PKI 14-22 amide,myristoylated

Modaﬁnil inhibits KCa3.1 currents and muscle contraction via a cAMP-dependent mechanism

Shinkyu Choia,1, Moon Young Kima,1,2, Ka Young Jooa, Seonghee Parka, Ji Aee Kima,
Jae-Chul Jungb, Seikwan Ohb, Suk Hyo Suha,∗
a Department of Physiology, Medical School, Ewha Womans University, Seoul, South Korea
b Department of Neuroscience, Medical School, Ewha Womans University, Seoul, South Korea

Abstract

Modaﬁnil has been used as a psychostimulant for the treatment of narcolepsy. However, its primary mechanism of action remains elusive. Therefore, we examined the effects of modaﬁnil on KCa3.1 chan- nels and vascular smooth muscle contraction. KCa3.1 currents and channel activity were measured using a voltage-clamp technique and inside-out patches in mouse embryonic ﬁbroblast cell line, NIH-3T3 ﬁbrob- lasts. Intracellular adenosine 3∗,5∗-cyclic monophosphate (cAMP) concentration was measured, and the phosphorylation of KCa3.1 channel protein was examined using western blotting in NIH-3T3 ﬁbroblasts and/or primary cultured mouse aortic smooth muscle cells (SMCs). Muscle contractions were recorded from mouse aorta and rat pulmonary artery by using a myograph developed in-house. Modaﬁnil was found to inhibit KCa3.1 currents in a concentration-dependent manner, and the half-maximal inhibi- tion (IC50) of modaﬁnil for the current inhibition was 6.8 ± 0.7 nM. The protein kinase A (PKA) activator forskolin also inhibited KCa3.1 currents. The inhibitory effects of modaﬁnil and forskolin on KCa3.1 currents were blocked by the PKA inhibitors PKI14–22 or H-89. In addition, modaﬁnil relaxed blood vessels (mouse aorta and rat pulmonary artery) in a concentration-dependent manner. Modaﬁnil increased cAMP con- centrations in NIH-3T3 ﬁbroblasts or primary cultured mouse aortic SMCs and phosphorylated KCa3.1 channel protein in NIH-3T3 ﬁbroblasts. However, open probability and single-channel current ampli- tudes of KCa3.1 channels were not changed by modaﬁnil. From these results, we conclude that modaﬁnil inhibits KCa3.1 channels and vascular smooth muscle contraction by cAMP-dependent phosphorylation, suggesting that modaﬁnil can be used as a cAMP-dependent KCa3.1 channel blocker and vasodilator.

1. Introduction

The psychostimulant modaﬁnil has been used to treat sleep disorders such as narcolepsy [1]. There are ongoing clinical tri- als to assess its use in other psychiatric disorders such as cocaine dependence, attention deﬁcit hyperactivity disorder, depression, seasonal affective disorder, bipolar depression, nicotine addiction, and schizophrenia [1–5]. Some preclinical evidence also indicates its possible use in the treatment of neurodegenerative diseases, Parkinson’s disease, and cancer-related fatigue [6–9]. The use of modaﬁnil for treating these psychiatric disorders is noteworthy, as other medications for treatment are yet to be approved.

Most research on the mechanism of action of modaﬁnil has focused on neuronal cells and its monoaminergic effects, showing that modaﬁnil stimulates the histamine, norepinephrine, sero- tonin, dopamine, and orexin pathways in the brain. Modaﬁnil binds to and modulates dopamine and norepinephrine transporters [10,11]. In addition, modaﬁnil ( 100 µM) is known to inhibit human hepatic cytochrome P450 activities [12] and has a neu- roprotective function [13–16]. However, its primary mechanism of action remains elusive, and the intracellular sites of action for modaﬁnil need to be studied to explain its stimulant and neuropro- tective effects. In addition, since most studies regarding the effects of modaﬁnil have focused on the central nervous system, further studies should be extended to cells other than neuronal cells.

Although many cellular functions are modulated by ion channels, little is known about the effects of modaﬁnil on ion channels. While modaﬁnil (1 mM) has been shown to have no binding afﬁnity to Cl− channels, low conduction K+ channels, or Ca2+ channels [17], it has been suggested that ion channels are involved in enhanced neuroelectrosecretory coupling by modaﬁnil [18]. Furthermore, modaﬁnil (0.01–1 mM) has been demonstrated to inhibit gamma- aminobutyric acid (GABA)-activated currents and is thought to modulate KATP channels [16]. We therefore examined the effect of modaﬁnil on ion channels and found that modaﬁnil inhibits the intermediate-conductance Ca2+-activated K+ (KCa3.1) channels in the mouse embryonic ﬁbroblast cell line NIH-3T3 ﬁbroblasts. During this study, modaﬁnil was found to increase intracellular 3∗,5∗-cyclic monophosphate (cAMP) content in various cells, includ- ing NIH-3T3 ﬁbroblasts and vascular smooth muscle (VSM). Since VSM relaxes via a cAMP-dependent mechanism, we examined the effect of modaﬁnil on VSM contraction and found that modaﬁnil relaxed VSMs. We therefore investigated the mechanism by which modaﬁnil inhibits KCa3.1 channels and relaxes VSMs.

2. Materials and methods

The investigation complied with the principles outlined in the Declaration of Helsinki and the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996) pub- lished by the US National Institute of Health and was approved by the Institutional Review Board for Human Research and Animal Care and Use Committee at Ewha Womans University in Seoul.

2.1. Cell isolation and culture

NIH-3T3 ﬁbroblasts (CRL-2795) were purchased from Ameri- can Type Culture Collection (Manassas, VA, USA) and cultured as a monolayer in Dulbecco’s modiﬁed Eagle medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 µg/ml streptomycin. All cells were maintained at 37 ◦C in humidiﬁed conditions under 5% CO2. Media were changed twice weekly, and cultures were passaged at a dilu- tion of 1:5 weekly. The medium was then removed and replaced with fresh medium, and the cells were maintained for the time periods indicated.

Single smooth muscle cells (SMCs) were isolated from mouse aorta. Three- to 4-month-old mice of either sex were anesthetized with pentobarbital sodium (50 mg/kg body weight) and sacri- ﬁced by cervical dislocation. Thoracic aortas were dissected out in Ca2+-free external solution and dissociated by incubation in papain (1 mg/ml) for 5 min followed by incubation in collagenase (1.5 mg/ml), bovine serum albumin (2 mg/ml), and dithiothreitol (1 mg/ml) for 10–20 min in Ca2+-free external solution at 37 ◦C. Segments were then transferred to fresh Ca2+-free external solution, and single mouse SMCs were dispersed with gentle agitation using a ﬁre-polished wide-bore glass pipette.Mouse aortic SMCs were grown in DMEM containing 10% FBS plus 1% minimum essential amino acids (Life Technologies, Grand Island, NY, USA). The cell culture was maintained at 37 ◦C in fully humidiﬁed air with 5% CO2.

2.2. Electrophysiology

The patch-clamp technique was used in whole-cell and excised- patch conﬁgurations at room temperature. Whole-cell currents were measured using ruptured patches. Currents were moni- tored using an appropriate ampliﬁer (EPC-10; HEKA, Lambrecht, Germany). In the whole-cell experiment, we applied a voltage ramp from 100 mV to +100 mV with a 10-s interval (650 ms duration) or voltage steps from a holding potential of 60 mV to potentials ranging from 100 mV to 100 mV in 20 mV increments (1 s dura- tion) with a 5-s interval. Currents were recorded at a sampling rate of 1–4 kHz. Inside-out voltage clamps were performed using glass electrodes with a tip resistance of 8–10 M▲. Data were ﬁltered at 1 kHz and stored in a computer for analysis using standard soft- ware (Axoscope 9.0; Axon Instruments, Foster City, CA, USA). For some experiments, channel activity was expressed as the product of the number of channels and the open probability, NPo, where NPo = [(open time number of channels open)/total recording time].

The standard external solution contained (in mM) 150 NaCl, 6 KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose; the solution was brought to pH 7.4 with NaOH. The osmolarity of this solu- tion, as determined by an osmometer (Fiske, Norwood, MA, USA), was 320 ± 5 mOsmol. In the inside-out mode, the bath solution con- tained (in mM) 150 KCl, 0.5 MgCl2, 10 HEPES, pH 7.2 with KOH, and the pipette solution contained (in mM) 150 NaCl, 6 KCl, 1 MgCl2, 10 HEPES, 10 glucose, and 5 EGTA; NaOH was used to bring the pH to 7.4. Free Ca2+ concentrations in the pipette or bath solu- tions were adjusted to 1 µM by adding appropriate amounts of Ca2+ (calculated using CaBuf software; G. Droogmans, Leuven, Belgium; ftp://ftp.cc.kuleuven.ac.be/pub/droogmans/cabuf.zip) in the presence of 5 mM EGTA.KCa3.1 currents were activated by loading 1 µM Ca2+ via a patch pipette in whole-cell clamped NIH-3T3 ﬁbroblasts and adding 1-ethyl-2-benzimidazolinone (1-EBIO; 100 µM) to the external solution.

2.3. Measurement of intracellular cAMP

NIH-3T3 ﬁbroblasts or cultured mouse aortic SMCs were incubated with modaﬁnil for 3 min in incomplete media. The con- centration of cAMP from the lysate was determined in each sample using a Parameter cAMP assay kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

2.4. Western blotting

After the treatment, cells were washed once in ice-cold phos- phate buffered saline (PBS) and lysed in protein-extraction buffer containing protease inhibitor cocktail. Protein concentration in the supernatant fraction was determined using the Bradford protein assay. For western blot analysis, 30 µg of protein was subjected to SDS-PAGE, and proteins were then transferred to a nitrocellulose membrane. Membranes were blocked for 1 h with TBST (10 mM Tris–HCl, 150 mM NaCl, and 1% Tween-20 (v/v), pH 7.6) containing 5% bovine serum albumin at room temperature. The blots were incubated for 3 h with rabbit anti-P- PKA substrate antibody, followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h. Bands were visualized by chemiluminescence. Data collection and processing were performed using a luminescent image analyzer, LAS- 3000, and IMAGE GAUSE software (Fuji Film, Minato-ku, Tokyo, Japan).

2.5. Contraction measurement on isolated arterial rings

Five- to 6-month-old mice or rats were anesthetized by an intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight). The mouse thoracic aorta and rat pulmonary artery were dissected and cut into rings of approximately 2–3 mm in length. Mechanical responses were recorded from the ring segments using a myograph developed in-house [19]. Each ring was threaded with 2 strands of tungsten wire (120 µm in diameter). One wire was anchored in the organ bath chamber (1 ml) and the other was connected to a mechanotransducer (FT-03; Grass Technolo- gies, West Warwick, RI, USA) mounted on a three-dimensional manipulator. The muscle chamber was perfused at a ﬂow rate of 2.5 ml/min with oxygenated (95% O2/5% CO2) Krebs/Ringer bicarbonate solution with a peristaltic pump. The composition (in mM) of the Krebs solution was as follows: NaCl, 118.3; KCl, 4.7; MgCl2, 1.2; KH2PO4, 1.22; CaCl2, 2.5; NaHCO3, 25.0; glucose, 11.1; pH 7.4. Endothelial cells were removed by gentle rubbing with a cotton ball, and NO production in endothelial cells was inhibited by pretreating with NG-nitro-L-arginine methyl ester (L- NAME). Rings were precontracted with 0.1 µM norepinephrine or 30 mM K+.

Fig. 1. Effect of modaﬁnil on KCa 3.1 currents. Cells were loaded with 1 µM Ca2+ in the pipette and exposed to the KCa 3.1 channel activator 1-EBIO (100 µM). (A–C) Data points were obtained at 50 mV during repetitive ramps (upper traces), and current–voltage (I–V) curves obtained at the points marked in upper traces are shown in lower traces. (D) KCa 3.1 currents recorded by applying voltage steps (left traces) and the current–voltage (I–V) curves of the currents measured at the middle of the steps (right traces). (E) The concentration–response relationship. The magnitude of KCa 3.1 current inhibition at each treatment was expressed as a percentage of initial KCa 3.1 current density at 50 mV (n = 7).

2.6. Chemicals

The PKA inhibitor H-89, PKA activator forskolin, and KCa3.1 channel blocker TRAM-34 were purchased from Sigma–Aldrich (St Louis, MO, USA); KCa3.1 channel activator 1-EBIO, from Tocris Bio- science (Ellisville, MO, USA); PKA inhibitor PKI14–22, from Enzo Life Sciences (Farmingdale, NY, USA); cAMP-dependent vasodila- tor iloprost, from Cayman Chemical Company (Ann Arbor, MI, USA); rabbit anti-P-PKA substrate, from Cell Signaling Technology (Danvers, MA, USA); rabbit anti-KCa3.1, from Santa Cruz Biotech- nology (Santa Cruz, CA, USA); and collagenase, from Wako (Japan). Modaﬁnil, TRAM-34, and 1-EBIO were ﬁrst dissolved in dimethyl- sulfoxide (DMSO). The ﬁnal concentration of DMSO was 0.1%; at this level, DMSO exhibited no effect.

2.7. Statistical analysis

Data are expressed as mean SEM. Statistical analysis of the data was performed using ANOVA. Values with p < 0.05 were con- sidered statistically signiﬁcant. 3. Results 3.1. Inhibition of KCa3.1 current by modaﬁnil When the NIH-3T3 ﬁbroblast was loaded with 1 µM Ca2+ via the patch pipette and exposed to 1-EBIO, outward currents devel- oped slowly (Fig. 1). The current–voltage curve by ramp or step pulses was linear at potentials negative to 50 mV and bent toward an abscissa at potentials positive to 50 mV. The currents were inhib- ited by the speciﬁc KCa3.1 channel blocker TRAM-34 (Fig. 1A). These results suggest that KCa3.1 currents are activated. We then examined the effect of modaﬁnil on KCa3.1 currents (Fig. 1B–D). Modaﬁnil inhibited KCa3.1 currents in a concentration- dependent manner. When current inhibition was ﬁtted for each modaﬁnil concentration using Hill’s equation, the concentration required to evoke half-maximal inhibition (IC50) of modaﬁnil for the current inhibition was found to be 6.8 ± 0.7 nM, and the con- centration of modaﬁnil for maximal inhibition was 100.0 ± 5.7 nM. The Hill coefﬁcient was 1.3 0.2. In addition, when KCa3.1 current was activated only by Ca2+ and without applying 1-EBIO, the IC50 and the Hill coefﬁcient of modaﬁnil were not changed signiﬁcantly (data not shown), suggesting that the effect of modaﬁnil on the KCa3.1 current is not affected by 1-EBIO. 3.2. PKA inhibitors block the inhibitory effect of modaﬁnil on KCa3.1 currents KCa3.1 channels were reported to be inhibited by PKA-induced phosphorylation of the channel protein [20], suggesting that modaﬁnil might inhibit KCa3.1 currents by activating PKA. Thus, we determined whether PKA inhibition by the PKA inhibitors H- 89 and PKI14–22 blocks the inhibitory effect of modaﬁnil on KCa3.1 currents (Fig. 2A and B). KCa3.1 currents were activated in cells pre- treated by H-89 (10 µM), and modaﬁnil was then applied to the cells by adding it to the external solution. The activated KCa3.1 cur- rents, which reached a steady state, were not inhibited by modaﬁnil in cells pretreated by H-89 (Fig. 2A). In addition, when PKI14–22 (10 µM) was applied to cells by adding it to the pipette solution, KCa3.1 currents were not affected by modaﬁnil (Fig. 2B). Further- more, we examined whether the PKA activator forskolin inhibits KCa3.1 currents. When the activated KCa3.1 currents reached a steady state, forskolin (10 µM) was added to the external solution; the KCa3.1 currents subsequently decreased (Fig. 2C). In contrast, when PKI14–22 (10 µM) was applied to the cell by adding it to the pipette solution, the KCa3.1 currents were not affected by forskolin (Fig. 2D). These results suggest that modaﬁnil-induced inhibition of KCa3.1 currents occurs via a PKA-mediated event. Fig. 2. Role of protein kinase A in modaﬁnil-induced KCa 3.1 current inhibition. KCa 3.1 currents were activated by loading Ca2+ (1 µM) and applying 1-EBIO (100 µM). Data points were obtained at 50 mV during repetitive ramps (left upper traces), and I–V curves obtained at the points marked in upper traces are shown in right lower traces. 3.3. Modaﬁnil relaxes arterial rings from mouse aorta and rat pulmonary artery Since PKA activation relaxes VSMs, we examined whether modaﬁnil relaxes VSMs. Mouse aortic rings or rat pulmonary arte- rial rings were contracted by norepinephrine (0.1 µM). When the contraction reached a steady state, modaﬁnil was applied. With the application, the contracted mouse aortic ring (Fig. 3A and B) or rat pulmonary arterial ring (Fig. 3C and D) was relaxed in a concentration-dependent manner. When the concentration- dependent response of rat pulmonary artery was ﬁtted to Hill’s equation, the IC50 of modaﬁnil for mouse aortic rings and rat pul- monary arterial rings was determined to be 11.9 ± 0.3 nM and 10.1 ± 0.1 nM, respectively. When the rat pulmonary artery was contracted by high K+ (30 mM), modaﬁnil relaxed the contracted artery in a concentration-dependent manner (Fig. 3E). When the concentration-dependent response of the rat pulmonary artery was ﬁtted to Hill’s equation, the IC50 of modaﬁnil for rat pulmonary arterial rings was determined to be 10.5 ± 0.4 µM (Fig. 3F). 3.4. Modaﬁnil increases intracellular cAMP concentration and phosphorylates KCa3.1 protein PKA is activated by cAMP and phosphorylates intracellular pro- teins such as KCa3.1 protein. We examined whether modaﬁnil increases cAMP and phosphorylates KCa3.1 protein. When NIH-3T3 cells (Fig. 4A and D) and cultured mouse aortic SMCs (Fig. 4B and C) were exposed to modaﬁnil for 3 min, intracellular cAMP concen- tration increased in a concentration-dependent manner. Iloprost (Fig. 4C) and forskolin (Fig. 4D) also increased cAMP concentration in cultured mouse aortic SMCs and NIH-3T3 cells. Modaﬁnil increased KCa3.1 phosphorylation in a concentration- dependent manner (Fig. 5), and phosphorylation was inhibited by H-89 (data not shown). The presence of KCa3.1 channels was conﬁrmed by western blotting (Fig. 5A, left panel). KCa3.1 phosphorylation was then measured by using the phospho-PKA substrate antibody, which detects peptides and proteins containing a phopho-serine/threonine residue with arginine at the -3 posi- tion [21,22]. If the KCa3.1 channel was phosphorylated by PKA, the phospho-form of the KCa3.1 channel could be detected by anti- phospho-PKA substrate antibody. KCa3.1 channel phosphorylation was shown in the resting state, and channel phosphorylation was increased by the treatment of modaﬁnil or forskolin for 5 min (Fig. 5A, right panel). The channel phosphorylation in the resting state (data not shown) and by forskolin (Fig. 5) was completely abolished by the pretreatment of H-89. In addition, it was previ- ously shown that cAMP phosphorylates myosin light chain kinase (MLCK) in VSMs and relaxes VSMs [23]. These results suggest that modaﬁnil activates PKA by increasing cAMP to phosphorylate tar- get proteins such as the KCa3.1 channels and thereby inhibits the channel and relaxes VSM. Fig. 3. Concentration-dependent relaxation of VSM by modaﬁnil. Endothelial cells were removed by gentle rubbing with a cotton ball and NO production in endothelial cells was inhibited by pretreating with L-NAME. Mouse aortic rings were contracted with 0.1 µM norepinephrine (A and B) and rat pulmonary arterial rings with 0.1 µM norepinephrine (C and D) or 30 mM K+ (E and F). The magnitude of relaxation at each treatment was expressed as a percentage of initial norepinephrine or K+-induced contraction (B, D, and F; n = 4). 3.5. Modaﬁnil does not modulate the activity of KCa3.1 channel directly Since KCa3.1 channels are activated by intracellular Ca2+, modaﬁnil might inhibit KCa3.1 currents by decreasing Ca2+ sen- sitivity. Thus, we examined whether modaﬁnil modulates KCa3.1 currents by altering Ca2+ sensitivity. Modaﬁnil (30 nM) did not affect the concentration required to evoke half-maximal activation, EC50, of Ca2+ on KCa3.1 currents (data not shown). To further show that KCa3.1 current inhibition by modaﬁnil is not the result of a direct interaction between KCa3.1 channels and modaﬁnil, we then examined whether modaﬁnil affects the activity of KCa3.1 channels. In inside-out patches, KCa3.1 channels were activated by clamping Ca2+ concentration of the bath solution to 1 µM and applying 1- EBIO. The holding potential was then changed from 70 to 50 mV (Fig. 6). The single-channel currents were inhibited by TRAM-34, suggesting that the activated currents were through KCa3.1 chan- nels (Fig. 6A). We then applied modaﬁnil (30 or 100 nM) to the bath solution. Modaﬁnil did not change single-channel activity of KCa3.1 channels. The open probability was also not changed by modaﬁnil (Fig. 6A and B). A linear ﬁt of the data between 50 and 70 mV yielded a slope conductance of 35.2 pS in the control and 35.6 pS in the presence of modaﬁnil (Fig. 6C). The single- channel conductance recorded in the control condition was very similar to that in the presence of modaﬁnil (30 µM), indicating that the conductance of the single channel was not changed by modaﬁnil. Fig. 4. Effect of modaﬁnil on intracellular cAMP content. Modaﬁnil affects cAMP levels in NIH-3T3 cells (A) and primary cultured mouse aortic SMCs (B and C). Iloprost (C) and forskolin (D) also increase cAMP levels in primary cultured mouse aortic SMCs and NIH-3T3 cells. Cells were treated with modaﬁnil for 3 min within incomplete media. Each point represents the mean ± SEM of 3 separate experiments (n = 3). **p < 0.01. 3.6. Iloprost inhibits KCa3.1 current and VSM contraction We examined the effect of the synthetic analog of prostacy- clin (prostaglandin I2) iloprost, which is used to treat pulmonary hypertension, on KCa3.1 currents and rat pulmonary arterial con- traction to compare modaﬁnil with iloprost in terms of inhibitory effects on KCa3.1 currents and vasocontraction (Fig. 7). When KCa3.1 currents were activated by intracellular Ca2+ (1 µM) and 1-EBIO, iloprost was applied. KCa3.1 currents were decreased by iloprost in a concentration-dependent manner, and the IC50 of iloprost on the current was 101.5 16.8 nM (Fig. 7A and B). In addition, iloprost inhibited rat pulmonary arterial contraction by norepinephrine (0.1 µM); the IC50 was 39.4 ± 5.1 nM (Fig. 7C and D). The IC50s of iloprost to inhibit KCa3.1 current and rat pulmonary arterial contraction were higher than those of modaﬁnil. These results sug- gest that modaﬁnil is at least as potent as iloprost in stimulating cAMP/PKA, thereby inhibiting KCa3.1 channels and VSM contraction. 4. Discussion Modaﬁnil inhibited KCa3.1 currents in NIH-3T3 cells and the con- tractions induced by norepinephrine in the isolated mouse aorta and rat pulmonary artery. The PKA activator forskolin and ilo- prost also inhibited KCa3.1 currents, and the inhibitory effect of modaﬁnil or forskolin was not demonstrated in the presence of the PKA inhibitors. In addition, modaﬁnil increased the cAMP content in a concentration-dependent manner and phosphorylated KCa3.1 channel protein. On the other hand, modaﬁnil did not modulate the single-channel activity of the KCa3.1 channel directly. From these results, we conclude that the inhibitory effect of modaﬁnil on KCa3.1 currents and VSM contraction is mediated by increasing cAMP content. The mechanisms of the cAMP-induced decrease in KCa3.1 cur- rents or in VSM contraction might be PKA dependent. It has been reported that PKA inhibits KCa3.1 channels by phosphorylation at its calmodulin-binding domain [20,24]. In addition, it has been suggested that MLCK is phosphorylated by PKA and that this phos- phorylation reduces the afﬁnity of MLCK for the Ca2+/calmodulin complex [23]. Therefore, PKA-dependent phosphorylation of these proteins might inhibit KCa3.1 currents and muscle contraction, and this might be the mechanism of action of modaﬁnil. KCa3.1 channels are found in microglia [25], dorsal root ganglion sensory neurons [26], erythrocytes, ﬁbroblasts, proliferating VSMs, and vascular endothelium [27] and are therefore regarded as tar- gets for various diseases involving these tissues/cell types. In the cardiovascular system, KCa3.1 channels are exclusively expressed in endothelial cells, and KCa3.1 channel dysfunction evokes endothe- lial dysfunction that is attributed to an increase in blood pressure [28,29]. On the other hand, KCa3.1 channels might be involved in the proliferation of T and B cells [30,31], ﬁbroblasts [32], cancer cells [33], and de-differentiated proliferative VSM cells [34]. Thus, KCa3.1 blockers or the channel disruption have been shown to suppress the proliferation of cancer cells [33] and to attenuate the development of atherosclerosis [35], renal ﬁbrosis [36], and post-angioplasty re- stenosis [37]. In addition, KCa3.1 channels might be involved in the development of inﬂammatory diseases, since the speciﬁc KCa3.1 channel blockers relieve symptoms in experimental autoimmune encephalomyelitis and in several models of cardiovascular diseases [33]. Among Ca2+-activated K+ channels (large-, intermediate-, and small-conductance Ca2+ activated K+ channels), small-conductance Ca2+ activated K+ channels (KCa2.1, KCa2.2, and KCa2.3) are expressed in the central and peripheral nervous systems [38]. Among them, the KCa2.2 channel has been reported to be regu- lated through phosphorylation by PKA [39]. Activation of PKA with forskolin causes a dramatic decrease in surface localization of the KCa2.2 channel subunit due to direct phosphorylation of the KCa2.2 channel subunit. In addition, the Ca2+ sensitivity of the KCa2.2 chan- nel is regulated through phosphorylation by protein kinase [40,41]. These results suggest that modaﬁnil might inhibit the KCa2.2 chan- nel in neuronal cells. Fig. 5. Modaﬁnil-induced KCa 3.1 channel phosphorylation. Modaﬁnil induces KCa 3.1 channel phosphorylation in NIH-3T3 cells. (A) Cells, which were not exposed to modaﬁnil or forskolin, were immunoblotted by anti-KCa 3.1 antibody to conﬁrm the presence of KCa 3.1 channels (left panel). The arrow indicates the presence of KCa 3.1 channels. Cells were pretreated with (or without) the PKA inhibitor H-89 for 30 min and then exposed to modaﬁnil or forskolin for 5 min within incomplete media (right panel). Lysates were subjected to immunoblot analysis as indicated. (B) Summary of modaﬁnil- and forskolin-induced phosphorylation of KCa 3.1 channels (n = 3). *p < 0.05; **p < 0.01. The action mechanism of modaﬁnil, cAMP/PKA stimulation, is very similar to that of the synthetic analog of prostacyclin, ilo- prost, which acts as a PKA activator by increasing cAMP content and thereby relaxes VSM [42,43]. Thus, we suggest that modaﬁnil might be a good alternative to iloprost and can be used to treat pulmonary hypertension, similar to iloprost. On the other hand, the effect of modaﬁnil on systemic blood pressure is unclear and complicated. cAMP/PKA stimulation and phosphorylation of MLCK might relax VSM by decreasing intracellular Ca2+ concentration and Ca2+ sensitivity of MLCK [44]. In addition, cAMP/PKA stimu- lation can increase NO production by increasing endothelial nitric oxide synthase activity [45,46]. However, cAMP/PKA stimulation can increase Ca2+ inﬂux by activating L-type Ca2+ channels [47] and Ca2+-permeable cyclic nucleotide-gated ion channels (CNGCs) [48,49], which might be involved in smooth muscle contraction. Furthermore, KCa3.1 channel inhibition can increase vascular con- tractility by inhibiting endothelial function. PKA-dependent phosphorylation might explain at least in part the mechanism underlying modaﬁnil-induced wakefulness and synaptic plasticity in hypocretin neurons. PKA activation by forskolin-induced long-term potentiation (LTP), and this forskolin- induced LTP was inhibited by modaﬁnil treatment in mice [50], suggesting that synaptic plasticity induced by modaﬁnil treat- ment shares a common pathway, cAMP/PKA stimulation, with LTP induced by forskolin. The maximal plasma concentration of modaﬁnil was reported to be approximately 4.3 mg/ml [51] or 4.79–5.15 mg/ml [52] with daily doses of 200 mg, equivalent to approximately 15.7 µM or 17.5–18.8 µM. For the treatment of narcolepsy, the recommended daily dose of modaﬁnil is 200–400 mg.Thus, the plasma concentration of modaﬁnil for narcolepsy treat- ment might be expected to be approximately 15–35 µM and therefore enough to activate PKA. Fig. 6. Effect of modaﬁnil on KCa 3.1 channel activity in NIH-3T3 cells. (A) In inside-out patches, single channel currents were activated by 1 µM Ca2+ and 1-EBIO, and the activated currents were abolished by the speciﬁc KCa 3.1 channel blocker TRAM-34, indicating that KCa 3.1 channel currents are activated. (A and B) The channel activity was not changed by modaﬁnil. (C) The single channel I–V relation under control conditions (●) and after the application of 30 nM modaﬁnil (Ⓧ) (n = 3). Fig. 7. Effect of iloprost on KCa 3.1 currents and VSM contraction. (A and B) KCa 3.1 currents were activated by loading Ca2+ (1 µM) and applying 1-EBIO (100 µM). Data points were obtained at 50 mV during repetitive ramps (upper traces) and I–V curves obtained at the points marked in upper traces are shown in lower traces (A). (B) The concentration–response relationship (B). The magnitude of KCa 3.1 current inhibition at each treatment was expressed as a percentage of initial KCa 3.1 current density at 50 mV (n = 7). (C and D) Iloprost inhibited rat pulmonary arterial contraction by norepinephrine (C) and the concentration-response relationship. The magnitude of relaxation at each treatment was expressed as a percentage of initial norepinephrine-induced contraction (n = 4). In addition, KCa3.1 channel inhibition and enhanced Ca2+ inﬂux by activation of Ca2+ permeable CNGCs or Ca2+ channels might explain the stimulant effect of modaﬁnil on neuronal cells. KCa3.1 channel inhibition and Ca2+-permeable channel activation by cAMP/PKA stimulation depolarizes neuronal cells, which can increase Ca2+ inﬂux, thereby increasing neurotransmitter release. This is consistent with the suggestion that modaﬁnil’s ability to enhance neurotransmitter release without actually stimulating neurons is due to enhanced neuroelectrosecretory coupling [18]. In conclusion, modaﬁnil increases cAMP in NIH-3T3 cells and primary cultured VSMs and phosphorylates intracellular pro- teins such as KCa3.1 channels. 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