Regulatory effects associated with changes in intracellular potassium level in susceptibility to mitochondrial depolarization and excitotoxicity

Journal Pre-proof Regulatory effects associated with changes in intracellular potassium level in susceptibility to mitochondrial depolarization and excitotoxicity Hiroshi Higashi, Toshihiko Kinjo, Kyosuke Uno, Nobuyuki Kuramoto PII:

S0197-0186(19)30501-7

DOI:

https://doi.org/10.1016/j.neuint.2019.104627

Reference:

NCI 104627

To appear in:

Neurochemistry International

Received Date: 2 September 2019 Revised Date:

16 November 2019

Accepted Date: 2 December 2019

Please cite this article as: Higashi, H., Kinjo, T., Uno, K., Kuramoto, N., Regulatory effects associated with changes in intracellular potassium level in susceptibility to mitochondrial depolarization and excitotoxicity, Neurochemistry International (2020), doi: https://doi.org/10.1016/j.neuint.2019.104627. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

SUR1

Kir6.2

Minoxidil Kir6.2

NMDA

NMDA Receptor

KATP channel

K+

Ca2+ [Ca2+]i ↑

PTP Mitochondrial depolarization

PTP: mitochondrial permeability transition pore

SUR1

[K+]i ↓

1

Regulatory effects associated with changes in intracellular potassium level in

2

susceptibility to mitochondrial depolarization and excitotoxicity

3 4

Hiroshi Higashi, Toshihiko Kinjo, Kyosuke Uno and Nobuyuki Kuramoto*,

5 6

1

7

University, Hirakata, Osaka 573-0101, Japan.

Laboratory of Molecular Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan

8 9 10

Running title: Minoxidil protected neurons by lowering intracellular potassium level.

11 12 13

*All correspondence should be addressed to Kuramoto Nobuyuki Ph.D., Laboratory of

14

Molecular Pharmacology, Setsunan University Faculty of Pharmaceutical Sciences,

15

45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan. Tel/Fax; 81-(0)72-866-3207

16

E-mail: [email protected]

17

18

Abstract

19 20

Excitotoxicity has been believed to be one of the causes of neurodegenerative diseases

21

such as Alzheimer’s disease and Huntington’s disease. So far, much research has been

22

done to suppress the neuronal excessive excitations, however, we still have not achieved

23

full control, which may be due to the lack of some factors. As a matter of course, there

24

is an urgent need to clarify all mechanisms that inhibit the onset and progression of

25

neurodegenerative diseases. We found that potassium ion level regulation may be

26

important in the sense that it suppresses mitochondrial depolarization rather than

27

hyperpolarization of cell membrane potential. Minoxidil, an opener of ATP-activated

28

potassium (KATP) channels decreased injury with middle cerebral artery occlusion in

29

vivo

30

N-methyl-D-aspartate (NMDA)-induced mitochondrial depolarization was suppressed

31

by minoxidil treatment. Minoxidil inhibited the increase in levels of cleaved caspase 3

32

and the release of cytochrome c into the cytosol, further reducing potassium ion levels.

33

It was observed decreased potassium levels in neurons by the treatment of minoxidil.

34

Those effects of minoxidil were blocked by glibenclamide. Therefore, it was suggested

35

that minoxidil, via opening of KATP channels, reduced intracellular potassium ion level

experiment

using

TTC

staining.

In

the

primary

cortical

neurons,

36

that

contribute

to

mitochondrial

depolarization,

and

suppressed

subsequent

37

NMDA-induced mitochondrial depolarization. Our findings suggest that the control of

38

ion levels in neurons could dominate the onset and progression of neurodegenerative

39

diseases.

40 41

Key words: minoxidil, mitochondrial depolarization, excitotoxicity, intracellular

42

potassium level.

43 44

Abbreviations

45 46

APG-2: asante potassium green-2 (AM), AUC: area under curve, [Ca2+]i: intracellular

47

Ca2+

48

3,3'-dipropylthiadicarbocyanine iodide, DIV: days in vitro, ECA: external carotid artery,

49

EDTA: Ethylene diamine tetraacetic acid, FluxOR: FluxOR potassium Ion Assay Kit,

50

GIRK: G protein-activated inwardly rectifying potassium channel, GABA: gamma

51

amino butyric acid, HRP: horseradish peroxidase, ICA: internal carotid artery, [K+]i:

52

intracellular K+ concentration, KATP channel: ATP sensitive potassium channel, MCA:

53

middle

concentration,

cerebral

artery,

CCA:

MCAO:

common

middle

carotid

cerebral

artery,

artery

DiSC3(5):

occlusion,

MTT:

54

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium

bromide,

NMDA:

55

N-methyl-D-aspartic acid, SDS: sodium lauryl sulfate, TBST: 0.05% Tween-20

56

containing Tris-buffer saline,

57

TNF-α: tumor necrosis factor alpha, TTC: 2,3,5-triphenyltetrazolium choloride, PTP:

58

permeability transition pore

59 60

Acknowledgments

61

The author thanks all collaborators in University of Setsunan for their efforts and

62

valuable comments.

63 64

Funding: This research did not receive any specific grant from funding agencies in

65

the public, commercial, or not-for-profit sectors.

66 67 68

Introduction

69 70

Cerebrovascular disease is one of the most patient-ridden diseases in the world. Cerebral

71

infarction, cerebral ischemia and cerebral hemorrhage are originally preventable

72

diseases, but unfortunately they are not perfectly disappeared (Nakayama et al., 1997;

73

Inoue et al., 2009; Kokubo et al., 2010; Baba et al., 2011; Hata et al., 2013; Ohsawa et

74

al., 2013; Konno and Munakata, 2015). In cerebral infarction and cerebral ischemia, the

75

central nervous system is impaired by oxygen and nutritional deficiencies, or by

76

bleeding associated with blood vessel collapse after reperfusion (Takarada et al., 2016).

77

One of the causes of the failure is accumulated oxidative stress (Murakami et al., 1998).

78

Edaravone have dramatic protective effect against ischemic damage, by acting as free

79

radical scavenger (Fujiwara et al., 2016) that is why it has been widely used in the

80

treatment of cerebrovascular disease. Vascular endothelial cells also degenerate due to

81

hypoxia and undernutrition, and that induces hemorrhage to the cerebral parenchyma.

82

During intracerebral hemorrhage, excess of glutamate in serum stimulates neurons and

83

causes neurodegeneration (Choi, 1987). Middle cerebral artery occlusion (MCAO)

84

operation to rodents is one of the most useful and general models for investigating

85

cerebral ischemia (Hatfield et al., 1991). In MCAO using mouse, not only damages of

86

the cerebral cortex, which is the responsible region of the middle cerebral artery, but

87

also damages can be confirmed to the peripheral region (Takarada et al., 2016).

88

Neuronal cell death in the peripheral region is caused not by direct oxygen and nutrient

89

deficiency, but by factors including glutamate and the like released from cells died in

90

the responsible area or released from blood vessels (Hardingham and Bading, 2003).

91 92

Glutamate receptors consist of ionotropic receptor, which is worked rapidly response,

93

and G protein coupled receptor, which is slowly worked indirect response due to

94

associated with G protein (Rojas and Dingledine, 2013). Ionotropic glutamate receptors

95

are more classified by N-methyl-D-aspartic acid (NMDA)-sensitivity those are called

96

NMDA receptor and non-NMDA receptor. The NMDA receptor is a calcium channel

97

type and the influx of calcium depolarizes neurons. Increase in intracellular calcium

98

level acts not only for excitation but also activation of intracellular enzymes involving

99

neural circuit formation in immature stage and forming synaptic plasticity which have

100

been seemed to base on memorization learning on cell level (Malenka and Nicoll, 1993;

101

Chittajallu et al., 2017). Excess of calcium influx, however, have been known to induce

102

neuronal apoptosis by activating caspase pathways (Leist et al., 1997). Apoptosis is

103

caused by various factors such as Fas, Tumor necrosis factor alpha (TNF-α), ultraviolet

104

(UV), γ-ray or by the removal of growth factors, and there are several checkpoints in

105

their signal pathways (Marchetti et al., 1996; Ghavami et al., 2014). Mitochondria

106

depolarization is one of the important checkpoints since it triggers the release of

107

cytochrome c, which activates caspase 9 and subsequently caspase 3 (Li et al., 1997).

108

Similar to the plasma membrane potential, the mitochondrial membrane potential is

109

formed by concentration gradients of ions, mainly proton, and also potassium ion,

110

calcium ion etc, which exist inside and outside the mitochondrial inner membrane. The

111

gradients fail with an uncoupler compounds such as 2,4-dinitrophenol, carbonyl cyanide

112

m-chlorophenyl hydrazine, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone and

113

valinomycin. The uncoupling agent such as 2,4-dinitrophenol, carbonyl cyanide

114

m-chlorophenyl hydrazine, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone and

115

valinomycin breaks this gradient and depolarizes the mitochondria. Uncoupling protein

116

or the opening of permeability transition pore (PTP) is responsible for an endogenous

117

regulatory mechanism that eliminates this gradient (Schinder et al., 1996; Nedergaard et

118

al., 2005). PTP opens with elevation of intracellular calcium ion level, permeating not

119

only calcium ions but also cations such as proton, sodium ion and potassium ion to the

120

mitochondrial matrix, resulting in loss of membrane potential (Schinder et al., 1996).

121

We have demonstrated mitochondrial depolarization associated with PTP opening with a

122

fluorescent indicator 3,3'-dipropylthiadicarbocyanine iodide [DiSC3(5)] (Higashi et al.,

123

2017 ; Kinjo et al. 2018). When NMDA was exposed to neurons, not only calcium

124

channel opening but a transient PTP opening as well as neuronal cell death were

125

observed, but gamma butyric acid

(GABA) B receptor agonist suppressed the opening

126

of PTP via opening of G protein-coupled potassium channel, and that subsequent

127

neuronal cell death (Kinjo et al., 2018). Intracellular potassium level is regulated to be

128

higher than extracellular level by sodium pumps (Pirahanchi and Aeddula, 2019). High

129

intracellular potassium level is important to form resting membrane potential on plasma

130

membrane. Opening of potassium channels causes hyperpolarization by efflux of

131

potassium ions and it would cancel the cellular excitation. However, it was

132

demonstrated that the chloride ion channel opening of the GABAA receptor causing the

133

same hyperpolarization failed to suppress NMDA-induced neuronal cell death (Kinjo et

134

al., 2018). Therefore, it was suggested that potassium channel opening on the cell

135

membrane and lowered potassium ion level may protect nerve cells from excitotoxicity.

136 137

Adenosine triphosphate (ATP) sensitive potassium channels (KATP channels) are one of

138

the inwardly-rectifying potassium channel, which are blocked by intracellular

139

interactions of ATP. Sulfonylureas such as glybenclaminde block the opening of the

140

channels and is used to treat diabetes, and minoxidil promotes the opening of them and

141

is used to treat baldness (Greenwood and Weston 1993). Each KATP channel is a

142

hetero-octamer consisting of four genes, Kir 6.1, Kir 6.2, SUR 1 and SUR 2

143

(Baukrowitz and Fakler, 2000). KATP channels are mainly expressed in heart, kidney,

144

blood vessel and brain (Yokoshiki et al., 1998). For example, an octamer consisting of

145

two subunits SUR1 and Kir6.2 (SUR1/Kir6.2) was found in pancreas and brain.

146

SUR2A/Kir6.2 in heart, and SUR2B/Kir6.1 or SUR2B/Kir6.2 in the smooth muscles

147

were also reported (Yokoshiki et al., 1998). The composition of these subunits is

148

believed to have functional differences, but this has not been fully elucidated. Minoxidil

149

opens KATP channels and had been developed hypotensive agent in the beginning.

150

However, the clinical application for that purpose has been postponed, while instead has

151

become widespread as a hair restorer (Tsoporis et al., 1993; Lachgar et al., 1998). On

152

the other hand, minoxidil has been studied on the possibility of reducing Stroke's

153

damage in the heart (Pompermayer et al., 2007; Sato et al., 2014). The mechanism of

154

the action is thought to be since potassium efflux associated with KATP channel’s

155

opening induced hyperpolarization (Pompermayer et al., 2007; Sato et al., 2014). In this

156

study, we investigated whether minoxidil, via reducing the intracellular potassium level,

157

suppresses the degree of mitochondrial depolarization, and therefore resulted in a

158

protection against excitotoxicity in the neurons.

159 160

161

Materials and Methods

162 163

Materials

164

Western Lightning Chemiluminescence Reagent Plus was obtained from PerkinElmer

165

(Waltham, MA). The X-ray film was from Fujifilm (Tokyo, Japan). PVDF membrane

166

was

167

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium

168

2,3,5-triphenyltetrazolium choloride (TTC) and Sepasol®-RNAⅠSuper G were from

169

Nacalai Tesque (Kyoto, Japan). NMDA was from Sigma-Aldrich Corp. (St. Louis, MO).

170

3,3′-Dipropylthiacarbocyanine iodide [DiSC3(5)] was from Anaspec Inc.(San Jose, CA).

171

GlutaMaxTM, MitoTracker® Green FM and FluxORTM Potassium Ion Channel Assay,

172

Fluo-4 AM was from Thermo Fisher Scientific (Waltham,, MA). Asante potassium

173

green-2 (AM) was from Texas Fluorescence Labs. (Austin, TX). NeuroBrew-21 is

174

produced by Miltenyi Biotec (Bergisch Gladbach, Germany). Edaraveone was from

175

Tocris (Ellisville, MO). Glibenclamide was from FUJIFILM Wako Pure Chemical

176

Corporation (Osaka, Japan). All other chemicals were of standard grade. ImageJ is an

177

application produced by Wayne Rasband at National Institutes of Health (Bethesda,

purchased

from

Millipore

(Billerica, bromide

MA). (MTT),

178

MD). FluoView™ FV1000 confocal microscope is product of Olympus (Tokyo, Japan).

179

ImageQuantTM 400 was product of GE (Chicago, IL).

180 181

Animals

182

The protocol used here met the guidelines of the Japanese Society for Pharmacology

183

and was approved by the Committee for Ethical Use of Experimental Animals at

184

Setsunan University. For MCAO experiments, adult male C57BL/6 mice weighing

185

18-20 g were used. For primary culture experiments pregnant Std-ddy mice were raised.

186

The animals were housed in standard breeding cages for mice with a light–dark cycle of

187

12–12 h and a humidity of 55% at 23ºC and given free access to food and water.

188

All efforts were made to minimize animal suffering, to reduce the number of animals

189

used and to utilized alternatives to in vivo techniques, if available.

190 191

Middle cerebral artery occlusion (MCAO)

192

Transient ischemia was conducted as previously report (Takarada et al., 2016) with

193

slight modification. Mouse was deeply anesthetized with medetomidine (0.75 mg/kg

194

b.w., i.p.), midazolam (4 mg/kg b.w., i.p.) and butorphanol (5 mg/kg b.w., i.p.). After

195

disinfecting the neck with 70% ethanol, the median cervix was incised and the left

196

common carotid artery (CCA) was exposed. Using a silk (0.15-199 mm dimeter), distal

197

external carotid artery (ECA) was tied and its branch between the tied site and the

198

branching site from the CCA was also tied. CCA was ligated temporarily and internal

199

carotid artery (ICA) just a distal site from the branching site from CCA was temporally

200

clipped to prevent reverse flow. A small hole at proximal ECA was made and silicon

201

coated fiber, which is a 0.05-0.069 mm dimeter fiber having a silicon embolus of ~400

202

µm in length and ~200 µm in thickness, was inserted into the hole. The fiber was then

203

passed from the ECA across the CCA branch to the ICA clipping site and the ECA side

204

of the CCA branch loosely tied by the silk and the clip on the ICA side was removed.

205

Silicon coated fiber was further inserted to reach the origin of MCA in the circle of

206

Willis located approximately 9-10 mm from the first hole and it occluded the MCA. The

207

ECA loosely tied up and the occlusion of the MCA was kept for 1 or 2 h. After this

208

period the silicon coated fiber was removed to provide reperfusion. To end the operation,

209

the neck scar was stitched with silk suture. Glibenclamide was administered

210

intraperitoneally 2 h prior to MCAO surgery. Minoxidil was administered immediately

211

after the MCAO, and in the beginning of occlusion, for 1 h.

212 213

TTC staining

214

Mice were decapitated 24 hours after reperfusion for assessment of the volume damaged

215

by cerebral infarction. Brains were removed quickly and placed in iced-cold saline, total

216

5 coronal slices were prepared with a thickness of each 1000 µm, 2 slices toward the

217

anterior and 3 toward posterior from the bregma using a brain slicer. The slices were

218

stained in a saline solution containing 0.8% 2,3,5-Triphenyl tetrazolium chloride (TTC)

219

for 10 min at 37℃. Photos were taken and the damaged areas in white were digitally

220

quantified by using a software Image J.

221 222

Cortical culture

223

Embryos (E14.5) were removed from a female mouse on day 15 of gestation and

224

dissected to obtain cerebral cortex. Cerebral cortexes were collected and incubated in

225

0.02 % EDTA solution for 10 min at room temperature and washed by phosphate

226

buffered saline containing 33 mM glucose. The tissues were then dispersed by a

227

narrowed hole of pasteur pipette in Neurobasal medium that is Neurobasal™ medium

228

containing 2 mM GlutaMaxTM, 100 U/ml penicillin-streptomycin and 1×NeuroBrew-21.

229

The cell suspension was centrifuged, fresh Neurobasal medium was added to the pellet

230

and the cells were resuspended. The cells were seeded 100,000 cells/200 mm2 for

231

fluorescence imaging or 300,000 cells/200 mm2 for else.

232 233

RT-PCR

234

Tissue of mouse cerebral cortex and the cortical cells at 9 days in vitro (DIV) were

235

lysed in the Sepasol®-RNAⅠSuper G and total RNA was prepared. Aliquots (1 µg) of

236

total RNA were subjected to reverse transcription and resultant cDNA products were

237

immediately stored at -30℃ until the use of poly chain reaction to detect the gene

238

expressions of KCNJ 8 as Kir 6.1, KCNJ11 as Kir6.2, ABCC8 as SUR1 and ABCC9 as

239

SUR2. The primers used are Kir6.1F: 5’-CACAAGAACATCCGAGAGCA-3’, Kir6.1R:

240

5’-TTCTCCATGGTGCCTTTCTC-3’, Kir6.2F: 5’-CTGGCCATCCTCATTCTCAT-3’,

241

Kir6.2R:

242

5’-CCCTCTACCAGCACACCAAT-3’,

SUR1R:

243

5’-CAGTCTGCATGAGGCAGGTA-3’,

SUR2F:

244

5’-CCGAGAGGTTGAAGAAGACG-3’,

245

5’-TGCGTTCATGAAGATGGAAA-3’. The PCR products were electrophoresed using

246

0.8% of TAE agarose gel and took photos by ImageQuantTM 400.

5’-CTCTTTCGGAGGTCCCCTAC-3’,

and

SUR1F:

SUR2R:

247 248

Fluorescence detection of mitochondria and mitochondrial membrane potential

249

As a recording medium, Hank’s balanced salt solution without Ca2+ nor Mg2+ which

250

consisted of 137 mM NaCl, 4.17 mM NaHCO3, 0.34 mM Na2HPO4, 5.37 mM KCl,

251

0.44 mM KH2PO4 and 5.55 mM D-glucose, and added with 20 mM HEPES-NaOH (pH

252

7.5) and 2.0 mM CaCl2 was used and called HHBSS. For detection of mitochondria,

253

cells were incubated with HHBSS supplemented with MitoTracker® Green FM and

254

3,3'-dipropylthiadicarbocyanine iodide [DiSC3(5)] for 30 min. To observe for

255

mitochondrial depolarization, cells were incubated with HHBSS supplemented with 250

256

nM DiSC3(5) for 30 min for loading the dye at 37 °C in 95% air/5% CO2. Pretreatment

257

with minoxidil, was usually carried out last 5 min of the loading. Photos or continuous

258

shootings were taken by FluoView™ FV1000 confocal microscope. In continuous

259

shooting experiments, 38 photos were taken every 5 s, which means 180 s in total. The

260

beginning of the shooting for 30 s was taken as background and then a drug such as

261

valinomycin or NMDA was added. The fluorescence intensity of region of interests

262

were quantified by free application ImageJ. Excitation wavelength was 473 nm for

263

MitoTracker® Green FM or 635 nm for DiSC3(5) and emission wavelength band was

264

485-585 nm for MitoTracker® Green FM or 650-750 nm for DiSC3(5).

265

266

Fluorescence detection of potassium channel opening and intracellular potassium

267

level

268

To support potassium channel opening, FluxORTM Potassium Ion Channel Assay was

269

used. Culture medium was replaced to loading buffer containing thallium indicator for

270

60 min-incubation and then to assay buffer for additional 30 min-incubation to ready to

271

stimulate. Photos or continuous shootings were taken by FluoView™ FV1000 confocal

272

microscope. In continuous shooting experiments, 50 photos were taken every 5 s, which

273

means 250 s in total. The beginning of the shooting for 30 s was taken as background

274

and then stimulation buffer containing thallium and a drug such as minoxidil was added.

275

With this kit, when thallium ions flows into the cells via the opened potassium channel,

276

thallium indicator become to show green fluoresce.

277

potassium level, culture medium was replaced to HHBSS without or with minoxidil and

278

incubated for 5 min and then added Asante potassium green-2 (AM) [APG-2] to make

279

final concentration at 2 µM for additional incubation for 30 min. Excitation wavelength

280

was 473 nm for APG-2 and the thallium indicator and emission wavelength band was

281

485-585 nm.

282 283

Fluorescence detection of intracellular calcium level

For detection of intracellular

284

To

measure

intracellular

calcium

level,

285

N-[4-[6-[(acetyloxy)methoxy]-2,7-difluoro-3-oxo-3H-xanthen-9-yl]-2-[2-[2-[bis[2-[(ace

286

tyloxy)methoxy]-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-N-[2-[(acetylox

287

y)methoxy]-2-oxoethyl]-, (acetyloxy)methyl ester (Fluo4 AM: Invitrogen) was used.

288

Culture medium was replaced to a HHBSS containing 2.5 mM probenecid, an organic

289

cation transporter inhibitor and Fluo-4 and incubated for 30 min. Pretreatment with

290

minoxidil, was usually carried out last 5 min of the loading. Photos or continuous

291

shootings were taken by FluoView™ FV1000 confocal microscope. In continuous

292

shooting experiments, 38 photos were taken every 5 s, which means 180 s in total. The

293

beginning of the shooting for 30 s was taken as background and then a drug such as

294

NMDA or glibenclamide was added. Excitation wavelength was 473 nm and emission

295

wavelength band 485-585 nm.

296 297

MTT assay

298

Drugs were prepared in HHBSS. The cells were incubated in HHBSS for 30 min,

299

followed by exposure to NMDA and additional incubation for 2 min. Pre-incubation

300

with minoxidil was 5 min before the exposure to NMDA. After the exposure to NMDA

301

for 2 min, the medium was changed to the fresh Neurobasal medium as described above

302

for further culture for 24 h. For 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium

303

bromide (MTT) assay, the medium was again replaced with a solution containing 0.5

304

mg/mL MTT and 33 mM glucose in phosphate buffered saline, followed by further

305

incubation for 2 h at 37°C under an atmosphere of 95% air/5% CO2. The produced blue

306

formazan dye as an indicator of living cells was solubilized with 0.04 mol/L HCl in

307

2-propanol for determining of the optical density at 570 nm.

308 309

Immunoblot analysis

310

Protein samples were incubated for 10 min at 100ºC in the buffer containing 2% SDS,

311

5% 2-mercaptoethanol, 10% glycerol, and 0.01% bromophenol blue and immediately

312

stored at -80ºC until used for immunoblot analysis as described previously (Kuramoto et

313

al., 2003). Briefly, aliquots (5 µg) of total protein were subjected to sodium dodecyl

314

sulfate polyacrylamide gel electrophoresis with 12.5% polyacrylamide gels and

315

transferred onto PVDF membranes. The membranes were blocked with 5% skim milk

316

in 0.05% Tween-20 containing Tris-buffered saline (TBST) for 1 h, incubated with the

317

desired primary antibodies for 2 h, and then with horseradish peroxidase

318

(HRP)-conjugated secondary antibodies for 1 h at room temperature. HRP on the

319

membrane were reacted with Western Lightning Chemiluminescence Reagent Plus and

320

resultant luminescence was detected by X-ray films or taken by ImageQuantTM 400.

321

GAPDH and β-tubulin were detected as internal standards for total lysate and cytosolic

322

fraction, respectively.

323 324

Preparation of the cytosol fraction

325

Cortical neurons were collected by silicon scraper and disrupted using Teflon

326

homogenizer (300 rpm, 1 min) on ice, then the cell suspension was centrifuged by

327

100,000 g, 60 min at 4 ºC. Resultant supernatant was used as the cytosol fraction.

328 329

Data analysis

330

Degree of TCC staining and fluorescent intensity of indicators were quantified by

331

ImageJ. The region of interest was defined to cover each hemisphere by quantification

332

of TCC staining and to cover all cells except nonspecific signal, such area as always

333

showing high signals, by quantification of fluorescence intensity. Densitometric analysis

334

for RT-PCR and immunoblotting was carried out by using ImageQuantTM 400. All data

335

were expressed as means ± SE, and statistical significance was determined using the

336

Student’s t-test or Bonferroni’s test.

337

338

339

Results

340 341

Minoxidil suppressed the loss of respirational activity after ischemic insults

342

The mice were operated to unilaterally middle cerebral artery occlusion (MCAO) for

343

maximum 2 h and their blood was then reperfused. Coronal sections nearby bregma

344

were prepared after 24 h from the reperfusion, after which were stained by

345

2,3,5-triphenyl tetrazoium chloride (TTC) (Fig. 1). TTC staining detects living cells in

346

red by converting TTC to producing a red colored metabolite, 1,3,5-triphenylformazan

347

(TPF) by cell respiration. Failure to stain and showing original white color of the tissue

348

means that in such areas cells are less (or loss of) respiration. The coronal sections in

349

sham operation were stained red in whole area, meaning neurons and the other neural

350

cells were survived after the surgery. In the unilateral MCAO operation, on the side

351

subjected to MCAO surgery (ipsilateral side: I), an area where was not stained red but

352

kept white was observed around the fields corresponding to the striatum and the

353

cerebral cortex. Compared to the contralateral side (C), the extent to which the region

354

was stained red was significantly reduced. It suggested that cells in that area were

355

damaged by ischemia and resulted into respiratory failure. The red area did not decrease

356

further even if the period for ischemic insult was changed from 1 h to 2 h. When

357

edaravone was intravenously administered immediately after the MCAO operation, the

358

extent of staining red was significantly increased compared to the condition only

359

subjected to the operation, demonstrating that edaravone protected the damage of the

360

surgery. Similarly, the extent of staining red was significantly increased compared to the

361

condition only subjected to the operation, by minoxidil in a concentration dependent

362

manner. Therefore, not only edaravone, but also minoxidil was suggested as a possible

363

agent for reducing ischemic brain damage. We had then investigated whether minoxidil

364

directly protected neuronal cells.

365 366

Expression of the genes constituting KATP channels in the cultured cortical neuron,

367

and which showed that KATP on cell membrane were activated by minoxidil to

368

decrease cellular potassium level.

369

The ATP-activated potassium channel (KATP channel) is an inward rectifier potassium

370

channel, which minoxidil opens (Greenwood and Weston, 1993). Primary cortical

371

neurons were prepared and expressions of genes constituting KATP channels were

372

estimated (Fig.2). KATP channels exist as a hetero-octamer which structures are

373

complexed from sulfonylurea receptor and Kir6.X. The major constituent genes are

374

KCNJ8 (Kir6.1), KCNJ11 (Kir6.2), ABCC8 (SUR1), ABCC9 (SUR2) (Takano M et al.,

375

1998). As a result, all genes of Kir6.1, Kir6.2, SUR1 and SUR2 were expressed in the

376

cerebral cortex, while only Kir6.2 and SUR1 were expressed in the primary neurons.

377

Although we have not confirmed the existence of each protein, it was suggested that

378

there might be KATP channels, which consisted of Kir6.2 and SUR1, and which

379

regulated extent of excitation and membrane potential in the culture cells. To confirm

380

the opening of the KATP channels associated with the exposure of minoxidil, we utilized

381

the other fluorescence techniques (Fig.2b-e). The alteration in intracellular potassium

382

levels in seconds by the minoxidil exposure was chased by using FluxOR Potassium Ion

383

Channel Assay kit (FluxOR) and the intracellular potassium levels after 30 minutes

384

incubation with minoxidil were measured by using Asante potassium green-2 (APG2).

385

The fluorescence of FluxOR was immediately increased by minoxidil. In the vehicle

386

treatment, slight increase in the fluorescence was observed due to thallium ions

387

permeation through potassium leak channels that open always (Fig.2b). The AUC

388

obtained from the minoxidil-induced spectrum of fluorescence intensity over time was

389

significantly higher than one of the vehicle (Fig.2c). Moreover, the fluorescence of

390

APG-2 meant a significant decrease in intracellular potassium level with the minoxidil

391

exposure (Fig.2d-e). Therefore, it was suggested that NMDA-induced apoptosis

392

depending on the degree of mitochondrial depolarization and that the extent of

393

depolarization might be suppressed by decreasing in the intracellular potassium levels.

394 395

Measuring alteration of the mitochondrial membrane potential by using DiSC3(5)

396

Mitochondrial membrane potentials during the drug exposure to primarily cultured

397

cortical

398

[DiSC3(5)], which is a fluorescence dye for imaging mitochondrial membrane potential

399

(Fig.3, 4). The red fluorescence of DiSC3(5) mostly merged with the green fluorescence,

400

which is of a mitochondrial marker (Fig.3a). Although GABA and minoxidil have been

401

known to induce hyperpolarization on cellular membrane potential, which were not

402

affect the fluorescence intensity in the cortical neurons (Fig.3b). However, valinomycin

403

as potassium ionophore that induces potassium efflux to extracellular space and also

404

providing potassium influx to mitochondria showed increase in fluorescence intensity. It

405

was therefore suggested that increasing fluorescence intensity was due to mitochondrial

406

depolarization.

neurons

were

measured

using

3,3'-dipropylthiadicarbocyanine

iodide

407 408

Minoxidil suppressed NMDA induced mitochondrial depolarization, cleavage of

409

caspase-3 and the release of cytochrome c into the cytosol

410

By pretreatment with minoxidil, the AUC obtained from the NMDA-induced spectrum

411

of fluorescence intensity of DiSC3(5) was significantly suppressed to about half, and the

412

NMDA-induced suppression of the ability of MTT reduction was partially but

413

significantly cancelled (Fig.4a-c). In addition, the release of cytochrome c into the

414

cytosol and the cleavation of caspase 3 were promoted by NMDA, but minoxidil

415

pretreatment suppressed it (Fig.4d-e). NMDA-induced intracellular calcium influx can

416

be detected with other fluorescent indicator Fluo-4 (Fig.5). NMDA-induced increase in

417

the fluorescence due to the calcium influx was not suppressed by pre-treatment with

418

minoxidil (Fig.5c-d).

419 420

Glibenclamide reversed the effects of minoxidil both in vitro and in vivo.

421

Glibenclamide, a sulfonylurea, is known to induce cellular depolarization by inhibiting

422

KATP channels and in fact glibenclamide, but minoxidil, caused intracellular calcium

423

influx (Fig.5a-b, e-f). Minoxidil suppressed the glibenclamide-induced calcium influx

424

(Fig.5e-f), glibenclamide suppressed the minoxidil-induced efflux of potassium

425

(Fig.6a-b), and minoxidil-lowered intracellular potassium (Fig.6c-d), since both

426

minoxidil and glibenclamide are antagonistic each other to the opening of the KATP

427

channels. At last, glibenclamide was intraperitoneally injected 2 h prior to the MCAO

428

surgery that was similar to that in Fig.1 and the effect of minoxidil on neuronal damage

429

was evaluated. The protective effect of minoxidil against neuronal damage was

430

cancelled by glibenclamide (Fig.6e). Therefore, it was confirmed that the effect of

431

minoxidil demonstrated by this study is an effect associated with KATP channel opening.

432 433

434

Discussion

435 436

We had previously reported that baclofen which was an activator of GABAB receptor

437

was suppressed NMDA-induced excitotoxicity by opening of G protein-coupled

438

inwardly-rectifying potassium channel (GIRK). However, muscimol causes chloride

439

influx by activating GABAA receptor was not shown to decrease NMDA-induced

440

excitotoxicity. Thus it suggested that neuroprotection by baclofen was not simply

441

occurring hyperpolarization, it seemed to arise from alteration intracellular potassium

442

levels (Kinjo et al., 2018). Therefore, we have investigated whether alteration of

443

intracellular potassium level would contribute to determine degree of mitochondrial

444

depolarization. As a result, we found out that decreasing of intracellular potassium

445

levels with opening of potassium channel on cell membrane inhibited that neuronal

446

apoptosis, which was probably due to less depolarization at mitochondrial membrane

447

and no activation of caspase pathway. Mitochondria work weakly as buffering calcium

448

compared to endoplasmic reticulum, it takes calcium into mitochondria when increasing

449

intracellular calcium levels (Perocchi et al., 2010). On the other hand, when calcium is

450

incorporated into mitochondria too much, it induces the opening of PTP, so the cation

451

gradient and the mitochondrial inner membrane potential are lost (Giorgio et al., 2017).

452

Uncoupling induces cytochrome c release and causes apoptosis via caspase 9 and

453

subsequent activation of caspase 3 (Kinnally and Antonsson, 2007). Over influx of

454

calcium occurred neurodegeneration with mitochondrial depolarization following to

455

open of mitochondrial PTP (Hajnóczky et al., 2006). In the inner mitochondrial

456

membrane, not only an ion gradient of protons is formed, ie, the potassium ion

457

concentration is lower in the mitochondrial matrix than in the cytosol (Laskowski et al.,

458

2016). PTP opening induces mitochondrial depolarization by also passing potassium

459

ions (Elustondo et al., 2016). Therefore, it suggests that decreased intracellular

460

potassium levels in neurons provide neuroprotection.

461 462

Potassium levels in neuronal axons of the cuttlefishes and intracellular in muscle cell

463

are each of 400 mM and 155 mM, extracellular of muscle cell is 4 mM (Schmidt RF

464

1985; Kandel ER et al., 2013). This concentration difference contributes to the

465

formation of the resting membrane potential. Hyperkalemia and hypokalemia are known

466

to induce dysfunction of muscle tissue such as cardiac muscle and skeletal muscle

467

(Jurkat-Rott et al., 2002; Parham et al., 2006; Spodick, 2008; Kang et al., 2008). There

468

have been few reports of nervous system dysfunction accompanied by changes in

469

potassium levels. This is considered to be due to the appearance of musculoskeletal

470

abnormalities prior to the appearance of nervous system ones. Therefore, a compound

471

that alters the potassium concentration in the nervous system, that is to say, lower it in

472

this study, while taking care not to affect the muscle system, will be a drug that protects

473

neurons. Over- or under-dose of potassium through the diet has little effect on the body

474

of healthy people except for long-term intake (Overview of Dietary Reference Intakes

475

for Japanese, 2015). It is serious that low potassium levels are caused by taking diuretic

476

medications or are associated with severe vomiting or diarrhea (Stokes, 1964; Unwin et

477

al., 2011; Dongilli et al., 2016). Conversely, as a cause of severe hyperkalemia, there is

478

excretion failure associated with renal dysfunction (Fried et al., 2011). Chronic over

479

intake of potassium is a cause of renal dysfunction, thus it is limited a daily intake. Even

480

if eating habits are normal, renal function declines with age, so potassium concentration

481

may tend to increase in elderly people compared to young people.

482 483

Transient increase in potassium concentrations around excitatory cells induce

484

depolarization of them (Raiteri et al., 2007). An increase in the pericellular potassium

485

level increases the intracellular potassium level, which is presumed not only to deepen

486

the resting membrane potential but also to increase the degree of mitochondrial

487

depolarization accompanied by the opening of PTP. That is, an increase in pericellular

488

potassium concentration is suggested to make neurons more vulnerable. Thus, a chronic

489

increase in systemic potassium levels, even a slight increase, can be a cause of

490

progressive neurodegenerative disease. While ischemic injury was completely

491

suppressed by edaravone, minoxidil suppressed ischemic injury significantly but not

492

completely but only the surrounding area. Whether higher concentrations of minoxidil

493

can reduce ischemic damage in the area of responsibility is a critical consideration.

494

Vascular endothelial cells nearby ischemic damages in the region of decreasing blood

495

flow are damaged with increasing reactive oxygen species following hypoxia (Spescha

496

et al., 2015). On these places with resuming blood flow induce to leak out blood plasma

497

components and stimulating cerebral parenchyma, become a wide obstacle (Szydlowska

498

and Tymianski, 2010). Our demonstration indicated same results as this fact (Fig.1).

499

While we could not exclude that the neuroprotections of minoxidil in vivo were

500

protections to vascular endothelial cells, our research was demonstrated that at least, the

501

data of in vitro showed us minoxidil has neuroprotections. As the brain parenchyma

502

contains glial cells containing astrocytes, the effects of minoxidil on these cells need to

503

be examined in the future (Griffith et al., 2016) .

504

KATP channels are known to configure different subunits each organs. For example,

505

vascular endothelial cells are SUR2B and Kir6.1, neuronal cells are SUR1 and Kir6.2,

506

myocardial cells are SUR2A and Kir6.2, and these are normally configured

507

hetero-octamer (Kawahito et al., 2011; Li et al., 2019). KATP channels are presence both

508

on cell membrane and mitochondrial inner membrane, however mitochondrial KATP

509

channel are not clearly understood as function and structure (Suzuki et al., 1997; Riess

510

et al., 2008). The primary cortical neurons in this study expressed a possibility

511

activating KATP channel, in addition we confirmed potassium flow into intracytoplasmic

512

by minoxidil (Fig.2). Minoxidil has not been shown to cause mitochondrial

513

depolarization (Fig.3). It is assumed that minoxidil almost abolishes membrane

514

permeability, or that minoxidil-responsive KATP channels are not present in

515

mitochondria, at least in the cells used in this study. In this report, the effect of

516

minoxidil was canceled with sulfonylurea, glibenclamide, which antagonizes each other

517

(Fig5 and 6). Our results are similar, as it has already been suggested that sulfonylurea

518

may increase the risk of cerebral ischemic injury (Liu R et al., 2016, Parkinson FE and

519

Hatch GM, 2016). In any case we could conclude that the neuroprotections by minoxidil

520

in vitro follow opening of KATP channel on cell membrane. Whole body administration

521

of minoxidil are shown to decrease blood pressure with atonicity vascular endothelial

522

cells. This study showed that minoxidil may have a suppressive effect on

523

neurodegeneration with acute ischemia-reperfusion injury. Anti-hypertensive agent such

524

as losartan protects against cerebral ischemia/reperfusion-induced apoptosis, we will

525

have needed to investigate whether suppression of cerebral ischemic/reperfusion

526

damage was caused by decreasing blood pressure. Minoxidil might be arrival to passage

527

on BBB by relaxed vascular endothelial cells. The development of potassium channel

528

activator crossing BBB is important to connect a novel treat mechanism against

529

neurodegeneration. However, KATP channel activator have an action to heart function,

530

we have an idea as needing to target to specific inhabiting receptor on central nervous

531

system such as large-conductance calcium-activated potassium channel (BK channel).

532 533

Figure legends

534 535

Fig.1 Effect of edaravone and minoxidil on loss of respirational activity after

536

middle cerebral artery occlusion.

537

Middle cerebral artery occlusion (MCAO) was performed on one side (Ipsilateral) on 6

538

weeks old C57/BL6 mice for 1 or 2 h. a) Edaravone or b) minoxidil was administered

539

immediately after the MCAO, and in the beginning of occlusion, for 1 h. Brains were

540

dissociated 24 h after the MCAO and a total of 5 coronary slices were prepared with a

541

thickness of each 1000 µm, 2 slices toward the anterior and 3 toward posterior from the

542

bregma using a brain slicer. The slices were stained with 2,3,5-triphenyl tetrazolium

543

chloride to indicate cellular respiration. The photos on the right side are representative

544

staining results. The degrees of staining in each side were quantified and the averages of

545

the degrees were calculated. The graphs in the left side are the results of comparison of

546

contra- and ipsilateral side under each condition.

547

#

548

test).

P<0.05 and

∗∗

P<0.01 vs ipsilateral of sham,

##

P<0.01 vs ipsilateral without minoxidil (0 mg/kg b.w.) (Bonferroni’s

549 550

Fig.2 Minoxidil surely opened potassium channels and lowered intracellular

551

potassium level.

552

Cortical tissue was dissected from 6 weeks old adult mouse. Primary neurons at 8 days

553

in vitro were prepared from cerebral cortex of mouse embryo. RT-PCR was performed

554

to detect genes constituting ATP-sensitive potassium channels (KATP) including Kir 6.1,

555

Kir6.2, SUR1 and SUR2. b,c) Cortical neurons were loaded with FluxOR, followed by

556

the treatment with 100 µM minoxidil containing thallium (Tl+) ions under confocal

557

microscope. In the vehicle treatment, slight increase in the fluorescence was observed

558

due to thallium ions permeation through potassium leak channels. c) Area under curve

559

(AUC) of the fluorescence intensity of b) was quantified. ∗P<0.05 vs vehicle (student’s

560

t-test). d,e) Cortical neurons were treated with 100 µM minoxidil for 5 min and

561

subsequent exposure to APG2 for additional 30 min. The photos of the fluorescence

562

were then taken by confocal microscope. e) Area under curve (AUC) of the fluorescence

563

intensity of d) was quantified. ∗∗P<0.01 vs vehicle (student’s t-test).

564 565

Fig.3 DiSC3(5) indicated the depolarization of the mitochondria inner membrane,

566

but not hyperpolarization of the plasma membrane in the cortical neurons.

567

a) Cortical neurons were co-stained with DiSC3(5) as red signal and MitoTracker as

568

green signal. Scale bar = 20 µm. b) Cortical neurons were loaded with 250 nM

569

DiSC3(5), followed by confocal microscopic measurement in either the presence or

570

absence of 2 µM valinomycin, 100 µM minoxidil or 500 µM GABA. Each bar means

571

the exposure period with each compound.

572 573

Fig.4 Suppression of increase in DiSC3(5) fluorescence intensity correlates with

574

suppression of reduction of cell viability and the expression levels of caspase3.

575

a,b) Cortical neurons were loaded with DiSC3(5), followed by pretreatment with 100

576

µM minoxidil for 5 min and subsequent exposure to 100 µM NMDA under confocal

577

microscope. b) Area under curve (AUC) of the fluorescence intensity of a) was

578

quantified. ∗P<0.05 vs vehicle (student’s t-test). c,d) After pretreatment with 100 µM

579

minoxidil for 5 min, the neurons were exposed to 100 µM NMDA for 2 min. The cells

580

were further cultured either c) for 24 h or d) 6 h, in a fresh medium to determine c) the

581

ability of MTT reduction to estimate the cell viabilities or d,e) to measure cytochrome c

582

in the cytosol fractions and cleavage of caspase 3 to detect apoptosis. b) +P<0.05 vs

583

NMDA alone c)∗∗P<0.01 vs control experiment; #P<0.05 vs 100 µM NMDA alone

584

(Bonferroni’s test). d,e) Alteration of levels of cytochrome c and caspase 3. After a

585

stimulation for 2 min, cells were further cultured for 6 h in fresh medium. ∗∗P<0.01 vs

586

control experiment; #P<0. 05, ##P<0.01 vs 100 µM NMDA (Bonferroni’s test).

587 588

Fig.5 The fluorescence of the intracellular calcium indicator Fluo-4 increased with

589

NMDA, while minoxidil did not suppress it.

590

a-f) Cortical neurons were loaded with Fluo-4, followed by confocal microscopic

591

measurement in either a) vehicle, or drugs of b) 100 µM minoxidil, c,d) 100 µM NMDA

592

or e,f) 100 µM glibenclamide. Each bar means the exposure period with each compound.

593

c-f) Pretreatment with either vehicle or 100 µM minoxidil was started 5 min prior to the

594

measurement.

595 596

Fig.6 Glibenclamide reversed the effects of minoxidil both in vitro and in vivo.

597

a,b) Cortical neurons were loaded with FluxOR, followed by the pretreatment with

598

either vehicle or 100 µM glibenclamide for 10 min and subsequent exposure to 100 µM

599

minoxidil containing thallium (Tl+) ions under confocal microscope. b) Area under

600

curve (AUC) of the fluorescence intensity of a) was quantified. ∗P<0.05 vs left side

601

column, without glibenclamide (student’s t-test). c,d) Cortical neurons were treated with

602

100 µM glibenclamide for 10 min and minoxidil was added at final concentration of

603

100 µM for additional incubation for 5 min, and were loaded with APG2 for further 30

604

min. The photos of the fluorescence were then taken by confocal microscope. d) Area

605

under curve (AUC) of the fluorescence intensity of c) was quantified. ∗P<0.05 vs left

606

side column, without glibenclamide (student’s t-test). b,d) Control means that both

607

pretreatment and treatment were done with vehicle. e) Pretreatment with glibenclamide

608

was performed 2 h prior to MCAO surgery for 1 h. Minoxidil was administered

609

immediately after the MCAO. After 24 h, brain slices were prepared and stained with

610

2,3,5-triphenyl tetrazolium chloride to indicate cellular respiration. The photos on the

611

right side are representative staining results. The degrees of staining in each side were

612

quantified and the averages of the degrees were calculated. The graphs in the left side

613

are the results of comparison of contra- and ipsilateral side under each condition.

614 615

∗∗

P<0.01 vs ipsilateral, #P<0.05 vs ipsilateral without glibenclamide (0 mg/kg b.w.)

(Bonferroni’s test).

616

617

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Figure 6

Highlights ◆Minoxidil reduced infarct area caused by MCAO. ◆Minoxidil reduced the NMDA-induced mitochondrial depolarization. ◆Minoxidil suppressed the NMDA-induced decrease in the ability of MTT reduction. ◆The effects of minoxidil above were blocked by glibenclamide. ◆Low intracellular potassium level may cause protective effect of neuronal damages.

CRediT author statement Higashi Hiroshi: Investigation, Formal analysis, Writing - Original Draft, Writing - Review & Editing, Visualization. Kinjo Toshihiko: Investigation, Formal analysis, Visualization. Uno Kyosuke: Investigation, Supervision, Formal analysis. Kuramoto Nobuyuki: Conceptualization, Writing - Original Draft, Writing - Review & Editing, Project administration, Supervision.