Implication of opioid receptors in the antihypertensive effect of a

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Bioactive Constituents, Metabolites, and Functions

Implication of opioid receptors in the antihypertensive effect of a casein hydrolysate and #s1-casein derived peptides Laura Sánchez-Rivera, Pedro Ferreira Santos, Maria Angeles Sevilla, Maria José Montero, Isidra Recio, and Beatriz Miralles J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03872 • Publication Date (Web): 11 Aug 2019 Downloaded from pubs.acs.org on August 18, 2019

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Journal of Agricultural and Food Chemistry

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Implication of opioid receptors in the antihypertensive effect of a casein hydrolysate and αs1-casein derived peptides

3 4 5

Laura Sánchez-Riveraa#, Pedro Ferreira Santosb#, M Angeles Sevillabc, M José Monterobc, Isidra Recioa, Beatriz Mirallesa*

6 7

a

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Nicolás Cabrera 9, 28049 Madrid, Spain.

9

b

Instituto de Investigación en Ciencias de la Alimentación (CIAL, CSIC-UAM).

Department of Physiology and Pharmacology, Faculty of Pharmacy, University

10

of Salamanca, 37007 Salamanca, Spain

11

c Cardiovascular

12

(IBSAL), Hospital Virgen de la Vega, 37007 Salamanca, Spain

Pharmacology. Institute for Biomedical Research of Salamanca

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# Both

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* Corresponding author: Beatriz Miralles

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Tel.: +34 910017932

17

Fax: +34 910017905

18

E-mail address: [email protected]

authors equally contributed to this work

19 20

1

ACS Paragon Plus Environment


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ABSTRACT

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The antihypertensive activity of two αs1-casein-derived peptides and a casein

23

hydrolysate containing these sequences was evaluated in the presence of

24

naloxone. The activity was abolished by this opioid antagonist at 2, 4 and 6 h

25

post-administration. Similarly, the antihypertensive effect of the αs1-casein

26

peptides

27

Hg) at 5 mg/kg body weight was antagonized by the co-administration of

28

naloxone. Because peptide 143AYFYPEL149 had recently shown opioid activity, a

29

molecular dynamic simulation of this peptide with human µ-opioid receptor was

30

performed to demonstrate its favorable structure and interaction energy despite

31

the presence of Ala at the N-terminus. Altogether, these results revealed that the

32

in vivo effect on systolic blood pressure of the studied αs1-casein peptides is

33

mediated by interaction with opioid receptors, and the antihypertensive activity of

34

the casein hydrolysate can be very likely ascribed to them with the possible

35

contribution of other mechanisms.

90RYLGY94

(-23.8 ± 2.5 mm Hg) and

143AYFYPEL149

(-21.1 ± 3.2 mm

36 37

Keywords: antihypertensive peptide, casein hydrolysate, naloxone, opioid

38

receptor, spontaneously hypertensive rat.

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INTRODUCTION Among bioactive peptides derived from food, antihypertensive peptides are 1-2

41

undoubtedly the group with more solid evidence in human trials.

Much work

42

has been done to evaluate the in vitro activity of peptides on the angiotensin-I-

43

converting enzyme (ACE), an enzyme which plays an important role in the

44

regulation of blood pressure.3 However, for many peptides, an important lack of

45

correlation has been found between the in vitro IC50 values and the observed

46

effects on arterial blood pressure. It has been postulated that this discordance

47

can be due to the degradation of peptides during gastrointestinal digestion, poor

48

bioavailability or due to the involvement of different mechanisms of action.4 The

49

in vivo plasma o tissue concentrations of peptides are in some cases too low to

50

support the observed antihypertensive activity.5 The pharmacokinetic behavior

51

has been evaluated for a small group of antihypertensive peptides

52

plasma concentration has been correlated with plasma ACE inhibition for some

53

tryptophan-containing dipeptides.8

6-7,

and the

54

The modes of action of food-derived peptides on blood pressure, apart from

55

ACE inhibition, include inhibition of renin activity or endothelin-converting

56

enzyme, interactions with bradykinin receptors, Ca2+ channels, opioid receptors,

57

and effects on the sympathetic nerve. Opioid receptor modulation has been

58

demonstrated for some antihypertensive food-derived peptides, such as, α-

59

lactorphin, α-La f(50-53), with sequence YGLF.9 This peptide has been found to

60

lower blood pressure in spontaneously hypertensive rats (SHR) and produce an

61

endothelium-dependent relaxation in mesenteric arteries which was reverted by

62

an eNOS inhibitor.10 The activity of this peptide was suggested to be mediated 3



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through interaction with opioid receptors from peripheral tissues.11 These

64

receptors are found in the nervous, endocrine and immune system. However,

65

they are also present in the gastrointestinal tract, and this points to the possible

66

interaction at this level.12 The activation of opioid receptors present in the

67

intestinal wall could result in the modulation of sympathetic nerve activity and

68

might explain certain observed antihypertensive effects despite low plasma

69

concentrations. Since several classes of opioid peptides and receptors are

70

involved in multiple anatomic circuits controlling blood pressure 13-14, the influence

71

of dietary peptides with opioid features on hypertension should be investigated.

72

In fact, the endomorphin analog [d-Ala(2)-endomorphin 2] lowered blood

73

pressure and enhanced NO concentration in tissues in anesthetized rats after

74

intravenous administration.15 Other opioid analogs like biphalin, an enkephalin

75

analog, have also demonstrated decreases in SHR blood pressure and blockage

76

of the response with naloxone.16 Several ongoing phase 3 trials conducted to

77

observe the effect of opioid agonists on blood pressure support their significant

78

role in hypertension, especially in forms related to sympathetic hyperactivity.17

79

Our group has reported the antihypertensive activity of a peptic casein 90RYLGY94

80

hydrolysate in SHR. Two αs1-casein-derived peptides,

and

81

143AYFYPEL149,

82

sequences responsible for the antihypertensive effect.18 The administration of

83

this casein hydrolysate for 6 weeks to SHR revealed that not only the

84

development of hypertension was attenuated in the treated group of animals, but

85

they showed improved aorta and mesenteric acetylcholine relaxations and

86

increased eNOS expression in aorta.19 One of the proposed active peptides

with antihypertensive activity, were identified as the main

4


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90RYLGY94,

87

found in this casein hydrolysate, αs1-casein fragment

88

within the sequences of previously reported αs1-casein-derived exorphins,

89

90RYLGYLE96, 90RYLGYL95

90

demonstrated by the displacement of enkephalins from rat brain membranes, by

91

naloxone inhibition in glioma hybrid cells and in preparations of mouse vas

92

deferens.20 The second one, αs1-casein 143AYFYPEL149 has more recently shown

93

opioid activity in guinea pig ileum assays.21 Other related fragments

94

144YFYPEL149

95

preparation and in mouse vas deferens but to a lower extent. In addition,

96

143AYFYPEL149

97

relative expression of MUC5AC, being this effect also mediated through the

98

interaction with µ-opioid receptors expressed in goblet cells.22

and

and 91YLGYLE96. The activity of these peptides was

144YFYPE148

and

is comprised

showed also opioid agonistic activity in this

144YFYPEL149

induced mucin secretion and increased

99

The aim of this work was to evaluate, by the use of naloxone, an opioid

100

antagonist, if the in vivo antihypertensive activity of these αs1-casein peptides,

101

and the casein hydrolysate containing the same, was mediated through the

102

interaction with opioid receptors. In order to better explain the opioid activity of

103

αs1-casein 143AYFYPEL149, that contains a residue different from Tyr at N-terminal

104

position, a molecular modelling of the receptor-peptide interaction was

105

performed.

5



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MATERIALS AND METHODS

107

Chemicals

108

HPLC-grade acetonitrile was from VWR (VWR International, Radnor,

109

Pennsylvania, USA), ultrapure water was obtained from a Milli-Q water filtration

110

station (Millipore Corporation, Bedford, Massachusetts, USA). Formic acid 98-

111

100% and naloxone hydrochloride (injectable solution, Kern Pharma, Barcelona,

112

Spain). Food-grade pepsin (E.C. 3.4.23.1) was from Biocatalysts Ltd (Cardiff,

113

UK), and food-grade KOH and HCl were from Aditio (Panreac Quimica, S.A.U.,

114

Castellar del Vallés, Spain). All other chemicals were obtained from commercial

115

sources and of high quality grade.

116

Peptide synthesis.

117

Peptides from αs1-casein, fragment (90-94), 90RYLGY94, and fragment (143-149),

118

143AYFYPEL149

119

methoxy-carbonyl chloride (Fmoc) solid-phase. A 431A peptide synthesizer

120

(Applied Biosystems Inc. Überlingen, Germany) was employed. Their purities

121

were determined by reversed phase liquid chromatography-UV and mass

122

spectrometry and were over 90%.

123

Preparation of the casein. The casein used was a commercial bovine milk

124

protein isolate (Promilk-85B Casein, Arras Cedex, France) naturally rich in

125

micellar caseins (92%). The moisture of the casein isolate was 5%, 5.5%

126

corresponded to lactose and 7.5% to mineral content. The preparation of the

127

casein hydrolysate was carried out with food-grade pepsin as previously

128

described.23 Briefly, a 6% aqueous solution of micellar casein adjusted to pH 2.0

were synthesized in-house by using the method fluorenyl-

6


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129

with food grade HCl 1 M and it was digested for 6 h at 37ºC with 2% porcine

130

pepsin (w/w substrate) added twice, at the beginning and after 3 h of hydrolysis.

131

The reaction was stopped by adjusting the pH to 7.0 by addition of food-grade

132

NaOH 1 M. The hydrolysate was clarified by cross flow ultrafiltration and the

133

filtrate was subsequently spray-dried. The inlet temperature of spray drying was

134

set at 140ºC and the outlet temperature ranged from 75 to 100ºC. The protein

135

concentration of the filtrate after hydrolysis was determined by Kjeldahl. Previous

136

to MS analysis, freeze-dried samples were prepared at 2.5 mg protein/mL.

137 138

Analysis by RP-HPLC-MS/MS. The casein hydrolysate was analyzed by HPLC

139

(Agilent Technologies, Waldbronn, Germany) coupled to an ion trap instrument

140

(Esquire 3000, Bruker Daltonik GmbH, Bremen, Germany). The analyses and

141

data processing were performed as previously described24 but the column used

142

was a Mediterranea Sea 18 150 mm × 2.1 mm column (Teknokroma, Barcelona,

143

Spain) with a precolumn Novapack C18 3.9 × 2 mm (Waters, Cerdanyola del

144

Vallés, Barcelona, Spain). Solvent A (water/formic acid, 1000:1 v/v), and solvent

145

B (acetonitrile/formic acid, 1000:1 v/v). A linear gradient was used from 0 to 55%

146

of solvent A and 45% of solvent B in 120 min. The UV-wavelength detector was

147

set at 214 nm. MS spectra were acquired at mass/charge (m/z) range of 100-

148

3000. The samples were run in duplicate at two different target mass: m/z of 750

149

and 1500. Data processing was done by using Data AnalysisTM (version 4.0,

150

Bruker Daltonilks, Gmbh, Germany). The peptide identification was performed by

151

MASCOT, using a homemade database which includes the main genetic variants

152

from bovine milk proteins. 7



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153 154

Quantification of peptides by RP-HPLC-MS

155

RP-HPLC separation of the hydrolysates were performed as described above.

156

The hydrolysate was injected at 2.5 mg/ml. Stock solutions of 1 mg/mL of the

157

synthetic peptides were prepared in Milli-Q deionized water (Millipore) and six

158

calibration points were injected. For quantitative purposes MS spectra were

159

recorded over the m/z range of 100-1500, by using a signal threshold of 10,000.

160

The quantification of peptides 90RYLGY94 and 143AYFYPEL149 was performed by

161

plotting the peak area of the molecular ions with m/z values of parental ions and

162

their sodium and potassium adducts. Curves from 0.5 to 2.5 µg/ml (linear

163

regression y = 55.99 106x – 4.03 106, R2 = 0.999) and 0.5 to 7.5 µg/ml (linear

164

regression y = 80.04 106x – 51.27 106, R2 = 0.992) for

165

143AYFYPEL149 were

90RYLGY94

and

used, respectively.

166 167

Animal assays and experimental design. All the animal trials were carried out

168

in agreement with the European Union guidelines for the ethical care and use of

169

laboratory animals (European Directive 86/609/CE). SHR aged from the 14th to

170

the 16th-week were purchased from Elevage Janvier (Le Genest, Saint Isle,

171

France). The animals were housed in groups of three rats with 12 h light/dark

172

cycles and kept at a controlled temperature of 23°C. Standard food (Global Diet

173

2014, Harlan, France) and water were available ad libitum. The synthetic peptides

174

and the casein hydrolysate dissolved in pure water, were orally administered to

175

rats by a cannula from mouth to stomach, at a single dose of 5 and 300 mg/kg 8


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176

body weight, respectively. These doses had been established in previous assays.

177

19, 25-26

178

injection, just after the administration of the peptide or the hydrolysate, at a dose

179

of 10 mg/kg body weight. Control trials were performed by oral administration of

180

water or casein (same dose as the hydrolysate, on protein basis); and by

181

subcutaneous injection of naloxone (10 mg/kg body weight). The animals were

182

deprived of solid food diet 12 h before experiments. During this period, they only

183

had access to NaCl (2 g/L) and sucrose (80 g/L) solution. The systolic blood

184

pressure (SBP) were measured using the CODA tail-cuff blood pressure system

185

(Kent Scientific, Torrington, CT, USA), as previously described.19 The SBP was

186

measured before each experiment to estimate the basal blood pressure. After

187

administration of the peptides, hydrolysate, casein or water, the SBP was

188

measured at 2, 4, 6, 8 and 24 h. However, after the administration of the peptide

189

with naloxone or only naloxone (control), the first 6 h post-administration were

190

recorded.

The opioid antagonist, naloxone, was administered by subcutaneous

191 192

Molecular modelling receptor-peptide. Homology model for the human µ-type

193

opioid receptor OPRM (UniprotKB P35372) was built with the software

194

MODELLER version 9.7, as previously described.21 The model was then

195

immersed in a complete membrane-aqueous system using the web-based

196

graphical interface CHARM-GUI membrane builder.27 The structure of the

197

bifunctional µ-opioid agonist and -opioid antagonist tetrapeptide Dmt-Tic-Phe-

198

Phe-NH2 (PDB entry 4RWA) was used as template for docking peptide αs1-casein

199

143AYFYPEL149

into the active site of the receptor model. 9



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200 201

Statistical analysis. The effects of casein hydrolysate, peptides, casein, water

202

or naloxone were calculated as changes in SBP (increase or decrease) from the

203

baseline values. Data were expressed as the mean values ± standard error of

204

mean (SEM). Two-way analysis of variance (ANOVA), using GraphPad Prism 5.0

205

(GraphPad software Inc., San Diego, USA) was carried out. A post-test

206

(Bonferroni) was applied to establish the significant differences between the

207

effect of the peptides or the hydrolysate vs the controls. Significant differences

208

were considered at p ≤ 0.05, p ≤ 0.01 and p ≤ 0.001.

209

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210

RESULTS

211

Characterization of casein and its hydrolysate. The nitrogen content of the

212

casein and the hydrolysate were 12.2 and 12.1%, respectively. The analysis of

213

the peptidomic profile derived from the casein hydrolysate is shown in

214

Supplementary Material, Table S1. Peptides from β, αs- and κ-casein could be

215

identified, with the vast majority of them belonging to β-casein (45%) and αs1-

216

casein (34%). It has to be noted, that the hydrolysate corresponds to the filtrate

217

fraction after an ultrafiltration step and therefore, non-digested casein and large

218

size peptides, like casein-derived phosphorylated peptides were removed.

219

Calibration curves were used to quantify the known antihypertensive peptides

220

from αs1-casein. The concentrations were 1.18 ± 0.07 and 3.58 ± 0.07 mg/g, for

221

90RYLGY94

222

determined in the hydrolysate used in previous studies in SHR at the same dose

223

(1.32 ± 0.04 and 4.11 ± 0.07mg/g, respectively).23

and 143AYFYPEL149, respectively. These values were similar to those

224 225

Antihypertensive activity of the peptic casein hydrolysate, its precursor

226

casein and the synthetic peptides. Reversion by naloxone. The effect on

227

blood pressure of the casein hydrolysate and the casein used as substrate in its

228

production was followed during 24 h after oral administration at 300 mg/kg body

229

weight (Figure 1). Non-hydrolysed casein produced no significant decrease of

230

the SBP in the animals (p > 0.05). The casein hydrolysate gave rise to significant

231

SBP decreases vs water or casein at 2, 4, 6 and 8 h post-administration, with

232

maximum decrease post-administration at 6 h (-14.3 ± 4.6 mm Hg). The

233

subsequent recovery occurred from 6 to 8 h. 11



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234

The casein hydrolysate was also administered together with naloxone, a

235

competitive antagonist of opioid receptors, and the antihypertensive effect was

236

antagonized from the first measurement and, later, throughout the experiment

237

(Figure 2A). Peptides αs1-casein 90RYLGY94 and 143AYFYPEL149, identified in the

238

active casein hydrolysate, were synthetized and orally administered to SHR. Both

239

peptides produced a significant decrease in the SBP of the animals at all times

240

recorded, i.e., 2, 4, 6 and 8 h (Figure 2 B, C). The maximum decrease after the

241

administration of

242

143AYFYPEL149

243

(Figure 2B). When naloxone was subcutaneously co-administered, the

244

antihypertensive effect was completely abolished at 2, 4 and 6 h post-

245

administration. In the case of 90RYLGY94, at 8 h, significant differences were still

246

found when co-administered with naloxone. Naloxone alone did not exert any

247

effect of the blood pressure of SHRs (Figure 2).

248

Molecular modelling of the µ-opioid receptor bound to peptide αs1-casein

249

143AYFYPEL149.

250

common structural characteristics of both exogenous or endogenous opioid

251

peptides, i. e., presence of a Tyr residue at the N-terminus and the occurrence of

252

other aromatic residue, Phe or Tyr in the third or fourth position

253

dynamics simulations of this sequence binding at the human µ-opioid receptor

254

were performed to explain the opioid activity of this αs1-casein-derived peptide

255

and aiming to elucidate the structure-activity relationship. The stability of the

256

complex receptor-peptide was assessed by monitoring the Root-Mean-Square

257

Deviation (RMSD) between different samples during the simulation. RMSD

90RYLGY94

(Figure 2A) was at 6 h (-23.8 ± 2.5 mm Hg); for

the maximum decrease was reached at 4 h (-21.1 ± 3.2 mm Hg)

Sequence

143AYFYPEL149

does not fully fit with the known

28.

Molecular

12


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258

values for the protein and the peptide along the 30 ns simulation indicated a jump

259

up to 3 Ǻ at the beginning due to the re-orientation of the side chains of residues

260

at the binding pocket of the protein. However, these positions remained stable

261

throughout the rest of the simulation showing the stability of the new orientation

262

(Supplementary Material). The fluctuation of the individual residues from the

263

receptor and the peptide were examined by looking at each α-carbon to

264

investigate the flexibility of the various segments in the complex. Intra and extra

265

loops exhibited the greatest variability, most noteworthy those between residues

266

100-125, 145-150, and 195-210 and the C-terminal domain of the receptor.

267

However, fluctuations of peptide residues showed that only the C-terminal

268

residue (Leu) changed its position, whereas the rest of peptide was stable at the

269

active site (Figure 3A).

270

The interaction energy of the

271

binding site was calculated along the trajectory (Supplementary Material).

272

Values ranged from -50 to -70 kcal/mol. Passing from the less favourable

273

microstate (-50 kcal/mol) to the most favourable (-70 kcal/mol) could be due to a

274

non-stable interaction with one residue of the protein. In order to have a more

275

illustrative picture of the behaviour, the distribution of energy among the peptide

276

residues within the binding cavity was determined (Table 1). The position of the

277

143AYFYPEL149

278

der Waals, hydrogen bond and charge-charge interactions. The strongest

279

hydrogen bond and charge-charge interactions with the peptide N-terminal

280

residue, Ala, involve receptor residues Asp84 and Tyr263. Residue at the second

281

position from the N-terminus, Tyr, stays stable because of strong hydrogen bond

143AYFYPEL149

sequence at the µ-opioid receptor

peptide at the base of the active site is stabilized by strong van

13



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282

interactions with Tyr263 and His234 and van der Waals interactions with Met88,

283

Ile259, Val173 and Trp230. This behaviour is in line with recent findings from

284

molecular docking of morphinan derivatives where the aromatic ring was

285

deduced to be embedded by hydrophobic residues.29 The rest of the van der

286

Waals interactions stabilizes the remainder peptide residues, keeping it in place

287

(Figure B). On the other hand, Tyr, Phe, Tyr, and Pro residues from

288

143AYFYPEL149,

289

and compatible µ-opioid receptor residues, also help Ala to maintain its right

290

conformation (Figure 3 B and C). It is remarkable the double hydrogen bond

291

interaction between the fourth residue of the peptide (Tyr) and receptor residues

292

Tyr12 and His256. Finally, at the top of the cavity, Glu and Leu establish strong

293

charge-charge and hydrogen bond interactions, building a network of

294

electrostatic interactions between the peptide and the last residues of the cavity

295

(Figure 3 D). To note are Lys170, Lys240 and Arg148 residues that form very strong

296

and stable charge-charge interactions. Thus, peptide

297

stable interaction with the µ-opioid receptor along the simulation with favorable

298

binding energy.

299

DISCUSSION

throughout strong van der Waals interactions between their rings

143AYFYPEL149

forms a

300

Opioid receptors have been reported to play a role in the heart and

301

cardiovascular system and it has been proposed that a deficiency of the κ-opioid

302

receptor may cause hypertension, since this receptor may be a component in the

303

central nervous system involved in the regulation of blood pressure.30 Several

304

studies have also indicated the contribution of endogenous opioid peptides in the

305

regulation of blood pressure. Endomorphin 2 and its homologous (D[Ala2]14


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306

endomorphin 2) endogenous ligands for the μ-opioid receptor, show

307

vasodepressor activity when peripherally administered. These responses were

308

mediated by a nitric oxide-dependent naloxone-sensitive mechanism within the

309

vascular endothelium.31 The food-derived peptide α-lactorphin, which is

310

described as a μ-opioid receptor ligand with low potency, exerted an effect not

311

related to central opioid receptors. Thus, nociception, locomotor activity, motor

312

coordination, rectal temperature and duration of pentobarbital anesthesia were

313

not influenced by the i.v. administration of the peptide.11 This led the authors to

314

consider that the blood-pressure lowering effect of α-lactorphin was mediated by

315

peripheral opioid receptor stimulation. Interestingly, this peptide improved the

316

endothelial function associated to an increase of nitric oxide.10 Similarly, the

317

active casein hydrolysate tested in this study had previously demonstrated an

318

improvement of endothelial function in aortic and mesenteric rings accompanied

319

by an increase of eNOS expression in aorta.19 Thus, both the hydrolysate and

320

the comprised αs1-casein fragments could exert their effect mediated by

321

peripheral opioid receptors following their oral administration. Recently, the role

322

of opioids in cardiovascular diseases has been reviewed, proposing µ and δ

323

opioids agonists as drug candidates in future for the treatment of hypertension.17

324

The hypotensive effect of endogenous opioid peptides, like β-endorphin, could

325

be mediated by the reduction in vasoconstrictive neurohormones and the

326

increase in plasmatic concentrations of vasorelaxing peptides.32 Interestingly, β-

327

endorphin produces also an increase in growth hormone and IGF-I levels which

328

caused a decrease in blood pressure and peripheral vascular resistance through

329

the nitric oxide pathway.33 Although the casein hydrolysate improved aorta and

330

mesenteric acetylcholine relaxations together with increases eNOS expression19, 15



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331

and here it is shown that the effect is mediated by interaction with opioid

332

receptors, it remains to be elucidated if this activity is mediated by similar

333

hormones and peptides as β-endorphin.

334

Because the enzymatically hydrolysed casein, but not casein, was shown to exert

335

antihypertensive activity after oral administration, the peptides present in the

336

hydrolysate and/or their concentrations seem to be crucial. However, to exert the

337

activity, the peptides should remain in the gastrointestinal tract in amounts

338

capable of confer the physiological action. Peptides

339

143AYFYPEL149

340

enzymes

341

143AYFYPE148

342

volunteers after consumption of casein.35 This implies that this peptide can

343

survive intact, at least, until the distal part of the small intestine. On the other

344

hand, the only sequences related to

345

jejunum are

346

production of the hydrolysate with pepsin implies a higher enzyme/substrate ratio

347

and for longer time than that taking place in the stomach. This explains the

348

different peptide profile and the lack of antihypertensive activity of the precursor

349

casein compared to the hydrolysate. In any case, the fact that opioid peptides

350

present in substantial proportion in the hydrolysate have proved gastrointestinal

351

resistance supports the hypothesis of their effect at this level. Regarding the

352

dosage and the stability of the active peptides to technological treatments, it has

353

been reported that the active peptides incorporated into a drinkable yogurt (4%

34.

90RYLGY94

and

have been shown to be partly resistant to gastrointestinal

More importantly, the sequence

143AYFYPEL149

and its analogue

have been identified in vivo, in jejunal digests from healthy

91YLGYLEQ97

e

90RYLGY94

91YLGYLEQL98.36

identified to date in human

It has to be noted that the

16


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354

w/w of hydrolysate containing 50% w/w of lactose as excipient) resist processes

355

of atomization, homogenization and pasteurization.

356

The common structural features between endogenous and exogenous

357

opioid peptides have been well defined: presence of Tyr at N-terminal end and

358

Phe or Tyr at third or fourth position. The already mentioned αs1-casein exorphins,

359

which contain an Arg as N-terminal residue, exemplify an exception. Indeed, αs1-

360

casein

361

YLGYLE and YLGYL, due to conformational flexibility changes evidenced by

362

NMR, showing that the Arg residue prior to the amino-terminal Tyr was not an

363

obstacle for optimal opioid activity.20 Other reported food-derived opioid agonists

364

with no Tyr at N-terminal position are oryzatensin, Allergen RA5B precursor

365

47GYPMYPLPR55 37

366

repeats) GYYPT 38, with a common Gly as N-terminal residue. To note that gluten

367

exorphin has a lower selectivity for the µ- than the -opioid receptor. Although, to

368

the best of our knowledge, the presence of Ala at N-terminal has not been

369

reported as a favorable feature for opioid receptor binding, it does not prevent the

370

activity of

371

behavior of this seven residues molecule within the µ-opioid receptor. The

372

structural stability and interaction energy between the protein and the peptide

373

were favorable, with a predominant role of hydrogen bonds between the fourth

374

peptide residue Tyr and two receptor hydrophobic residues. The participation of

375

the phenolic hydroxyl group at position 3 in water-mediated hydrogen bonding

376

network with His is recognized as a conserved interaction between potent opioid

377

ligands and the binding pocket of the µ-opioid receptor.39 A similar effect was

90RYLGYLE96,

was more potent than three of its analogs, RYLGYL,

and gluten exorphin, high molecular weight glutenin (various

143AYFYPEL149.

The molecular docking study characterized the

17



144YFYPEL149

Page 18 of 28

378

found for the interaction between peptide

379

receptor.21 This supports the activity of a peptide carrying Ala as N-terminal

380

residue and raises the possibility of other peptides exhibiting a residue different

381

from Tyr, Arg, or Gly at N-terminal position to act as opioid ligands, concretely as

382

µ-opioid ligands.

383

and the µ-opioid

Our results demonstrated that the two previously identified αs1-casein 90RYLGY94

and

143AYFYPEL149,

384

peptides

and the casein hydrolysate containing

385

them, exert their effect mediated by the opioid system in a naloxone-

386

antagonizable manner. Nonetheless, considering these results, it cannot be

387

excluded that other peptides present in the hydrolysate, or the synergism

388

between peptides, could also promote its antihypertensive activity. Moreover, this

389

study confirms the favorable interaction of αs1-casein-derived peptide

390

143AYFYPEL149

391

Acknowledgments

392

This work was supported by project AGL2015-66886R from the Spanish Ministry

393

of Science, Innovation and Universities.

with the human µ-opioid receptor.

394

18


Page 19 of 28


395

Figure captions

396

Figure 1. Changes in SBP after oral administration of the casein hydrolysate (300 mg/

397

kg body weight) vs administration of water or casein (300 mg/kg body weight). Data are

398

expressed as mean values ± SEM (n=4-6). Significant differences after administration of

399

the casein hydrolysate vs water are indicated at P≤ 0.05(*) and P≤ 0.01 (**). Significant

400

differences after administration of casein hydrolysate vs the control of casein were found

401

at P≤ 0.01 (##), P≤ 0.001 (###).

402

Figure 2. Changes in SBP after oral administration of (A) casein hydrolysate (300 mg/kg

403

body weight), and (B) peptide RYLGY, and (C) peptide AYFYPEL (5 mg/kg body weight),

404

vs co-administration thereof with naloxone or control of naloxone (10 mg/kg body weight,

405

subcutaneous). Data are expressed as mean values ± SEM (n = 4-6). Significant

406

differences after administration of the peptide vs the peptide with naloxone at P≤ 0.05(*),

407

P≤ 0.01 (**) and P≤ 0.001 (***). Significant differences between the administration of the

408

synthetic peptide and water control were at P≤ 0.05(a), P≤ 0.01 (b) and P≤ 0.001(c).

409

Figure 3. A. Mean residue fluctuations of the peptide AYFYPEL. Both represented as

410

ribbons where the most flexible regions are thicker and colored in red-orange, while static

411

regions are thinner and colored in blue. Final Molecular Dynamics structure of peptide

412

AYFYPEL bound to the mu-opioid receptor. Main residues in the protein are highlighted

413

in yellow and those from the peptide in pink (both are shown in sticks). B. Ala1, Tyr2

414

interactions (N-terminal group), C. Phe3 interactions, D. Tyr4 interactions and E. Pro5,

415

Glu6 and Leu7 interactions (C-terminal group). Three-letter code is used for amino acid

416

residues. HID indicates histidine protonated at nitrogen .

417

19



Page 20 of 28

418

Table 1. Binding free energy for µ-opioid receptor-AYFYPEL complex decomposed by

419

protein and peptide residues. vdW: Van der Waals, HB: hydrogen bond; q-q: charge-

420

charge. Peptide (AYFYPEL) Ala Tyr

Phe

Tyr

Pro Glu

Leu

µ-Opioid receptor Asp 84 Tyr 263 His234 Tyr263 Met88 Ile259 Val173 Trp230 Trp255 Val237 Lys170 Val173 Leu179 Ile233 Ile259 His256 Trp255 Tyr12 Gln61 Ile259 Trp255 Lys240 Lys170 Lys240 Phe158 Arg148 Lys146 Thr155 Asp153

E (kcal/mol)

Interaction type

-15.6 -4.19 -4.27 -2.75 -2.62 -1.27 -1.21 -1.14 -3,25 -1,76 -1,50 -1,39 -1.24 -1.19 -1.08 -4.03 -2,53 -1,80 -1,76 -1,68 -2.11 -1.01 -11,28 -9.69 -1.38 -5.31 -4.72 -1.91 -1.25

q-q HB+VdW HB HB vdW vdW vdW vdW HB vdW vdW vdW vdW vdW Vdw HB+vdW vdW HB HB vdW vdW vdW q-q q-q vdW HB q-q HB vdW

421 422

20


Page 21 of 28


423

Supplementary Material.

424

Table S1. Identified peptides from αS1-, αS2-, β - and κ-casein in the casein hydrolysate.

425

One letter code is used to denote amino acids.

426

Figure S1. RMSD along the simulation for the µ-opioid receptor and the peptide

427

(AYFYPEL) relative to the starting structure. The Y axis shows the RMSD values and

428

the X axis the time in nanoseconds. RMSD values are represented in red for the µ-opioid

429

receptor and in blue for the peptide.

430

Figure S2. Binding free energy values for mu-opioid receptor-peptide AYFYPEL

431

complex. The red line shows the energy value at each step of the simulation. The X axis

432

shows time in nanoseconds, the left Y axis shows global energy values (kcal/mol), and

433

the right Y axis shows the density energy values achieved by the complex during the

434

simulation.

21



435

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Page 26 of 28

Figure 2

ΔSBP (mm Hg)

10

Hydrolysate

0

Hydrolysate –Naloxone -10

** a

-20 10

ΔSBP (mm Hg)

A

*** b

B

a

Water

*** c RYLGY

0

RYLGY-Naloxone -10

Naloxone Water

* c

-20

*** c

-30 10

*** c

** c

C

0 ΔSBP (mm Hg)

Naloxone

AYFYPEL AYFYPEL-Naloxone

-10

Naloxone *** c

-20

** c

b

6

8

Water

*** c

-30 0

2

4 Time (hours)

26


Page 27 of 28


Figure 3

27



Page 28 of 28

TOC graphic

28


Implication of opioid receptors in the antihypertensive effect of a

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