Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

Label_Title

Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

Label_Title

Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

Endothelium-Dependent Vasorelaxant Effect of a Bioactive Tripeptide, Valine-Proline-Proline, on Rat Aortic Rings

Jair Lozano-Cuenca, Oscar Alberto López-Canales, Ángel Miliar-García, María de los Ángeles Martínez–Godínez, Osiris Teran-Gallardo, María Cristina Paredes-Carbajal, Ruth Mery López-Mayorga, Enrique Fernando Castillo-Henkel, Héctor Flores-Herrera, Jorge Skiold López-Canales*

Departamento de Fisiología y Desarrollo Celular, Instituto Nacional de Perinatología, Montes Urales 800, Col. Lomas Virreyes, Deleg. Miguel Hidalgo, C.P. 11000, Ciudad de México, México

Abstract: The aim of this study was to gain further insights into the mechanisms responsible for the Val-Pro-Pro-induced vasodilation of rat aortic rings. The vasorelaxant effect was found at 10⁻⁹-10⁻⁴ M of the tripeptide on phenylephrine-precontracted rings. It was endothelium-dependent and concentration-dependent at the higher concentrations. The vasodilator response was not modified by preincubation with 3.1 x 10⁻⁷ M glibenclamide, 10⁻³ M 4-aminopyridine (4-AP), 10⁻⁵ M indomethacin or 10⁻⁵ M cycloheximide. However, this response was significantly diminished by preincubation with 10⁻⁵ M L-NG-Nitroarginine methyl ester (L-NAME), 10⁻⁷ M 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 10⁻⁶ M (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i]benzodiazocine-10-carboxylic acid, methyl ester (KT 5823), 10⁻² M tetraethylammonium (TEA) and 10⁻⁷ M apamin plus 10⁻⁷ M charybdotoxin, as well as by the removal of the vascular endothelium. Apparently, Val-Pro-Pro directly produced vasorelaxation of phenylephrine-precontracted rat aortic rings by stimulating the vascular endothelium and activating the NO/cGMP/PKG/Ca²⁺-activated K⁺ channel pathway.

Keywords: bioactive peptides, nitric oxide, potassium channels, rat aorta, vascular endothelium.

Received July 08, 2023; Revised August 09, 2023; Accepted August 16, 2023

Citation: Jair Lozano-Cuenca, Oscar Alberto López-Canales, Ángel Miliar-García, María de los Ángeles Martínez–Godínez, Osiris Teran-Gallardo, María Cristina Paredes-Carbajal, Ruth Mery López-Mayorga, Enrique Fernando Castillo-Henkel, Héctor Flores-Herrera, Jorge Skiold López-Canales, Endothelium-Dependent Vasorelaxant Effect of a Bioactive Tripeptide, Valine-Proline-Proline, on Rat Aortic Rings. Journal of Food and Nutrition Research, 11 (8). 519-524. January 2023. doi:10.12691/jfnr-11-8-1

1. Introduction

Hypertension is the most common chronic disease and the most important risk factor for cardiovascular disease. It affects nearly 1 billion adults, accounts for about 9% of global disability-adjusted life years, and is associated with over 9 million deaths annually [1]

Hypertension is treated non-pharmacologically and pharmacologically. The former method involves lifestyle modifications such as losing weight in cases of obesity, adopting a diet rich in fruits and vegetables, consuming sodium below the recommended threshold, avoiding a high level of alcohol consumption, and refraining from smoking. These strategies have been effective for preventing or controlling stage 1 hypertension [2]. Pharmacological treatment with angiotensin-converting enzyme inhibitors (ACEIs), AT1 receptor blockers (ARBs), calcium channel blockers (CCBs) is necessary for more severe cases.

Different clinical studies have demonstrated that the consumption of macronutrients can play a key role in the management of high blood pressure. According to clinical trials, the partial replacement of carbohydrates with either protein or monounsaturated fat can reduce high blood pressure, and as a consequence, the risk of coronary heart disease [3].

The active peptide fragments (bioactive peptides) encrypted in the structure of food proteins exert beneficial effects on human health above and beyond their expected nutritional value. They are released from their parent proteins by gastrointestinal digestion, fermentation, or food processing [4]. Food-derived bioactive peptides have a wide range of potential applications as functional foods and nutraceuticals for the prevention and management of hypertension.

Hence, an evaluation has been made of various bioactive peptides in the proteins of distinct types of foods. Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) peptides are known to inhibit the angiotensin-converting enzyme [5], increase nitric oxide production in human umbilical vein endothelial cells (HUVECs), and generate an endothelial-dependent vasodilation in ex vivo and in vitro studies [6]. There is evidence that mechanisms of VPP-induced vasodilation involve nitric oxide and the endothelium-derived hyperpolarizing factor (EDHF) via the activation of the bradykinin B2 receptor [7]. The aim of the current contribution was to carry out a more in-depth examination of the mechanisms of VPP-induced vasodilation, especially the possible participation of the NO/cGMP/PKG/Ca²⁺ -activated K⁺ channel pathway.

2. Material and Methods

2.1. Animals

Experiments were performed on thoracic aortic rings isolated from adult male Wistar rats (body weight 250-300 g), which were purchased (n=52) from the bioterium of the Escuela Superior de Medicina. Animals were housed in plastic cages in a special temperature-controlled room (22±2 °C, 50% humidity) on a 12:12 h light/dark cycle (lights on at 7 am). The protocol was approved by the Animal Care Committee of the Escuela Superior de Medicina (Mexico City) and is in agreement with the 1986 Animals (Scientific Procedures) Act of the British Parliament: http://www.legislation.gov.uk/ukpga/1986/14/contents, accessed on January 12, 2023.

2.2. Preparation of Aortic Rings

Animals were euthanized by decapitation and the aortas were immediately excised and placed in cold buffer before being cleaned and freed from surrounding connective tissue. The isolated arteries were cut into rings (4-5 mm long) and placed in 10 ml tissue chambers filled with Krebs-Henseleit bicarbonate buffer (118 mM NaCl ; 4.7 mM KCl; 1.2 mM KH₂PO₄; 1.2 mM MgSO₄.7H₂O; 2.5 mM CaCl₂•2H₂O; 25 mM NaHCO₃; 11.7 mM dextrose; and 0.026 mM calcium disodium EDTA). Tissue baths, maintained at 37 °C and pH 7.4, were bubbled with a mixture of 95% O₂ and 5% CO₂.

Aortic rings were mounted on two stainless steel hooks, one fixed to the bottom of the chamber and the other to a BIOPAC TSD125C-50g pressure transducer connected to a BIOPAC MP100A-CE data acquisition system (BIOPAC Systems, Inc., Santa Barbara, CA, USA) in order to record the isometric tension. Optimal tension, selected from preliminary experiments, was that which afforded the greatest response to phenylephrine (10⁻⁶ M). The rings were given 2 g (100%) of initial tension and allowed to equilibrate for 2 h. Thirty minutes after setting up the organ bath, tissues were first contracted with 10⁻⁶ M phenylephrine to test their contractile responses. Aortic were denuded of endothelium by turning the rings gently several times on the distal portion of small forceps. Endothelial integrity was pharmacologically assessed with acetylcholine-induced vasodilatation (10⁻⁶ M). Segments showing no relaxation to acetylcholine were considered to be endothelium-denuded. After applying 10⁻⁶ M phenylephrine or 10⁻⁶ M acetylcholine, tissues were rinsed three times with Krebs solution to restore basal tension.

2.3. Drugs

All drugs except VPP were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The lactotripeptide was acquired from Productos y Equipos Biotecnológicos S.A. de C.V laboratories. All compounds were dissolved in distilled water. Fresh solutions were elaborated for each experiment.

2.4. Experimental Protocols

Two sets of experiments were conducted to establish the mechanism responsible for the VPP-induced relaxant effect on phenylephrine-precontracted rat aortic rings.

First set of experiments: Thirty minutes after the restoration of basal tension (see preparation of aortic rings), 10⁻⁶ M phenylephrine was added to endothelium-intact and -denuded rat aortic rings, and 20 min later a steady contraction began. Thirty minutes after adding phenylephrine, 10⁻⁹ - 10⁻⁴ M VPP or 10⁻⁹ - 10⁻⁵ M acetylcholine (a positive control of vasorelaxation) were cumulatively added in intervals of ∼10 min. Tension was expressed as a percentage of the aortic contraction resulting from the application of phenylephrine alone (3.54 ± 0.25 g), considered as 100%.

Second set of experiments: Since VPP produced moderate concentration-dependent vasorelaxation on phenylephrine-precontracted rat aortic segments, an attempt was made to determine the mechanism involved. Thirty minutes before adding 10⁻⁶ M phenylephrine (see first set of experiments), endothelium-intact aortic rings were preincubated for 30 min with various compounds: (i) the vehicle (distilled water); (ii) 10⁻⁵ M L-NAME (a direct inhibitor of NO synthase); (iii) 10⁻⁷ M ODQ (an inhibitor of nitric oxide-sensitive guanylyl cyclase); (iv) 10⁻⁶ M KT 5823 (an inhibitor of protein kinase G); (v) 10⁻² M TEA (a Ca²⁺-activated K⁺ channel blocker and nonspecific voltage-activated K⁺ channel blocker); (vi) 10⁻⁷ M apamin plus 10⁻⁷ M charybdotoxin (blockers of small- and large-conductance Ca²⁺-activated K⁺ channels, respectively); (vii) 10⁻³ M 4-AP (a voltage-activated K⁺ channel blocker); (viii) 3.1 X 10-⁷ M glibenclamide (an ATP-sensitive K⁺ channel blocker); (ix) 10⁻⁵ M indomethacin (a prostaglandin synthesis inhibitor) and (x) 10⁻⁵ M cycloheximide (a general protein synthesis inhibitor). Following preincubation with each compound, evaluation of the VPP-induced vasorelaxant response (with 10⁻⁹ – 10⁻⁴ M of the peptide) indicated whether the effect was significantly different. Accordingly, it was possible to gain insights into the mechanism of action of VPP.

2.5. Data Analysis and Statistics

Data are expressed as the mean SEM. In all experiments, the aortic segments were obtained from 6 animals. The vasorelaxant effect of VPP or acetylcholine on endothelium-intact aortic rings precontracted with phenylephrine was examined with one-way analysis of variance (ANOVA) of repeated measurements. In relation to the influence of the endothelium, the antagonist, inhibitors, and blockers on the VPP-induced relaxation of phenylephrine-precontracted aortic segments, significant differences were assessed with two-way ANOVA of repeated measurements. Each ANOVA was followed by the Student-Newman-Keuls post hoc test. The statistical analysis was performed on the SigmaPlot 12 program (Systat Software Inc., San Jose, CA, USA), with significance considered at P<0.05 [8].

3. Results

Effect of the Val-Pro-Pro tripeptide on phenylephrine-precontracted rat aortic rings.

The effects of cumulative concentrations of VPP on phenylephrine-precontracted aortic rings of rats (endothelium-intact or -denuded) were recorded (Figure 1). The concentration-dependent relaxation observed when applying 10⁻⁹-10⁻⁴ M VPP to intact aortic rings was not found with denuded aortic rings. The maximum relaxation (Emax) of phenylephrine-precontracted aortic rings was 80.05 ± 0.95 % (EC50 10-4.94 M) for those with intact endothelium and 1.54 ± 0.46 % (EC50 10-6.67 M) for those with the endothelium denuded.

Effect of L-NAME or TEA on the Val-Pro-Pro-induced relaxation of phenylephrine-precontracted rat aortic rings.

Preincubation of phenylephrine-precontracted aortic rings with 10⁻⁵ M L-NAME or 10⁻² M TEA was examined (Figure 2). The maximum vasorelaxant effect produced by 10⁻⁹ - 10⁻⁴ M VPP was decreased as a result of preincubation with 10⁻⁵ M L-NAME (Figure 2A; 80.20 ± 1.27 % vs 29.65 ± 0.50 %, respectively) and 10⁻² M TEA (Figure 2B; 88.20 ± 4.88 % vs 24.70 ± 1.35 %, respectively).

Effect of apamin plus charybdotoxin, glibenclamide, and 4-AP on the Val-Pro-Pro-induced relaxation of phenylephrine-precontracted rat aortic rings.

Preincubation of phenylephrine-precontracted aortic rings with glibenclamide, 4-AP, or 10⁻⁷ M apamin plus 10⁻⁷ M charybdotoxin was evaluated (Figure 3). The maximum vasorelaxant effect elicited by 10⁻⁹ - 10⁻⁴ M VPP was significantly diminished following preincubation with apamin plus charybdotoxin (Figure 3A; 84.35 ± 1.47 % vs 19.80 ± 1.32 %, respectively), but was unaffected by glibenclamide (Figure 3B; 79.20 ± 5.00 % vs 79.32 ± 4.61 %, respectively) or 4-AP (Figure 3C; 81.26 ± 4.68 % vs 80.15 ± 4.09 %, respectively).

Effect of ODQ or KT 5823 on Val-Pro-Pro-induced vasorelaxation of phenylephrine-precontracted rat aortic rings.

Pretreatment of phenylephrine-precontracted aortic rings with 10⁻⁷ M ODQ or 10⁻⁶ M KT 5823 was assessed (Figure 4). The maximum vasorelaxation provided by 10⁻⁹ - 10⁻⁴ M VPP was significantly reduced after pretreatment with ODQ (Figure 4A; 85.11 ± 2.97 % vs 11.66 ± 1.74 %, respectively) and KT 5823 (Figure 4B; 78.60 ± 1.16 % vs 17.59 ± 0.05 %, respectively).

Effect of indomethacin or cycloheximide on the Val-Pro-Pro-induced vasorelaxation of phenylephrine-precontracted rat aortic rings.

Pretreatment of phenylephrine-precontracted aortic rings with 10⁻⁵ M indomethacin or cycloheximide was examined (Figure 5). The maximum vasorelaxant effect afforded by 10⁻⁹ - 10⁻⁴ M VPP was unchanged following preincubation with indomethacin (Figure 5A; 91.19 ± 4.31 vs. 89.77 ± 4.20, respectively) or cycloheximide (Figure 5B; 97.11 ± 5.56 % vs. 98.66 ± 9.15, respectively).

Figure 1. Concentration-dependent relaxation of 10⁻⁹ - 10⁻⁴ M Val-Pro-Pro on endothelium-intact (closed circles) and endothelium-denuded (open circles) phenylephrine-precontracted rat aortic rings. Data are expressed as the mean ± SEM of n observations (n= 6). (*) P<0.001 vs (+) Endothelium.

Figure 2. Effects of 10⁻⁵ M L-NG-Nitroarginine Methyl Ester (L-NAME) (A) and 10⁻² M tetraethylammonium (TEA) (B) on Val-Pro-Pro-induced vasorelaxation in functional endothelium rat aortic rings precontracted with 10⁻⁶ M phenylephrine (PE). Data are expressed as the mean ± SEM of n observations. (*) P<0.001 vs control.

Figure 3. Effects of: (A) 10⁻⁷ M apamin plus 10⁻⁷ M charybdotoxin, (B) 3.1 x 10⁻⁷ M glibenclamide and (C) 10⁻³ M 4-aminopyridine (4-AP) on Val-Pro-Pro-induced vasorelaxation in functional endothelium rat aortic rings precontracted with 10⁻⁶ M phenylephrine (PE). Data are expressed as the mean ± SEM of n observations (n=6). * P<0.001 vs control (two-way ANOVA).

Figure 4. Effects of 10⁻⁷ M ODQ (A) and 10⁻⁶ M KT 5823 (B) on Val-Pro-Pro-induced vasorelaxation in functional endothelium rat aortic rings precontracted with 10⁻⁶ M phenylephrine (PE). Data are expressed as the mean ± SEM of n observations. (*) P<0.001 vs control (two-way ANOVA).

Figure 5. Effects of 10⁻⁵ M Indomethacin (A) and 10⁻⁵ M cycloheximide (B) on Val-Pro-Pro-induced vasorelaxation in functional endothelium rat aortic rings precontracted with 10⁻⁶ M phenylephrine (PE). Data are expressed as the mean ± SEM of n observations.

4. Discussion

The results of the present study clearly show that the VPP tripeptide causes vasodilation of endothelium-intact rat aortic rings precontracted with phenylephrine, which is in agreement with other studies that have evaluated the vascular effects of valyl prolyl proline (VPP) and isoleucyl prolyl proline (IPP). [7]

According to previous reports, peptides have diverse types of activity, including antimicrobial [9,10], antidiabetic [11,12], immunomodulatory [13,14], and antihypertensive [15,16]. With regard to the antihypertensive effect, the vasodilatory properties of bioactive peptides (e.g., VPP and IPP) are known to involve the inhibition of the angiotensin-converting enzyme [17], a decrease in the level of endothelin-1 [18], and an increase in the production of nitric oxide in human umbilical vein endothelial cells [6].

The vasorelaxant effect of VPP on phenylephrine-precontracted, endothelium-intact rat aortic rings is consistent with such activity described in the literature [7]. Nevertheless, to our knowledge, there are no studies on the possible role of the NO / cGMP / PKG / Ca²⁺ -activated K⁺ channel pathway in the vasodilation generated by VPP.

Precontraction of aortic rings with phenylephrine makes vasodilator responses to different agents (e.g., acetylcholine, bradykinin and statins) more evident [19,20]. Thus, the present experimental protocols were carried out on phenylephrine-precontracted rat aortic rings, finding that the application of 10⁻⁹ to 10⁻⁴ M of VPP caused a concentration-dependent vasodilation.

Interestingly, we found that the vascular endothelium plays a very important role in the relaxant effect of VPP. The above is due to the absence of endothelium or in the presence of L-NAME, the relaxant effect of this peptide was strongly inhibited, which clearly demonstrates that the endothelium participates in this relaxation through the liberation of NO.

The significant reduction in the VPP-induced vasorelaxation of rat aortic rings by 10⁻⁵ M L-NAME (a direct inhibitor of NOS; Figure 3A) [21] , 10⁻² M TEA (a Ca²⁺-activated K⁺ channel blocker and non-specific voltage-activated K⁺ channel blocker; Figure 3B) [22,23] , and 10⁻⁷ M apamin plus 10⁻⁷ M charybdotoxin (blockers of small- and large-conductance Ca²⁺-activated K⁺ channels, respectively; Figure 3C) [24,25] suggests the involvement of the release of nitric oxide by the endothelium, Ca²⁺- activated K⁺ channels, and to a lesser extent the voltage-activated K⁺ channel blocker. In this sense, VPP has been described to upregulate eNOS gene expression in spontaneously hypertensive rats [26]. On the other hand, the combination of apamin plus charybdotoxin was used because it was previously reported that a complete blockage of Ca²⁺-activated K⁺ channels is necessary to produce a pharmacological response [27]. According to a pilot experiment in our lab, apamin or charybdotoxin alone does not modify the vasorelaxant responses to VPP (data not shown). Hence, the combination of apamin plus charybdotoxin was presently employed. It is possible that VPP produces vascular hyperpolarization attributable to the release of the endothelium-dependent hyperpolarizing factor, as has been described for acetylcholine [28,29]. However, the aforementioned proposal is speculative. Future research, which is beyond the scope of the present study, will be required to further investigate the contribution of this factor to the vascular effects generated by peptides.

The current findings about the participation of NO and K⁺ channels in the VPP-induced vasorelaxation of rat aortic rings corroborate several reports suggesting that NO and K⁺ channels play an important role in regulating vascular tone [30,31,32,33,34,35].

Since the vasorelaxant response to VPP did not differ significantly after preincubation with 4-AP (a voltage-activated K⁺ channel blocker) or glibenclamide (an ATP-sensitive K⁺ channel blocker), the voltage-activated and ATP-sensitive K⁺ channels were not involved in the mechanism of action. Future research is necessary to gain greater insights into the specific subtype of Ca²⁺-activated K⁺ channels implicated in the vasorelaxant effect produced by the tripeptide.

Given that the VPP-induced vasorelaxation was not modified by indomethacin (a prostaglandin synthesis inhibitor) or cycloheximide (a general protein synthesis inhibitor), neither prostacyclins or protein synthesis contributes to the endothelium-mediated vasodilation. This is in contrast to previous findings obtained with small mesenteric arteries isolated from spontaneously hypertensive rats, in which prostacyclin and the IP receptor played a role in the vasodilation generated by the Arg-Ile-Tyr peptide [36]. A possible explanation for this apparent discrepancy may be at least partially related to the blood vessel and the peptide used. Similar to the current results, the vasodilation produced in small mesenteric arteries by another antihypertensive peptide, Leu-Arg-Ala was not mediated by PGI₂ [37]. Finally, cycloheximide, a general protein synthesis inhibitor, did not alter the VPP-induced vasorelaxation of aortic rings, thus excluding the involvement of protein synthesis.

5. Conclusion

The present study demonstrates that the vasodilator effect of VPP on phenylephrine-precontracted rat aortic rings was unaltered by pretreatment with 4-AP, glibenclamide or indomethacin, but was notably decreased by L-NAME, TEA, ODQ, KT5823, apamin + charybdotoxin and by the removal of the endothelium. Hence, VPP-induced vasodilation seems to depend on the stimulation of the vascular endothelium and involve activation of the NO / cGMP / PKG / Ca²⁺ -activated K⁺ channel pathway. The bioactive peptide herein studied could possibly have therapeutic benefit in the high blood pressure management.

List of Abbreviations

Angiotensin-converting enzyme inhibitors(ACEIs)

AT1 receptor blockers(ARBs)

Calcium channel blockers(CCBs)

Val-Pro-Pro(VPP)

Ile-Pro-Pro(IPP)

Human umbilical vein endothelial cells(HUVECs)

Endothelium-derived hyperpolarizing factor(EDHF)

4-aminopyridine(4-AP)

L-NG-Nitroarginine methyl ester(L-NAME)

1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one(ODQ)

Tetraethylammonium(TEA)

Acknowledgements

The authors greatly appreciate the technical assistance of Oscar Martín Boche Olivan. We thank the National Institute of Perinatology for the support to carry out this research related to the project 2021-1-18.

Statement of Competing Interests

The authors declare no conflicts of interest.

References

[1]   | World Health Organization. A global brief on Hypertension: silent killer, global public health crises (World Health Day 2013). Geneva: WHO 2013. [Online]
[2]   | Ozemek C, Tiwari SC, Sabbahi A, Carbone S, Lavie CJ. Impact of therapeutic lifestyle changes in resistant hypertension. Prog Cardiovasc Dis. 2019; S0033-0620(19)30146-X. doi:10.1016/j.pcad.2019.11.012.
[3]   | Carey, V.J.; Bishop, L.; Charleston, J.; Conlin, P.; Erlinger, T.; Laranjo, N.; McCarron, P.; Miller, E.; Rosner, B.; Swain, J.; et al. Rationale and Design of the Optimal Macro-Nutrient Intake Heart Trial to Prevent Heart Disease (OMNI-Heart). Clin. Trials 2005, 2, 529–37.
[4]   | Hartmann, R.; Meisel, H. Food-Derived Peptides with Biological Activity: From Research to Food Applications. Curr. Opin. Biotechnol. 2007, 18, 163–69.
[5]   | Nakamura Y, Yamamoto N, Sakai K, Okubo A, Yamazaki S, Takano T. Purification and characterization of angiotensin I-converting enzyme inhibitors from sour milk. J Dairy Sci 1995; 78: 777–83.
[6]   | Hirota T, Nonaka A, Matsushita A, Uchida N, Ohki K, Asakura M, Kitakaze M. Milk casein-derived tripeptides, VPP and IPP induced NO production in cultured endotelial cells and endothelium-dependent relaxation of isolated aortic rings. Heart Vessels 2011; 26: 549–56.

[7]  
Hirota Tatsuhiko, Nonaka Atsuko, Matsushita Akiko, Uchida Naoto, Ohki Kohji, Asakura Masanori, Kitakaze Masafumi. Milk casein-derived tripeptides, VPP and IPP induced NO production in cultured endothelial cells and endothelium-dependent relaxation of isolated aortic rings. Heart Vessels (2011) 26: 549–56 DOI 10.1007/s00380-010-0096-y.

[8]   | Steel RGD, Torrie JH. Principles and procedures of statistic: a biomedical approach.: McGraw-Hill, NY.; 1997.
[9]   | Taveira G.B.; Carvalho A.O.; Rodrigues R.; Trindade F.G.; Da Cunha M.; Gomes V.M. Thionin-like peptide from Capsicum annuum fruits: Mechanism of action and synergism with fluconazole against Candida species. BMC Microbiol. 2016, 16: 12.
[10]   | Sousa D.A.; Porto W.F.; Silva M.Z.; da Silva T.R.; Franco O.L. Influence of cysteine and tryptophan substitution on DNA-binding activity on maize α-hairpinin antimicrobial peptide. Molecules 2016, 21, 1062.
[11]   | Huang S.-L.; Jao C.-L.; Ho K.-P.; Hsu K.-C. Dipeptidyl-peptidase IV inhibitory activity of peptides derived from tuna cooking juice hydrolysates. Peptides 2012, 35, 114–21.
[12]   | Li-Chan E.C.; Hunag S.-L.; Jao C.-L.; Ho K.-P.; Hsu K.-C. Peptides derived from atlantic salmon skin gelatin as dipeptidyl-peptidase IV inhibitors. J. Agric. Food Chem. 2012, 60, 973–78.
[13]   | Xiao M.; Ding L.; Yang W.; Chai L.; Sun Y.; Yang X.; Li D.; Zhang H.; Li W.; Cao Z. St20, a new venomous animal derived natural peptide with immunosuppressive and anti-inflammatory activities. Toxicon 2017, 127, 37–43.
[14]   | Hou, H.; Fan, Y.; Wang, S.; Si, L.; Li, B. Immunomodulatory activity of Alaska pollock hydrolysates obtained by glutamic acid biosensor—Artificial neural network and the identification of its active central fragment. J. Funct. Foods 2016, 24: 37–47.
[15]   | Xu, X.; Gao, Y. Purification and identification of angiotensin I-converting enzyme-inhibitory peptides from apalbumin 2 during simulated gastrointestinal digestion. J. Sci. Food Agric. 2015, 95, 906–14.
[16]   | Castellano, P.; Aristoy, M.-C.; Sentandreu, M.Á.; Vignolo, G.; Toldrá, F. Peptides with angiotensin I converting enzyme (ACE) inhibitory activity generated from porcine skeletal muscle proteins by the action of meat-borne lactobacillus. J. Proteom. 2013, 89, 183–90.
[17]   | Nakamura Y, Yamamoto N, Sakai K, Takano T.J. Antihypertensive effect of sour milk and peptides isolated from it that are inhibitors to angiotensin I-converting enzyme. Dairy Sci. 1995, 78(6):1253-57. doi: 10.3168/jds.S0022-0302(95)76745-5.
[18]   | Zheng, Y.; Li, Y.; Zhang, Y.; Ruan, X.; Zhang, R. Purification, characterization, synthesis, in vitro ACE inhibition and in vivo antihypertensive activity of bioactive peptides derived from oil palm kernel glutelin-2 hydrolysates. J. Funct. Foods 2017, 28, 48–58.
[19]   | Lopez J, Mendoza R, Cleva Villanueva G, Martinez G, Castillo EF, Castillo C. Participation of K+ channels in the endothelium-dependent and endothelium-independent components of the relaxant effect of rosuvastatin in rat aortic rings. J. Cardiovasc. Pharmacol. Ther. 2008; 13: 207–13.
[20]   | Anozie O, Ross R, Oyekan AO, Yakubu MA. Differential modulation of bradykinin-induced relaxation of endothelin-1 and phenylephrine contractions of rat aorta by antioxidants. Acta Pharmacol Sin. 2007 28(10):1566-72. doi: 10.1111/j.1745-7254.2007.00631.x.
[21]   | Palacios M, Knowles RG, Palmer RM, Moncada S. Nitric oxide from L-arginine stimulates the soluble guanylate cyclase in adrenal glands. Biochem. Biophys. Res. Commun. (1989) 165: 802-09.
[22]   | Abderrahmane A, Salvail D, Dumoulin M, Garon J, Cadieux A, Rousseau E (1998) Direct activation of K(Ca) channel in airway smooth muscle by nitric oxide: involvement of a nitrothiosylation mechanism?. Am. J. Respir. Cell Mol. Biol. 19: 485-97.
[23]   | Mathie A, Wooltorton JR, Watkins CS. Voltage-activated potassium channels in mammalian neurons and their block by novel pharmacological agents. Gen. Pharmacol. 1998, 30: 13-24
[24]   | Zografos P, Li JH, Kau ST (1992) Comparison of the in vitro effects of K+ channel modulators on detrusor and portal vein strips from guinea pigs. Pharmacology 45: 216-30.
[25]   | Ishii TM, Maylie J, Adelman JP. Determinants of apamin and d-tubocurarine block in SK potassium channels. J. Biol. Chem. 1997, 272: 23195-00.
[26]   | Yamaguchi N, Kawaguchi K, Yamamoto N (2009) Study of the mechanism of antihypertensive peptides VPP and IPP in spontaneously hypertensive rats by DNA microarray analysis. Eur J Pharmacol 620(1–3):71–77
[27]   | Qiu Y, Quilley J. Apamin/charybdotoxin-sensitive endothelial K+ channels contribute to acetylcholine-induced, NO-dependent vasorelaxation of rat aorta. Med Sci Monit. 2001;7(6):1129-36.
[28]   | Corriu C, Feletou M, Canet E, Vanhoutte PM (1996) Endothelium-derived factors and hyperpolarization of the carotid artery of the guinea-pig. Br. J. Pharmacol. 119: 959-64.
[29]   | Tottrup A, Kraglund K. Endothelium-dependent responses in small human mesenteric arteries. Physiological research / Academia Scientiarum Bohemoslovaca. (2004) 53: 255-263.
[30]   | Moncada S, Higgs EA, Palmer RM. Characterization and biological significance of endothelium-derived relaxing factor. Biochem. Soc. Trans. (1988a) 16: 484-86.
[31]   | Moncada S, Palmer RM, Higgs EA. The discovery of nitric oxide as the endogenous nitrovasodilator. Hypertension (1988b) 12: 365-72.
[32]   | Moncada S, Radomski MW, Palmer RM. Endothelium-derived relaxing factor. Identification as nitric oxide and role in the control of vascular tone and platelet function. Biochem. Pharmacol. (1988c) 37: 2495-01.
[33]   | Leblanc N, Wan X, Leung PM. Physiological role of Ca(2+)-activated and voltage-dependent K+ currents in rabbit coronary myocytes. Am. J. Physiol. 1994, 266: C1523-37.
[34]   | Knot HJ, Nelson MT. Regulation of membrane potential and diameter by voltage-dependent K+ channels in rabbit myogenic cerebral arteries. Am. J. Physiol. 1995, 269: H348-55.
[35]   | Nelson MT, Quayle JM (1995) Physiological roles and properties of potassium channels in arterial smooth muscle. Am. J. Physiol. 268: C799-22.

[36]  
Yamada Yuko , Iwasaki Masashi, Usui Hachiro, Ohinata Kousaku, Marczak Ewa D, Lipkowski Andrzej W, Yoshikawa Masaaki. Rapakinin, an anti-hypertensive peptide derived from rapeseed protein, dilates mesenteric artery of spontaneously hypertensive rats via the prostaglandin IP receptor followed by CCK(1) receptor. Peptides. 2010, 31(5):909-14. doi: 10.1016/j.peptides.2010.02.013. Epub 2010 Feb 25.

[37]  
Shobako Naohisa, Ishikado Atsushi , Ogawa Yutaro , Sono Yoko, Kusakari Takashi , Suwa Makoto, Matsumoto Motonobu , Ohinata Kousaku. Vasorelaxant and Antihypertensive Effects That Are Dependent on the Endothelial NO System Exhibited by Rice Bran-Derived Tripeptide. J Agric Food Chem. 2019, 67(5):1437-42. doi: 10.1021/acs.jafc.8b06341. Epub 2019 Jan 28.