surgery.

Melatonin Receptor Agonist Ramelteon Reduces Ischemia-Reperfusion Injury Through Activation of Mitochondrial Potassium Channels.

Activation of melatonin receptors induces cardioprotection. Mitochondrial potassium channels (mKCa and mKATP) are involved in the signaling cascade of preconditioning. The melatonin receptor agonist ramelteon is an approved oral medication for treatment of insomnia, but nothing is known about possible cardioprotective properties. We investigated whether (1) ramelteon induces cardioprotection mediated by the melatonin receptor; (2) this effect is concentration-dependent; and (3) mKCa and/or mKATP channels are critically involved in ramelteon-induced cardioprotection. Hearts of male Wistar rats were randomized and placed on a Langendorff system, perfused with Krebs-Henseleit buffer at a constant pressure of 80 mm Hg. All hearts were subjected to 33 minutes of global ischemia and 60 minutes of reperfusion. Before, ischemic hearts were perfused with different concentrations of ramelteon (0.01-5 µM) for determination of a concentration-effect curve. In subsequent experiments, the lowest protective concentration of ramelteon was administered together with paxilline (mKCa channel inhibitor) and 5-hydroxydecanoate (mKATP channel inhibitor). To determine whether the reduction of ischemia and reperfusion injury by ramelteon is mediated by melatonin receptor, we combined ramelteon with luzindole, a melatonin receptor antagonist. Infarct size was determined by triphenyltetrazolium chloride staining. In control animals, infarct size was 58% ± 6%. Ramelteon in a concentration of 0.03 µM reduced infarct size to 28% ± 4% (P < 0.0001 vs. Con). A lower concentration of ramelteon did not initiate cardioprotection, and higher concentrations did not further decrease infarct size. Paxilline, 5-hydroxydecanoate, and luzindole completely blocked the ramelteon-induced cardioprotection. This study shows for the first time that (1) ramelteon induces cardioprotection through melatonin receptor; (2) the effect is not concentration-dependent; and (3) activation of mKCa and mKATP channels is involved.

Endogenous and Agonist-induced Opening of Mitochondrial Big Versus Small Ca2+-sensitive K+ Channels on Cardiac Cell and Mitochondrial Protection.

Both big (BKCa) and small (SKCa) conductance Ca-sensitive K channels are present in mammalian cardiac cell mitochondria (m). We used pharmacological agonists and antagonists of BKCa and SKCa channels to examine the importance of endogenous opening of these channels and the relative contribution of either or both of these channels to protect against contractile dysfunction and reduce infarct size after ischemia reperfusion (IR) injury through a mitochondrial protective mechanism. After global cardiac IR injury of ex vivo perfused Guinea pig hearts, we found the following: both agonists NS1619 (for BKCa) and DCEB (for SKCa) improved contractility; BKCa antagonist paxilline (PAX) alone or with SKCa antagonist NS8593 worsened contractility and enhanced infarct size; both antagonists PAX and NS8593 obliterated protection by their respective agonists; BKCa and SKCa antagonists did not block protection afforded by SKCa and BKCa agonists, respectively; and all protective effects by the agonists were blocked by scavenging superoxide anions (O2) with Mn(III) tetrakis (4-benzoic acid) porphyrin (TBAP). Contractile function was inversely associated with global infarct size. In in vivo rats, infusion of NS8593, PAX, or both antagonists enhanced regional infarct size while infusion of either NS1619 or DCEB reduced infarct size. In cardiac mitochondria isolated from ex vivo hearts after IR, combined SKCa and BKCa agonists improved respiratory control index and Ca retention capacity compared with IR alone, whereas the combined antagonists did not alter respiratory control index but worsened Ca retention capacity. Although the differential protective bioenergetics effects of endogenous or exogenous BKCa and SKCa channel opening remain unclear, each channel likely responds to different sensing Ca concentrations and voltage gradients over time during oxidative stress-induced injury to individually or together protect cardiac mitochondria and myocytes.

A pharmacologic activator of endothelial KCa channels enhances coronary flow in the hearts of type 2 diabetic rats.

Endothelial dysfunction is a common early pathogenic event in patients with type 2 diabetes (T2D) who exhibit cardiovascular disease. In the present study, we have examined the effect of SKA-31, a positive modulator of endothelial Ca(2+)-activated K(+) (KCa) channels, on total coronary flow in isolated hearts from Goto-Kakizaki rats, a non-obese model of T2D exhibiting metabolic syndrome. Total coronary flow and left ventricular developed pressure were monitored simultaneously in isolated, spontaneously beating Langendorff-perfused hearts. Acute administrations of bradykinin (BK) or adenosine (ADO) increased coronary flow, but responses were significantly blunted in diabetic hearts at 10-12 and 18-20weeks of age compared with age-matched Wistar controls, consistent with the presence of endothelial dysfunction. In contrast, SKA-31 dose-dependently (0.01-5µg) increased total coronary flow to comparable levels in both control and diabetic rat hearts at both ages. Flow responses to sodium nitroprusside were not different between control and diabetic hearts, suggesting normal arterial smooth muscle function. Importantly, exposure to a sub-threshold concentration of SKA-31 (i.e. 0.3µM) rescued the impaired BK and ADO-evoked vasodilatory responses in diabetic hearts. Endothelial KCa channel activators may thus help to preserve coronary flow in diabetic myocardium.

Tuning electrical conduction along endothelial tubes of resistance arteries through Ca(2+)-activated K(+) channels.

RATIONALE: Electrical conduction through gap junction channels between endothelial cells of resistance vessels is integral to blood flow control. Small and intermediate-conductance Ca(2+)-activated K(+) channels (SK(Ca)/IK(Ca)) initiate electrical signals in endothelial cells, but it is unknown whether SK(Ca)/IK(Ca) activation alters signal transmission along the endothelium. OBJECTIVE: We tested the hypothesis that SK(Ca)/IK(Ca) activity regulates electrical conduction along the endothelium of resistance vessels. METHODS AND RESULTS: Freshly isolated endothelial cell tubes (60 µm wide; 1-3 mm long; cell length, ≈35 µm) from mouse skeletal muscle feed (superior epigastric) arteries were studied using dual intracellular microelectrodes. Current was injected (±0.1-3 nA) at site 1 while recording membrane potential (V(m)) at site 2 (separation distance=50-2000 µm). SK(Ca)/IK(Ca) activation (NS309, 1 µmol/L) reduced the change in V(m) along endothelial cell tubes by ≥50% and shortened the electrical length constant (λ) from 1380 to 850 µm (P<0.05) while intercellular dye transfer (propidium iodide) was maintained. Activating SK(Ca)/IK(Ca) with acetylcholine or SKA-31 also reduced electrical conduction. These effects of SK(Ca)/IK(Ca) activation persisted when hyperpolarization (>30 mV) was prevented with 60 mmol/L [K(+)](o). Conversely, blocking SK(Ca)/IK(Ca) (apamin+charybdotoxin) depolarized cells by ≈10 mV and enhanced electrical conduction (ie, changes in V(m)) by ≈30% (P<0.05). CONCLUSIONS: These findings illustrate a novel role for SK(Ca)/IK(Ca) activity in tuning electrical conduction along the endothelium of resistance vessels by governing signal dissipation through changes in membrane resistance. Voltage-insensitive ion channels can thereby tune intercellular electrical signaling independent from gap junction channels.

Cerebellar ataxia by enhanced Ca(V)2.1 currents is alleviated by Ca2+-dependent K+-channel activators in Cacna1a(S218L) mutant mice.

Mutations in the CACNA1A gene are associated with neurological disorders, such as ataxia, hemiplegic migraine, and epilepsy. These mutations affect the pore-forming α(1A)-subunit of Ca(V)2.1 channels and thereby either decrease or increase neuronal Ca(2+) influx. A decreased Ca(V)2.1-mediated Ca(2+) influx has been shown to reduce the regularity of cerebellar Purkinje cell activity and to induce episodic cerebellar ataxia. However, little is known about how ataxia can be caused by CACNA1A mutations that increase the Ca(2+) influx, such as the S218L missense mutation. Here, we demonstrate that the S218L mutation causes a negative shift of voltage dependence of Ca(V)2.1 channels of mouse Purkinje cells and results in lowered thresholds for somatic action potentials and dendritic Ca(2+) spikes and in disrupted firing patterns. The hyperexcitability of Cacna1a(S218L) Purkinje cells was counteracted by application of the activators of Ca(2+)-dependent K(+) channels, 1-EBIO and chlorzoxazone (CHZ). Moreover, 1-EBIO also alleviated the irregularity of Purkinje cell firing both in vitro and in vivo, while CHZ improved the irregularity of Purkinje cell firing in vitro as well as the motor performance of Cacna1a(S218L) mutant mice. The current data suggest that abnormalities in Purkinje cell firing contributes to cerebellar ataxia induced by the S218L mutation and they advocate a general therapeutic approach in that targeting Ca(2+)-dependent K(+) channels may be beneficial for treating ataxia not only in patients suffering from a decreased Ca(2+) influx, but also in those suffering from an increased Ca(2+) influx in their Purkinje cells.

Exchange protein activated by cAMP (Epac) induces vascular relaxation by activating Ca2+-sensitive K+ channels in rat mesenteric artery.

Vasodilator-induced elevation of intracellular cyclic AMP (cAMP) is a central mechanism governing arterial relaxation but is incompletely understood due to the diversity of cAMP effectors. Here we investigate the role of the novel cAMP effector exchange protein directly activated by cAMP (Epac) in mediating vasorelaxation in rat mesenteric arteries. In myography experiments, the Epac-selective cAMP analogue 8-pCPT-2-O-Me-cAMP-AM (5 µM, subsequently referred to as 8-pCPT-AM) elicited a 77.6 ± 7.1% relaxation of phenylephrine-contracted arteries over a 5 min period (mean ± SEM; n = 6). 8-pCPT-AM induced only a 16.7 ± 2.4% relaxation in arteries pre-contracted with high extracellular K(+) over the same time period (n = 10), suggesting that some of Epac's relaxant effect relies upon vascular cell hyperpolarization. This involves Ca(2+)-sensitive, large-conductance K(+) (BK(Ca)) channel opening as iberiotoxin (100 nM) significantly reduced the ability of 8-pCPT-AM to reverse phenylephrine-induced contraction (arteries relaxed by only 35.0 ± 8.5% over a 5 min exposure to 8-pCPT-AM, n = 5; P < 0.05). 8-pCPT-AM increased Ca(2+) spark frequency in Fluo-4-AM-loaded mesenteric myocytes from 0.045 ± 0.008 to 0.103 ± 0.022 sparks s(-1) µm(-1) (P < 0.05) and reversibly increased both the frequency (0.94 ± 0.25 to 2.30 ± 0.72 s(-1)) and amplitude (23.9 ± 3.3 to 35.8 ± 7.7 pA) of spontaneous transient outward currents (STOCs) recorded in isolated mesenteric myocytes (n = 7; P < 0.05). 8-pCPT-AM-activated STOCs were sensitive to iberiotoxin (100 nM) and to ryanodine (30 µM). Current clamp recordings of isolated myocytes showed a 7.9 ± 1.0 mV (n = 10) hyperpolarization in response to 8-pCPT-AM that was sensitive to iberiotoxin (n = 5). Endothelial disruption suppressed 8-pCPT-AM-mediated relaxation in phenylephrine-contracted arteries (24.8 ± 4.9% relaxation after 5 min of exposure, n = 5; P < 0.05), as did apamin and TRAM-34, blockers of Ca(2+)-sensitive, small- and intermediate-conductance K(+) (SK(Ca) and IK(Ca)) channels, respectively, and N(G)-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase (NOS). In Fluo-4-AM-loaded mesenteric endothelial cells, 8-pCPT-AM induced a sustained increase in global Ca(2+). Our data suggest that Epac hyperpolarizes smooth muscle by (1) increasing localized Ca(2+) release from ryanodine receptors (Ca(2+) sparks) to activate BK(Ca) channels, and (2) endothelial-dependent mechanisms involving the activation of SK(Ca)/IK(Ca) channels and NOS. Epac-mediated smooth muscle hyperpolarization will limit Ca(2+) entry via voltage-sensitive Ca(2+) channels and represents a novel mechanism of arterial relaxation.

Hydrogen sulfide preconditioning or neutrophil depletion attenuates ischemia-reperfusion-induced mitochondrial dysfunction in rat small intestine.

The objectives of this study were to determine whether neutrophil depletion with anti-neutrophil serum (ANS) or preconditioning with the hydrogen sulfide (H(2)S) donor NaHS (NaHS-PC) 24 h prior to ischemia-reperfusion (I/R) would prevent postischemic mitochondrial dysfunction in rat intestinal mucosa and, if so, whether calcium-activated, large conductance potassium (BK(Ca)) channels were involved in this protective effect. I/R was induced by 45-min occlusion of the superior mesenteric artery followed by 60-min reperfusion in rats preconditioned with NaHS (NaHS-PC) or a BK(Ca) channel activator (NS-1619-PC) 24 h earlier or treated with ANS. Mitochondrial function was assessed by measuring mitochondrial membrane potential, mitochondrial dehydrogenase function, and cytochrome c release. Mucosal myeloperoxidase (MPO) and TNF-α levels were also determined, as measures of postischemic inflammation. BK(Ca) expression in intestinal mucosa was detected by immunohistochemistry and Western blotting. I/R induced mitochondrial dysfunction and increased tissue MPO and TNF-α levels. Although mitochondrial dysfunction was attenuated by NaHS-PC or NS-1619-PC, the postischemic increases in mucosal MPO and TNF-α levels were not. The protective effect of NaHS-PC or NS-1619-PC on postischemic mitochondrial function was abolished by coincident treatment with BK(Ca) channel inhibitors. ANS prevented the I/R-induced increase in tissue MPO levels and reversed mitochondrial dysfunction. These data indicate that neutrophils play an essential role in I/R-induced mucosal mitochondrial dysfunction. In addition, NaHS-PC prevents postischemic mitochondrial dysfunction (but not inflammation) by a BK(Ca) channel-dependent mechanism.

Structure-Activity Relationship Study of Subtype-Selective Positive Modulators of KCa2 Channels.

A series of modified N-cyclohexyl-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-methylpyrimidin-4-amine (CyPPA) analogues were synthesized by replacing the cyclohexane moiety with different 4-substituted cyclohexane rings, tyrosine analogues, or mono- and dihalophenyl rings and were subsequently studied for their potentiation of KCa2 channel activity. Among the N-benzene-N-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine derivatives, halogen decoration at positions 2 and 5 of benzene-substituted 4-pyrimidineamine in compound 2q conferred a ∼10-fold higher potency, while halogen substitution at positions 3 and 4 of benzene-substituted 4-pyrimidineamine in compound 2o conferred a ∼7-fold higher potency on potentiating KCa2.2a channels, compared to that of the parent template CyPPA. Both compounds retained the KCa2.2a/KCa2.3 subtype selectivity. Based on the initial evaluation, compounds 2o and 2q were selected for testing in an electrophysiological model of spinocerebellar ataxia type 2 (SCA2). Both compounds were able to normalize the abnormal firing of Purkinje cells in cerebellar slices from SCA2 mice, suggesting the potential therapeutic usefulness of these compounds for treating symptoms of ataxia.

CyPPA, a positive modulator of small-conductance Ca(2+)-activated K(+) channels, inhibits phasic uterine contractions and delays preterm birth in mice.

Organized uterine contractions, including those necessary for parturition, are dependent on calcium entry through voltage-gated calcium channels in myometrial smooth muscle cells. Recent evidence suggests that small-conductance Ca(2+)-activated potassium channels (K(Ca)2), specifically isoforms K(Ca)2.2 and 2.3, may control these contractions through negative feedback regulation of Ca(2+) entry. We tested whether selective pharmacologic activation of K(Ca)2.2/2.3 channels might depress uterine contractions, providing a new strategy for preterm labor intervention. Western blot analysis and immunofluorescence microscopy revealed expression of both K(Ca)2.2 and K(Ca)2.3 in the myometrium of nonpregnant (NP) and pregnant (gestation day 10 and 16; D10 and D16, respectively) mice. Spontaneous phasic contractions of isolated NP, D10, and D16 uterine strips were all suppressed by the K(Ca)2.2/2.3-selective activator CyPPA in a concentration-dependent manner. This effect was antagonized by the selective K(Ca)2 inhibitor apamin. Whereas CyPPA sensitivity was reduced in D10 and D16 versus NP strips (pIC(50) 5.33 ± 0.09, 4.64 ± 0.03, 4.72 ± 0.10, respectively), all contractions were abolished between 30 and 60 µM. Blunted contractions were associated with CyPPA depression of spontaneous Ca(2+) events in myometrial smooth muscle bundles. Augmentation of uterine contractions with oxytocin or prostaglandin F(2α) did not reduce CyPPA sensitivity or efficacy. Finally, in an RU486-induced preterm labor model, CyPPA significantly delayed time to delivery by 3.4 h and caused a 2.5-fold increase in pup retention. These data indicate that pharmacologic stimulation of myometrial K(Ca)2.2/2.3 channels effectively suppresses Ca(2+)-mediated uterine contractions and delays preterm birth in mice, supporting the potential utility of this approach in tocolytic therapies.

Selective toxicity of the anthelmintic emodepside revealed by heterologous expression of human KCNMA1 in Caenorhabditis elegans.

Emodepside is a resistance-breaking anthelmintic of a new chemical class, the cyclooctadepsipeptides. A major determinant of its anthelmintic effect is the calcium-activated potassium channel SLO-1. SLO-1 belongs to a family of channels that are highly conserved across the animal phyla and regulate neurosecretion, hormone release, muscle contraction, and neuronal network excitability. To investigate the selective toxicity of emodepside, we performed transgenic experiments in which the nematode SLO-1 channel was swapped for a mammalian ortholog, human KCNMA1. Expression of either the human channel or Caenorhabditis elegans slo-1 from the native slo-1 promoter in a C. elegans slo-1 functional null mutant rescued behavioral deficits that otherwise resulted from loss of slo-1 signaling. However, worms expressing the human channel were 10- to 100-fold less sensitive to emodepside than those expressing the nematode channel. Strains expressing the human KCNMA1 channel were preferentially sensitive to the mammalian channel agonists NS1619 and rottlerin. In the C. elegans pharyngeal nervous system, slo-1 is expressed in neurons, not muscle, and cell-specific rescue experiments have previously shown that emodepside inhibits serotonin-stimulated feeding by interfering with SLO-1 signaling in the nervous system. Here we show that ectopic overexpression of slo-1 in pharyngeal muscle confers sensitivity of the muscle to emodepside, consistent with a direct interaction of emodepside with the channel. Taken together, these data predict an emodepside-selective pharmacophore harbored by SLO-1. This has implications for the development of this drug/target interface for the treatment of helminth infections.

Opening of small and intermediate calcium-activated potassium channels induces relaxation mainly mediated by nitric-oxide release in large arteries and endothelium-derived hyperpolarizing factor in small arteries from rat.

This study was designed to investigate whether calcium-activated potassium channels of small (SK(Ca) or K(Ca)2) and intermediate (IK(Ca) or K(Ca)3.1) conductance activated by 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) are involved in both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in large and small rat mesenteric arteries. Segments of rat superior and small mesenteric arteries were mounted in myographs for functional studies. NO was recorded using NO microsensors. SK(Ca) and IK(Ca) channel currents and mRNA expression were investigated in human umbilical vein endothelial cells (HUVECs), and calcium concentrations were investigated in both HUVECs and mesenteric arterial endothelial cells. In both superior (∼1093 µm) and small mesenteric (∼300 µm) arteries, NS309 evoked endothelium- and concentration-dependent relaxations. In superior mesenteric arteries, NS309 relaxations and NO release were inhibited by both N(G),N(G)-asymmetric dimethyl-l-arginine (ADMA) (300 µM), an inhibitor of NO synthase, and apamin (0.5 µM) plus 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) (1 µM), blockers of SK(Ca) and IK(Ca) channels, respectively. In small mesenteric arteries, NS309 relaxations were reduced slightly by ADMA, whereas apamin plus an IK(Ca) channel blocker almost abolished relaxation. Iberiotoxin did not change NS309 relaxation. HUVECs expressed mRNA for SK(Ca) and IK(Ca) channels, and NS309 induced increases in calcium, outward current, and NO release that were blocked by apamin and TRAM-34 or charybdotoxin. These findings suggest that opening of SK(Ca) and IK(Ca) channels leads to endothelium-dependent relaxation that is mediated mainly by NO in large mesenteric arteries and by EDHF-type relaxation in small mesenteric arteries. NS309-induced calcium influx appears to contribute to the formation of NO.

Twenty-four-hour exposure to altered blood flow modifies endothelial Ca2+-activated K+ channels in rat mesenteric arteries.

We tested the hypothesis that changes in arterial blood flow modify the function of endothelial Ca2+-activated K+ channels [calcium-activated K+ channel (K(Ca)), small-conductance calcium-activated K+ channel (SK3), and intermediate calcium-activated K+ channel (IK1)] before arterial structural remodeling. In rats, mesenteric arteries were exposed to increased [+90%, high flow (HF)] or reduced blood flow [-90%, low flow (LF)] and analyzed 24 h later. There were no detectable changes in arterial structure or in expression level of endothelial nitric-oxide synthase, SK3, or IK1. Arterial relaxing responses to acetylcholine and 3-oxime-6,7-dichlore-1H-indole-2,3-dione (NS309; activator of SK3 and IK1) were measured in the absence and presence of endothelium, NO, and prostanoid blockers, and 6,12,19,20,25,26-hexahydro-5,27:13,18:21,24-trietheno-11,7-metheno-7H-dibenzo [b,n] [1,5,12,16]tetraazacyclotricosine-5,13-diium dibromide (UCL 1684; inhibitor of SK3) or 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; inhibitor of IK1). In LF arteries, endothelium-dependent relaxation was markedly reduced, due to a reduction in the endothelium-derived hyperpolarizing factor (EDHF) response. In HF arteries, the balance between the NO/prostanoid versus EDHF response was unaltered. However, the contribution of IK1 to the EDHF response was enhanced, as indicated by a larger effect of TRAM-34 and a larger residual NS309-induced relaxation in the presence of UCL 1684. Reduction of blood flow selectively blunts EDHF relaxation in resistance arteries through inhibition of the function of K(Ca) channels. An increase in blood flow leads to a more prominent role of IK1 channels in this relaxation.

Differential regulation of calcium-activated potassium channels by dynamic intracellular calcium signals.

Calcium (Ca(2+))-activated K(+) (K(Ca)) channels regulate membrane excitability and are activated by an increase in cytosolic Ca(2+) concentration ([Ca(2+)](i)), leading to membrane hyperpolarization. Most patch clamp experiments that measure K(Ca) currents use steady-state [Ca(2+)] buffered within the patch pipette. However, when cells are stimulated physiologically, [Ca(2+)](i) changes dynamically, for example during [Ca(2+)](i) oscillations. Therefore, the aim of the present study was to examine the effect of dynamic changes in [Ca(2+)](i) on small (SK3), intermediate (hIK1), and large conductance (BK) channels. HEK293 cells stably expressing each K(Ca) subtype in isolation were used to simultaneously measure agonist-evoked [Ca(2+)](i) signals, using indo-1 fluorescence, and current/voltage, using perforated patch clamp. Agonist-evoked [Ca(2+)](i) oscillations induced a corresponding K(Ca) current that faithfully followed the [Ca(2+)](i) in 13-50% of cells, suggesting a good synchronization. However, [Ca(2+)](i) and K(Ca) current was much less synchronized in 50-76% of cells that exhibited Ca(2+)-independent current events (55% of SK3-, 50% of hIK1-, and 53% of BK-expressing cells) and current-independent [Ca(2+)](i) events (18% SK3- and 33% of BK-expressing cells). Moreover, in BK-expressing cells, where [Ca(2+)](i) and K(Ca) current was least synchronized, 36% of total [Ca(2+)](i) spikes occurred without activating a corresponding K(Ca) current spike, suggesting that BK(Ca) channels were either inhibited or had become desensitized. This desynchronization between dynamic [Ca(2+)](i) and K(Ca) current suggests that this relationship is more complex than could be predicted from steady-state [Ca(2+)](i) and K(Ca) current. These phenomena may be important for encoding stimulus-response coupling in various cell types.

Openers of SKCa and IKCa channels enhance agonist-evoked endothelial nitric oxide synthesis and arteriolar vasodilation.

Recent data have led us to hypothesize that selective activation of endothelial small- and/or intermediate-conductance, calcium-activated potassium channels (SK(Ca) and IK(Ca) channels, respectively) by the opener compounds NS309 and DCEBIO would augment stimulated nitric oxide (NO) synthesis and vasodilation in resistance arteries. Experimentally, ATP-evoked changes in membrane potential, cytosolic Ca(2+), and NO synthesis were recorded by patch clamp and microfluorimetry in single human endothelial cells. Agonist-evoked inhibition of myogenic tone in isolated, pressurized arterioles from rat cremaster skeletal muscle was analyzed by video microscopy. NS309 and DCEBIO enhanced ATP-evoked membrane hyperpolarization and cytosolic Ca(2+) transients, along with acute NO synthesis in isolated endothelial cells. The acetylcholine-mediated inhibition of myogenic tone (IC(50)=237 nM) was left-shifted in the presence of NS309 and DCEBIO (10, 100, and 1000 nM) to IC(50) values of 101, 78, and 43 nM; endothelial denudation inhibited this drug effect. L-NAME attenuated the acetylcholine-induced inhibition of myogenic tone but did not interfere with NS309 and DCEBIO-evoked vasodilation. Collectively, our data demonstrate that drug-induced enhancement of endothelial SK(Ca) and IK(Ca) channel activities represents a novel cellular mechanism to increase vasodilation of small-resistance arterioles, thereby highlighting these channels as potential therapeutic targets in cardiovascular disease states associated with compromised NO signaling.

Morphine induces preconditioning via activation of mitochondrial K(Ca) channels.

PURPOSE: Mitochondrial calcium sensitive potassium (mK(Ca)) channels are involved in cardioprotection induced by ischemic preconditioning. In the present study we investigated whether morphine-induced preconditioning also involves activation of mK(Ca) channels. METHODS: Isolated rat hearts (six groups; each n = 8) underwent global ischemia for 30 min followed by a 60-min reperfusion. Control animals were not further treated. Morphine preconditioning (MPC) was initiated by two five-minute cycles of morphine 1 microM infusion with one five-minute washout and one final ten-minute washout period before ischemia. The mK(Ca) blocker, paxilline 1 microM, was administered, with and without morphine administration (MPC + Pax and Pax). As a positive control, we added an ischemic preconditioning group (IPC) alone and combined with paxilline (IPC + Pax). At the end of reperfusion, infarct sizes were determined by triphenyltetrazoliumchloride staining. RESULTS: Infarct size was (mean +/- SD) 45 +/- 9% of the area at risk in the Control group. The infarct size was less in the morphine or ischemic preconditioning groups (MPC: 23 +/- 8%, IPC: 20 +/- 5%; each P < 0.05 vs Control). Infarct size reduction was abolished by paxilline (MPC + Pax: 37 +/- 7%, P < 0.05 vs MPC and IPC + Pax: 36 +/- 6%, P < 0.05 vs IPC), whereas paxilline alone had no effect (Pax: 46 +/- 7%, not significantly different from Control). CONCLUSION: Cardioprotection by morphine-induced preconditioning is mediated by activation of mK(Ca) channels.

High ability of zileuton ((±)-1-(1-benzo[b]thien-2-ylethyl)-1-hydroxyurea) to stimulate IK(Ca) but suppress IK(DR) and IK(M) independently of 5-lipoxygenase inhibition.

Zileuton (Zyflo®) is regarded to be an inhibitor of 5-lipoxygenase. Although its effect on Ca2+-activated K+ currents has been reported, its overall ionic effects on neurons are uncertain. In whole-cell current recordings, zileuton increased the amplitude of Ca2+-activated K+ currents with an EC50 of 3.2 µM in pituitary GH3 lactotrophs. Furthermore, zileuton decreased the amplitudes of both delayed-rectifier K+ current (IK(DR)) and M-type K+ current (IK(M)). Conversely, no modification of hyperpolarization-activated cation current (Ih) was demonstrated in its presence of zileuton, although the subsequent addition of cilobradine effectively suppressed the current. In inside-out current recordings, the addition of zileuton to the bath increased the probability of large-conductance Ca2+-activated K+ (BKCa) channels; however, the subsequent addition of GAL-021 effectively reversed the stimulation of channel activity. The kinetic analyses showed an evident shortening in the slow component of mean closed time of BKCa channels in the presence of zileuton, with minimal change in mean open time or that in the fast component of mean closed time. The elevation of BKCa channels caused by zileuton was also observed in hippocampal mHippoE-14 neurons, without any modification of single-channel amplitude. In conclusion, except for its suppression of 5-lipoxygenase, our results indicate that zileuton does not exclusively act on BKCa channels, and its inhibitory effects on IK(DR) and IK(M) may combine to exert strong influence on the functional activities of electrically excitable cells in vivo.

Calcium-activated potassium channels: implications for aging and age-related neurodegeneration.

Population aging, as well as the handling of age-associated diseases, is a worldwide increasing concern. Among them, Alzheimer's disease stands out as the major cause of dementia culminating in full dependence on other people for basic functions. However, despite numerous efforts, in the last decades, there was no new approved therapeutic drug for the treatment of the disease. Calcium-activated potassium channels have emerged as a potential tool for neuronal protection by modulating intracellular calcium signaling. Their subcellular localization is determinant of their functional effects. When located on the plasma membrane of neuronal cells, they can modulate synaptic function, while their activation at the inner mitochondrial membrane has a neuroprotective potential via the attenuation of mitochondrial reactive oxygen species in conditions of oxidative stress. Here we review the dual role of these channels in the aging phenotype and Alzheimer's disease pathology and discuss their potential use as a therapeutic tool.

Mitochondrial potassium channels.

Mitochondrial potassium channels are believed to contribute to cytoprotection of injured cardiac and neuronal tissues. The following potassium channels have been described in the inner mitochondrial membrane: the ATP-regulated potassium channel, the large conductance Ca(2+)-activated potassium channel, the voltage-gated Kv1.3 potassium channel, and the twin-pore domain TASK-3 potassium channel. The putative functional roles of these channels include changes in mitochondrial matrix volume, mitochondrial respiration, and membrane potential. In addition, the activity of these channels modulates the generation of reactive oxygen species by mitochondria. In this article, we discuss recent observations on three fundamental issues concerning mitochondrial potassium channels: (i) their molecular identity, (ii) their interaction with potassium channel openers and inhibitors, and (iii) their functional properties.