Korean J Physiol Pharmacol 2022; 26(4): 255-262
Published online July 1, 2022 https://doi.org/10.4196/kjpp.2022.26.4.255
Copyright © Korean J Physiol Pharmacol.
Qian Xu, Kunping Zhuo, Xiaotian Zhang, Yaoxia Zhang, Jiaojiao Xue, and Ming-Sheng Zhou*
Department of Physiology, Shenyang Medical University, Shenyang 110034, P.R. China
Correspondence to:Ming-Sheng Zhou
Author contributions: Q.X. performed experiments and data analysis, wrote the draft of the manuscript. K.P.Z., X.T.Z., Y.X.Z., and J.J.X. performed experiment and data analysis. M.S.Z. contributed to the concept and experimental design, wrote the manuscript. All authors read and approved the final manuscript.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Oxytocin is a neuropeptide produced primarily in the hypothalamus and plays an important role in the regulation of mammalian birth and lactation. It has been shown that oxytocin has important cardiovascular protective effects. Here we investigated the effects of oxytocin on vascular reactivity and underlying the mechanisms in human umbilical vein endothelial cells (HUVECs) in vitro and in rat aorta ex vivo. Oxytocin increased phospho-eNOS (Ser 1177) and phospho-Akt (Ser 473) expression in HUVECs in vitro and the aorta of rat ex vivo. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited oxytocin-induced Akt and eNOS phosphorylation. In the rat aortic rings, oxytocin induced a biphasic vascular reactivity: oxytocin at low dose (10-9–10-8 M) initiated a vasorelaxation followed by a vasoconstriction at high dose (10-7 M). L-NAME (a nitric oxide synthase inhibitor), endothelium removal or wortmannin abolished oxytocin-induced vasorelaxation, and slightly enhanced oxytocin-induced vasoconstriction. Atosiban, an oxytocin/vasopressin 1a receptor inhibitor, totally blocked oxytocin-induced relaxation and vasoconstriction. PD98059 (ERK1/2 inhibitor) partially inhibited oxytocin-induced vasoconstriction. Oxytocin also increased aortic phospho-ERK1/2 expression, which was reduced by either atosiban or PD98059, suggesting that oxytocin-induced vasoconstriction was partially mediated by oxytocin/V1aR activation of ERK1/2. The present study demonstrates that oxytocin can activate different signaling pathways to cause vasorelaxation or vasoconstriction. Oxytocin stimulation of PI3K/eNOS-derived nitric oxide may participate in maintenance of cardiovascular homeostasis, and different vascular reactivities to low or high dose of oxytocin suggest that oxytocin may have different regulatory effects on vascular tone under physiological or pathophysiological conditions.
Keywords: Endothelial nitric oxide synthase, Oxytocin, Phosphatidylinositol 3-kinase, Vascular reactivity
Oxytocin is a hypothalamus neuropeptide, which has been shown to play an important role in the regulation of mammalian birth and lactation and a variety of social behaviors . Oxytocin is considered as a typical stress hormone that responds to various acute and chronic stress stimuli, and plays a significant role in a wide array of physiological activities . The cardiovascular system is one of oxytocin’s important targets [2,3]. Oxytocin and its receptors are expressed in the heart and its vascular cells . In the cultured brain microvascular endothelial cells (BMEC), oxytocin inhibits ox-LDL-induced monocyte adhesion to BMEC and the expression of adhesion molecules . Chronic administration of oxytocin reduces atherosclerotic plaque and inflammation in the aorta of ApoE-/- mouse . It has been proposed that the intrinsic oxytocin system may play a critical role in the maintenance of cardiovascular homeostasis [2,4,7].
The cardiovascular effects of oxytocin include natriuresis, negative inotropy, chronotropy, and the regulation of vascular tone or blood pressure . It has been reported that the systemic administration of oxytocin in rats significantly affects blood pressure, vascular tone and cardiovascular regulation [8,9]. Short term intravenous administration of oxytocin to women can enhance uterine contraction or decrease blood loss during labor or caesarean delivery . Oxytocin’s hypotensive response may be mediated by decreasing total vascular resistance .
Early studies suggest that oxytocin induces vasodilatory effects in the canine basilar artery which may be mediated through the endothelium and vasopressin 1 receptors [12,13]. Furthermore, Oyama
Six-week-old male Sprague–Dawley male rats were purchased from Liaoning Laboratory Animal Center and maintained under controlled conditions of light, temperature, and humidity. Animals were housed in animal facilities accredited by The Chinese Association for Accreditation of Laboratory Animal Care. All studies were approved by the Institutional Animal Care and Use Committee of Shenyang Medical University. After 2 weeks of accommodation to the new environment, the rats were sacrificed by overdose of sodium pentobarbital (100 mg/kg. i.p.). The aorta was removed and cleaned of adherent connective tissue. The thoracic aorta (from 0.5 cm below aortic arch) was cut into 3 mm ring in length and immediately placed into ice-cold modified Krebs-Ringer bicarbonate solution (composition in mM: NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, and glucose 11.1). In some rings, endothelium was mechanically removed by gentle rubbing of the intimate surface with a hair. The aortic rings were mounted horizontally between 2 steel wires in an organ bath (MO-720; DMT A/S, Aarhus, Denmark) with 5 ml of Krebs’ buffer bubbled with the mixed gas of 95% O2 and 5% CO2 at 37°C. One wire was connected to an isometric force transducer. The rings were equilibrated under a resting tension of 1 gr for 60 min and were exposed 2 times to KSS solution containing 60 mM KCl at 30 min intervals to induced contraction. In some experiments, the aortic tissues were preincubated with 100 nM wortmannin (a specific phosphatidylinositol 3-kinase [PI3K] inhibitor; Selleck, Houston, TX, USA), 100 nM atosiban (an oxytocin receptor antagonist), or 50 mM PD98059 (an inhibitor of ERK1/2 activation) for 30 min before oxytocin incubation. Aortic tissues used for Western blot were incubated with oxytocin (100 nM) for 15 min at 37°C, using cell culture medium bubbled with the mixed gas of 95% O2 and 5% CO2. The tissues were harvested and snap frozen in liquid nitrogen.
Oxytocin-induced vasorelaxation was examined by organ bath as previously described . Briefly, the aortic rings were precontracted with 70% of maximal norepinephrine-induced vasoconstriction (about 30 nM norepinephrine). A cumulative dose of oxytocin (10-11–10-7 M) was then added into an organ chamber. In some experiments, the aortic rings were incubated with the following chemicals for 30 min:100 μM NG-nitric-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor), 100 nM wortmannin, or 100 nM atosiban before norepinephrine and oxytocin were added into organ bath. Wortmannin and atosiban were dissolved in dimethylsulfoxide (DMSO). Final concentration of DMSO was less than 0.1%. The aortic rings in the control oxytocin group were preincubated with 0.1% DMSO for 30 min. Oxytocin-induced vasorelaxation was expressed as the percentage inhibition of 30 nM norepinephrine-induced vasoconstriction.
Oxytocin-induced vasoconstriction was examined in the endothelium-intact rings. A cumulative dose of oxytocin (10-11–10-7 M) was added in the unstimulated aortic rings at resting tension of 1 g. In some experiments, the aortic rings were preincubated with following reagents for 30 min, including 100 nM L-NAME, 100 nM atosiban, 50 μM PD98059, or 1 μM SQ29584 (a prostaglandin H2 [PGH2]/thromboxane A2 [TXB A2] receptor antagonist) to block PGH2/TXB A2, PGH2/TXB A2 is one of the most common endothelium-derived constricting factor. Oxytocin-induced vasoconstricting response was also examined in the aortic rings with endothelium removal. PD98059 or SQ29584 was dissolved in DMSO solvent, in these experiments, the aortic rings in control group were preincubated with 0.1% DMSO for 30 min. Oxytocin-induced vasoconstriction was expressed as percentage of 60 mM KCl-induced vasoconstriction.
HUVECs (ATCC TIB 202; ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified eagle medium (DMEM) medium supplemented with 10% fetal bovine serum. The cells were cultured at 37°C, 95% humidity and 5% CO2 and used between passages 4 and 16. The cells were seeded in six-well plates (106 cells/well) and starved in serum free DMEM medium for 24 h before the experiments were performed. The cells were incubated with oxytocin (10-10–10-7 M) for 1 to 15 min to determine a time course or dose-dependent peNOS or pAkt expressions. For all other experiments, the cell was treated with 10 nM oxytocin for 5 min. In some experiments, the cells were preincubated with 10 mM wortmannin or 100 nM atosiban for 30 min to block PI3K activity or oxytocin receptor. The cells in control group were incubated with 0.1% DMSO for 30 min.
The aortic tissues were homogenized with lysis buffer containing protease inhibitors including 10 μg/ml aprotinin, 10 μg/ml leupeptin and 1 mM phenylmethylsulphonyl fluoride (PMSF). The protein concentration was determined by the Bio-Rad protein assay. Thirty μg of proteins of the supernatants were loaded and separated by SDS-PAGE. The proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 5% (w/v) milk in the 0.1% Tween/Tris-Buffered Saline (TBS) buffer for 1 h at room temperature. The membrane was incubated with specific primary antibodies against eNOS (1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA), peNOS (Ser 1177, 1:1,000; Cell Signaling Technology), Akt (1:2,000; Cell Signaling Technology), pAkt (Tyr 473, 1:2,000; Cell Signaling Technology), ERK1/2 (1:1,000) and pERK1/2 (1:1,000; Cell Signaling Technology) at 4°C overnight. After washing, the membranes were incubated with the appropriate secondary antibody against the primary antibody for 1 h at room temperature. The signal for labeled proteins were detected by luminal chemiluminescence. Using the Bio-Rad system, the images were quantified by Image J software. β-actin (1:500; Santa Cruz Biotech, Santa Cruz, CA, USA) was used as an equal protein loading control. The data was normalized to β-actin or correspondence unphosphorylated proteins and expressed as fold increase versus control group.
Statistical analyses were performed by ANOVA with Bonferroni’s correction for multiple comparisons using GraphPad statistical software package (GraphPad Software Inc., San Diego, CA, USA).
All experimental results were expressed as mean ± SEM. Statistical analyses were performed by ANOVA with Bonferroni’s correction for multiple comparisons. Values were considered significant when p < 0.05.
To investigate the effect of oxytocin on eNOS/phospho-eNOS expression, HUVECs were incubated with oxytocin (10-10 to 10-7 M) for 1 to 15 min. As shown in Fig. 1, oxytocin (10 nM) significantly increased the expression of phospho-eNOS (Ser 1177) in a time-dependent manner, starting at the first minute, reaching a peak at 5 min and continuing a plateau at 15 min (Fig. 1A). Incubation of oxytocin for 5 min also dose-dependently increased phospho-eNOS expression with a maximal effect at 100 nM (Fig. 1C). However, oxytocin did not affect the protein expression of total eNOS (Fig. 1B, D). To investigate the signaling pathway that oxytocin promotes eNOS phosphorylation, we determined Akt and pAkt (Tyr473) expression in HUVECs
To investigate whether oxytocin induced eNOS phosphorylation
In the aortic rings precontracted with 30 nM norepinephrine, oxytocin induced a biphasic vascular response: vasorelaxation at low dose (10-9 to 10-8 M) of oxytocin with maximal relaxation of about 23% inhibition of norepinephrine-induced vasocontraction. However, oxytocin at a high dose (10-7 M) evoked a constriction response. Either L-NAME or endothelial removal abolished oxytocin-induced vasorelaxation and slightly increased oxytocin-induced vasoconstriction (Fig. 4A). Wortmannin also inhibited oxytocin-induced vasorelaxation and slightly enhanced oxytocin-induced vasoconstriction. Oxytocin receptor antagonist atosiban abolished oxytocin-induced vasorelaxation or vasoconstriction (Fig. 4). These results suggest that oxytocin induces vasorelaxation by stimulation of PI3K/eNOS pathway.
Oxytocin evoked a dose-dependent contraction response in the unstimulated aortic rings. Oxytocin-induced vasoconstriction started at 10 nM oxytocin and reached a maximal vasocontraction response at 100 nM, which is about 20% of 60 mM KCl-induced contraction. Both L-NAME and endothelium removal slightly but significantly enhanced the contraction response. PGH2/TXB A2 receptor antagonist SQ29584 did not affect the vasoconstriction. Oxytocin receptor antagonist atoshiban completely abolished the contraction, and ERK1/2 activation inhibitor PD98059 partially attenuated oxytocin-induced vasocontraction (Fig. 5A–E). Furthermore, we determined phospho-ERK1/2 in the aorta which was incubated with 100 nM oxytocin for 15 min
It has been shown that oxytocin has a great number of effects on the cardiovascular system. Major findings for the present study are: 1) in the isolated aortic rings, oxytocin induces biphasic effects on vascular reactivities, oxytocin in low dose (1–10 nM) induces vasorelaxation and high dose of oxytocin (100 nM) triggers vasoconstriction; 2) oxytocin induces vasorelaxation
It has been proposed that oxytocin may participate in the maintenance of normal homeostatic functions of cardiovascular system [1,16,17]. Both the heart and the vessels can synthesize oxytocin and express oxytocin receptor. Oxytocin may increase NO generation in cardiovascular system and has antioxidant effects to reduce reactive oxygen species synthesis [18,19]. Oxytocin may exert therapeutic effects on cardiovascular diseases, chronic administration of oxytocin reduces atherosclerotic lesion formation and vascular inflammation in Watanabe Heritable Hyperlipidemic Rabbits . Long-term treatment with oxytocin can protect against ischemia/reperfusion-induced cardiac injury
Vascular oxytocin may play an important role in the regulation of vascular tone and blood pressure. It has been reported that oxytocin can induce either vasorelaxation or vasoconstriction, depending on vascular beds and species [14,27]. Systemic administration of oxytocin has been reported to decrease blood pressure in hypertensive rats . In the present study, we demonstrate that oxytocin induces a biphasic vascular response in the rat aorta, vasorelaxation in low dose and vasoconstriction in high dose. Oxytocin induces vasorelaxation
In summary, in the present study, we demonstrate that oxytocin induces eNOS phosphorylation and vasorelaxation
This work was supported by the grants from the National Natural Science Foundation of China (Nos.81670384, 81970357) to MSZ, and the scientific research projects (20219027) of the college students to QX.
The authors declare no conflicts of interest.
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