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eNOS pathway
Biochemical and Biophysical Research Communications 518 (2019) 554e559
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Dopamine D4 receptor protected against hyperglycemia-induced endothelial dysfunction via PI3K /eNOS pathway He Wang a, b, 1, Yonggang Yao c, 1, Juncheng Liu b, 1, Yingjie Cao b, Chunying Si b, Rongfei Zheng b, Chunyu Zeng d, e, Huaimin Guan b, **, Ling Li a, * a
Department of Cardiology, The First Affiliated Hospital of Zhengzhou University, Henan, PR China Department of Cardiology, The First Affiliated Hospital of Henan University of Chinese Medicine, Henan, PR China Department of Critical Care Medicine, Chongqing Hospital of Traditional Chinese Medicine, Chongqing, PR China d Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing, PR China e Chongqing Institute of Cardiology, Chongqing Key Laboratory for Hypertension Research, Chongqing, PR China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 August 2019 Accepted 13 August 2019 Available online 22 August 2019
Hyperglycemia-induced endothelial dysfunction is generally believed to be the basis of diabetic vascular complications. Dopamine receptors is known to play an important protective role in diabetes. However, the protective effect of dopamine receptors against hyperglycemia-induced endothelial damage in diabetic rats is still unknown. In the present study, we established a cell model of hyperglycemia-induced endothelial dysfunction by treating human umbilical vein endothelial cells (HUVEC) with high glucose. MTT and lactate dehydrogenase assays results showed that high glucose treatment significantly reduced the cell viability and down-regulated dopamine D4 receptor. Pre-treatment with PD168077, a specific D4 receptor agonist, greatly improved endothelial cell viability and decreased apoptosis. Furthermore, pharmacological inhibition of phosphoinositide 3-kinase (PI3K) and endothelial nitric oxide synthase (eNOS) eliminated the protective effect of D4 receptor against endothelial injury. More importantly, the expression level of D4 receptor was also dramatically down-regulated in the arterial endothelium of rats with streptozotocin-(STZ)-induced diabetes, and the STZ-induced impairment of acetylcholine-induced vasodilation was reversed by activation of D4 receptor. In conclusion, our results indicated that dopamine D4 receptor protected against hyperglycemia-induced endothelial dysfunction via the PI3K/eNOS pathway, which may provide a novel strategy in the treatment of diabetes. © 2019 Elsevier Inc. All rights reserved.
Keywords: Dopamine D4 receptor Endothelial dysfunction HUVEC PI3K eNOS
1. Introduction Diabetes mellitus has become a serious threat to human health and causes the rapidly increasing cardiovascular events worldwide [1e3]. Cardiovascular complications are the leading causes of morbidity and mortality in diabetes mellitus patients [2]. In addition to being a physical barrier between the blood and tissues, the endothelium also serves other functions, including the regulation and maintenance of vascular tone, coagulation, fibrinolysis, cell
* Corresponding author. NO.1, Jianshe East Road, 2.7 District, Zhengzhou city, Henan Province, PR China. ** Corresponding author. No.19, Renmin Road, Jinshui District, Zhengzhou city, Henan Province, PR China. E-mail addresses: [email protected] (H. Guan), liling20181010@ 163.com (L. Li). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bbrc.2019.08.080 0006-291X/© 2019 Elsevier Inc. All rights reserved.
growth and platelet and leukocyte adherence [4,5]. Endothelial dysfunction is the earliest pathologic event and contributes significantly to the initiation and progression of diabetic vascular complications [6]. Therefore, improving endothelial function is an attractive therapeutic intervention in the prevention and treatment of diabetic vascular complications [7]. Dopamine, a neurotransmitter in the central nervous system, is also an important modulator of renal and adrenal function, sodium and fluid balance, and blood pressure [8]. Dopamine receptors are classified into two subfamilies depending on their ability to activate or inhibit adenylyl cyclase: the D1-like receptor subfamily includes D1 and D5 receptors, while D2, D3 and D4 receptors belong to the D2like receptor subfamily [9,10]. Kim MO et al. found that D1- like receptor mRNAs localized mainly in the smooth muscle cells and D2-like receptor mRNAs were mainly found in the arterial endothelium [11]. The D2-like receptors have been showed to play an important role in diabetes. Dopamine D2 receptor agonist, such as
H. Wang et al. / Biochemical and Biophysical Research Communications 518 (2019) 554e559
bromocriptine, could inhibit insulin secretion in isolated islets of rodents [12] and has recently been approved for the treatment of type 2 diabetes [13]. Mutant mice lacking the D2 receptor are glucose intolerant and have abnormal insulin secretion [14]. In humans, administration of neuroleptic drugs, which block dopamine receptors, can cause hyperinsulinemia, increase weight gain and glucose intolerance [15]. Our previous work found that D4 receptor agonists reduced the vascular smooth muscle cell proliferation and migration by down-regulating insulin receptors [16]. However, although expressed on endothelial cells [17], the effect of D4 receptor on endothelial dysfunction in diabetes is still unknown. In the present study, we found that dopamine D4 receptor was down-regulated on a cell model of hyperglycemia-induced endothelial injury and on the arterial endothelium of STZ-induced diabetic rats. However, whether this down-regulation of D4 receptor expression is of pathological importance still needs to be elucidated. Therefore, the purpose of this study is to clarify the role of the D4 receptor on hyperglycemia-induced endothelial dysfunction, and its underlying mechanisms.
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assay kit (Zhongshan Company, Beijing, China). 2.6. Detection of LDH releasing in HUVEC Lactate dehydrogenase (LDH) concentration of HUVEC was determined using the LDH releasing assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. The level of LDH was expressed as micromoles per liter supernatant. The absorbance of LDH was detected at wavelength of 490 nm. 2.7. Western blot The total proteins were extracted from HUVEC or aortic endothelium of rats using RIPA lysis buffer at 4 C. Protein concentration was determined using a bicinchoninic acid (BCA) protein Assay. Primary antibody: D4 receptor (1:500, Abcam), eNOS (1:800, Cell Signaling Technology, CST), phosphor (p)-eNOS (1:800, CST), protein kinase B (Akt; 1:1000, CST), phosphorylated-Akt (1:1000, CST), casepase-3 (1:800, CST) and b-actin (1:10000, CST).
2. Material and methods 2.8. Apoptosis in HUVECs 2.1. Animal All the procedures on rats were conformed to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publication No.85-23), and was approved at Daping Hospital. 30 healthy male Sprague-Dawley (SD) were randomly divided into three groups (10 animals/group): control group, STZ group and STZ þ PD168077 group. See supplemental material. 2.2. Blood sample and aorta preparation SD rats were fasted for 12 h before surgery and were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg). Blood samples were collected from the heart in ethylene diamine tetra acetic acid (EDTA)-coated tubes for measurement of plasma glucose and insulin at the end of the experiment. In the endothelium-denuded aorta group, the endothelial layer was removed by abrading the lumen using a rough-surfaced needle. The dissected endothelium was collected and used for immunoblotting.
A fluorescein in situ cell death detection kit was used according to the manufacturer's instructions for terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay (Roche Applied Science, Mannheim, Germany). 2.9. Evaluation of intracellular NO levels in HUVEC with DAF-2DA The concentration of NO in cells was measured using a DAF-2 DA fluorescence assay. Fluorescence was measured with the excitation wavelength set at 495 nm and the emission wave length at 515 nm, using fluorescence microscopy (Olympus America, Inc., Melville, NY). 2.10. Evaluation of vasorelaxation The third-order branches of the mesenteric arteries were dissected and cut in segments of 2 mm in length and mounted on 40 mm stainless-steel wires in an isometric Mulvany-Halpern smallvessel myograph (model 91 M610; J.P. Trading, Aarhus, Denmark). See supplemental material.
2.3. Blood glucose and plasma insulin concentration analysis 2.11. Statistical analysis Blood (5 mL) was centrifugated at 1800g for 10min and the serum was extracted and collected. Blood glucose values were determined by using the Accu-Chek Advantage glucose monitoring system (Roche Diagnostics GmbH, Sandhofer, Germany). Plasma insulin levels were measured by a rat insulin 96 well plate assay from Millipore Corporation (Billerica, MA). 2.4. Cell culture of human umbilical vein endothelial cell Human umbilical vein endothelial cell (HUVEC) line was purchased from ATCC Company (Manassas, VA, USA). HUVEC were cultured in DMEM or DMEM high glucose medium (Gibco) supplemented with 10% fetal bovine serum, in a humidified incubator at 37 C with 95% air/5% CO2. The cells were serum-starved overnight before the experiment. 2.5. MTT assay of cell viability HUVEC viability was measured by 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2-H-tetrazolium bromide (MTT) cell proliferation
Data are expressed as mean ± SEM. Relaxation in each arterial segment is expressed as the percentage of the contraction induced by PHE (10 mmol/L). Comparison within groups was made by repeated-measures ANOVA (or paired t-test when only 2 groups were compared), and comparison among groups was made by factorial ANOVA with the Holm-Sidak ad hoc test (or t-test when only 2 groups were compared). A value of P < 0.05 was considered significant. 3. Results 3.1. Hyperglycemia-induced endothelial injury and decreased expression of dopamine D4 receptor in HUVEC To establish a cell model of hyperglycemia-induced endothelial injury, HUVEC was treated with different concentration of Dglucose (10, 25, 30 mM) in basal HUVEC media with 5% fetal calf serum for 24 h. As showed in Fig. 1A, the cell viability determined by MTT was significantly decreased in a does-dependent manner
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L745870. As shown in Supplemental Fig. 1B and 1C, the protective effects of PD168077 were completely eliminated by L745870 treatment. 3.3. Activation of the D4 receptor protected against high-glucoseinduced cell apoptosis To determine whether the protective effect of D4 receptor on high-glucose-induced cytotoxicity is via the effect on cell apoptosis, TUNEL staining was performed to measure the rate of cell apoptosis. As shown in Fig. 2AeB, incubation with high glucose induced a significant increase in the number of TUNEL-positive cells. However, the number of apoptotic cells was greatly decreased by pre-treatment with PD168077 (107M,48 h). Here, the activity of pro-apoptotic caspase-3 was also measured using a caspase 3 colorimetric assay Kit. We found that the caspase 3 activity was significantly increased by high glucose treatment, but was inhibited by PD168077 treatment (Fig. 2C). Consistent with this result, high-glucose-induced up-regulation of cleaved caspase 3 was prevented by PD168077 treatment (Fig. 2D and E). The specificity of PD168077 was also confirmed by L745870 treatment, in
Fig. 1. Activation of D4 receptor protected against high-glucose-induced injury on endothelial cells. (A) and (B) Cell viability and LDH release were determined by MTT and LDH release assays. (C) and (D) D4 receptor expression level in HUVEC was estimated by Western blot. (E) The specificity of D-glucose (30 mM) on D4 receptor expression in endothelial cells. (F) MTT assay showing activation of D4 receptor by PD168077 increased the cell viability. *P < 0.05 vs. Control, #P < 0.05 vs. HG groups. HG: high glucose. PD: PD168077. L: L745870.
after D-glucose treatment, consistent with the increased level of LDH levels (Fig. 1B), suggesting a gyperglycemia-induced endothelial injury in HUVEC. Moreover, we found that D-glucose treatment dramatically down-regulated D4 receptor expression in timedependent and does-dependent manners (Fig. 1CeD). However, 30 mM L-glucose and 30 mM mannitol both can not alter the expression level of D4 receptor (Fig. 1E), showing the specificity of D-glucose on D4 receptor expression. 3.2. Activation of D4 receptor protected against high-glucoseinduced injury on endothelial cells To investigate the effect of D4 receptor on high-glucose-induced endothelial cells injury, we treated the HUVEC with a selective dopamine D4 receptor agonist, PD168077, for 48 h after 24 h high glucose treatment. MTT assay showed that in the presence of PD168077 (108-106 M), high-glucose-induced endothelial cells injury was partly prevented (Fig. 1F). Similar result was observed in LDH release assay (Supplemental figure 1A), indicating that activation of D4 receptor protected HUVEC against high-glucoseinduced injury. To rule out the side effect of PD168077, we treated HUVEC simultaneously with D4 receptor antagonist,
Fig. 2. Activation of the D4 receptor protected against high-glucose-induced cell apoptosis. (A) Apoptotic cells were evaluated by TUNEL-staining. Representative TUNEL staining images ( 200). DAPI: blue. TUNEL staining: green. (B) The percentage of apoptotic cells (C) Caspase-3 enzyme activity (D) and (E) Protein levels of cleaved caspase-3 (17kd and 19kd) were assessed by Western blot. *P < 0.05 vs. Control, # P < 0.05 vs. HG group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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which pretreatment with L745870 (107M) abrogated the antiapoptotic effect of PD168077 in high-glucose-treated endothelial cells (Fig. 2BeE).
3.4. PI3K/Akt/eNOS/NO pathway involved in the protective effects of D4 receptor against high-glucose-induced endothelial cells injury To explore whether PI3K/Akt/eNOS pathway involve in the protective effects of D4 receptor, we first detected the phosphorylation of Akt and eNOS (Ser1177) after activating D4 receptor in high-glucose-treated HUVEC. We found that treatment with 107M PD168077 dramatically increased the phosphorylation of Akt and eNOS (Fig. 3A and B), indicating activation of D4 receptor by PD168077 activated the downstream PI3K/Akt/eNOS pathway. To confirm the involvement of PI3K/Akt/eNOS pathway in the protective effects of D4 receptor, Akt inhibitor, LY294002, and eNOS inhibitor, L-NAME, was pre-treated with HUVEC prior to PD168077 treatment. MTT and LDH assays showed that the protective effect of D4 receptor on high-glucose-induced endothelial cell injury was eliminated by 105M LY294002 or 104M L-NAME pre-treatment (Fig. 3C and D). Moreover, the effect of D4 receptor activation on
Fig. 3. Role of PI3K/Akt/eNOS/NO signal pathway in the protective effect of D4 receptor on HUVEC after high-glucose-induced injury. (A) and (B) Western blot results showing Akt and p-Akt, eNOS and p-eNOS expression levels. *P < 0.05 vs. HG, #P < 0.05 vs. HG þ PD group. (C) and (D) MTT assay and LDH release assay. *P < 0.05 vs. Control, # P < 0.05 vs. HG, & P < 0.05 vs. HG þ PD group. (E) Intracellular NO production was measured by using DAF-2DA. Representative images of NO detection ( 10). (F) The summary of fluorescence intensity data. *P < 0.05 vs. HG groups, #P < 0.05 vs. HG þ PD (n ¼ 6). HG: high glucose. PD: PD168077. L: L745870. *P < 0.05 vs. HG groups, #P < 0.05 vs. HG þ PD.
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NO production was also determined by DAF-2DA fluorescence. As shown in Fig. 4EeF, PD168977 treatment greatly elevated the NO production. However, the NO production was completely returned to control level by Akt inhibitor, LY294002, indicating that activation of D4 receptor induces NO production via PI3K/Akt/eNOS pathway in endothelial cells.
3.5. Down-regulation of D4 receptor in arterial endothelium of STZinduced diabetic rats To investigate the protective effects of dopamine D4 receptor against hyperglycemia-induced endothelial dysfunction in vivo, we established a diabetic rat model by injecting intraperitoneally with STZ. Three days after 60 mg/kg STZ injection [18], these rats showed a significant increase in their plasma glucose (Fig. 4A), accompanied with decreased plasma insulin (Fig. 4B), indicating the successful establishment of a type I diabetic rat model. To evaluate the expression level of D4 receptor in the arterial endothelium, endothelia was denuded from the thoracic aorta. Western bolt result showed that the protein level of D4 receptor was significantly down-regulated in the arterial endothelium of STZ-induced diabetic rats (Fig. 4C), consistent with the above cell model of hyperglycemia-induced endothelial injury.
Fig. 4. Activation of D4 receptor improved endothelium-dependent vasodilation of mesenteric arteries in STZ-induced diabetic rats. (A) and (B). The concentrations of blood glucose (A) and plasma insulin (B) in STZ-induced diabetic rats. (C) The protein expression of D4 receptors in the aortic endothelium of STZ-induced diabetic and control rats. (D) and (E) The vascular endothelium cumulative concentration-response curves for Ach (A) or SNP (B) in aortic rings. Relaxation of aortic rings is expressed as a percentage of contraction induced by PHE (10-5 M). *P < 0.05 vs. control rats, #P < 0.05 vs. STZ.
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3.6. Activation of D4 receptor improved endothelium-dependent vasodilation of mesenteric arteries in STZ-induced diabetic rats To determine the effect of the D4 receptor on endothelial dysfunction in STZ-induced diabetic rats, PD168077 (6 mg/kg/day) were intraperitoneally injected for 2 weeks. Compared to control, the weight in STZ-induced diabetic rats was greatly reduced, accompanied with significantly increased plasma glucose level and decreased insulin level. However, treatment with PD168077 in the STZ-induced diabetic rats did not improve the body weight, plasma glucose and insulin levels (Table 1). Consistent with other reports [19], the acetylcholine (Ach, 109105M)-induced vasodilation was impaired in the mesenteric arteries of STZ-induced diabetic rats. This impairment was partially reversed by PD168077 treatment in the STZ-induced diabetic rats (Fig. 4D). However, sodium nitroprusside (SNP)-induced vasodilation did not change in STZ-induced diabetic rats with or without PD168077 treatment (Fig. 4E), indicating that activation of D4 receptor improved endothelium-dependent vasodilation of mesenteric arteries in STZ-induced diabetic rats. 4. Discussion Endothelial dysfunction is the earliest pathologic event and contributes significantly to the initiation and progression of diabetic vascular complications [20,21]. Glucose intolerance and hyperglycemia are the main metabolic disorders found in diabetes mellitus and are important risk factors for cardiovascular diseases [22,23]. Reducing the injury of glucose on endothelial cells may provide new insights in the prevention of diabetic vascular complications. The salient feature in the present study is that the dopamine D4 receptor agonist was proven to be a remarkable endothelial protective agent in the diabetic animal model. Accumulating evidence indicate an interaction between glucose and dopamine receptors in various tissues and cells [12,14,17,24,25]. The renal dopaminergic system is impaired in hyperglycemic rats and type-2 diabetes patients [26,27]. In pancreatic beta cells, the D2-like receptor agonist, quinpirole, inhibits glucose-stimulated insulin secretion [28]. Lawford et al. have found that the D2 receptor gene is associated with raised blood glucose in cases of schizophrenia [29]. KumarV et al. have found that the D2 receptor agonist, bromocriptine, could ameliorate hyperglycaemia [30]. Our previous studies have found that hyperglycemia decreases arterial D1 receptor expression and increases D1 receptor phosphorylation in obese Zucker rats [31], thereby causing D1 receptor dysfunction. Zarei et al. found that endothelial cells only express dopamine D2like receptors, and dopamine modulates von Willebrand factor secretion in endothelial cells [17]. Our present study showed that the expression of the D4 receptor was reduced in the endothelium of rats with STZ-induced diabetes and high-glucose-treated HUVEC. The decreases D4 receptor expression is due to high Dglucose, but not the hyperosmolarity, since L-glucose and mannitol both did not change the expression level of D4 receptor. High glucose, apoptosis, and endothelial dysfunction were reported to play an important role in the pathophysiology of diabetic disease. Accelerated endothelial cell apoptosis induced by
Table 1 The basal index of rats (mean ± SEM, n ¼ 6).
Body weight(g) Blood glucose(mmol/L) Insulin(ng/mL) *P < 0.05 vs. Control.
Control
STZ
PD þ STZ
284 ± 9.0 5.50 ± 0.3 0.799 ± 0.056
235 ± 6.0* 26.20 ± 0.8* 0.421 ± 0.086*
248 ± 7.0* 24.8 ± 0.7* 0.478 ± 0.032*
hyperglycemia is an important event in the process of diabetesassociated vascular complications [32e34]. It is well established that endothelial cells cultured under the high glucose environment led to a significant increase in apoptosis [35,36]. Similar to those studies, our results showed that high glucose induced a significant decrease in cell viability and a significant increase in the number of apoptotic cells. Interestingly, we found that stimulation of the D4 receptor by PD168077 could alleviate high-glucose-induced endothelial cell injury by promoting endothelial cell survival and inhibiting cell apoptosis. Endothelial cells regulate vascular homeostasis by generating paracrine factors that regulate vascular tone, inhibit platelet function, prevent adhesion of leukocytes, and limit proliferation of vascular smooth muscle [38]. The most important weapon of endothelial cells to fight vascular diseases is eNOS, an enzyme that generates the vasoprotective molecule NO [39]. Endothelial dysfunction characterized by inactivation or reduced synthesis of NO, alone or in combination, is implicated in the effect of diabetes induced cardiovascular injury [40]. Evidence has shown that the PI3K/Akt/eNOS pathway plays an important role in preventing high glucose-induced endothelial cell injury [41]. Previous study has also shown that D4 dopamine receptor agonist, PD168077, activates Akt in time-an dose-dependent manner in D4MN9D cells [19]. Similarly, our study showed that activation of the D4 receptor by PD168077 could increase PI3K, Akt and eNOS phosphorylation and NO production. Moreover, the protective effect of PD168077 on high-glucose-induced endothelial cell injury was blocked by the PI3K inhibitor LY294002 and eNOS inhibitor L-NAME, indicating that PI3K/Akt-dependent eNOS pathway is involved in the D4-receptor-mediated protective effect on endothelial cells. Based on our findings, we concluded that dopamine D4 receptor has protective effects on endothelial cells of STZ-induced diabetic rats, partially by reducing apoptosis via the PI3K/Akt/eNOS/NO pathway. The protective effect is of pathological significance, as pretreatment with activators of the D4 receptor could reverse the impaired acetylcholine-induced vasodilation, which may lead to a novel strategy in the treatment of diabetic vascular complications. Declaration of interest No conflicts of interest, financial or otherwise, are declared by the authors. Acknowledgment These studies were supported in part by grants from the National Natural Science Foundation of China (81603432,81603431). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.08.080. References [1] S.D. de Ferranti, I.H. de Boer, V. Fonseca, C.S. Fox, S.H. Golden, C.J. Lavie, S.N. Magge, N. Marx, D.K. McGuire, T.J. Orchard, B. Zinman, R.H. Eckel, Type 1 diabetes mellitus and cardiovascular disease: a scientific statement from the American Heart Association and American Diabetes Association, Circulation 130 (2014) 1110e1130. [2] B.M. Leon, T.M. Maddox, Diabetes and cardiovascular disease: epidemiology, biological mechanisms, treatment recommendations and future research, World J. Diabetes 6 (2015) 1246e1258. [3] A.M. Schmidt, Diabetes mellitus and cardiovascular disease, Arterioscler. Thromb. Vasc. Biol. 39 (2019) 558e568. [4] P.E. Szmitko, C.H. Wang, R.D. Weisel, J.R. de Almeida, T.J. Anderson, S. Verma, New markers of inflammation and endothelial cell activation: Part I, Circulation 108 (2003) 1917e1923.
H. Wang et al. / Biochemical and Biophysical Research Communications 518 (2019) 554e559 [5] P.E. Szmitko, C.H. Wang, R.D. Weisel, G.A. Jeffries, T.J. Anderson, S. Verma, Biomarkers of vascular disease linking inflammation to endothelial activation: Part II, Circulation 108 (2003) 2041e2048. [6] M. Brownlee, Biochemistry and molecular cell biology of diabetic complications, Nature 414 (2001) 813e820. [7] M.A. Potenza, S. Gagliardi, C. Nacci, M.R. Carratu, M. Montagnani, Endothelial dysfunction in diabetes: from mechanisms to therapeutic targets, Curr. Med. Chem. 16 (2009) 94e112. [8] I. Armando, V.A. Villar, P.A. Jose, Dopamine and renal function and blood pressure regulation, Comp. Physiol. 1 (2011) 1075e1117. [9] A.A. Banday, M.F. Lokhandwala, Dopamine receptors and hypertension, Curr. Hypertens. Rep. 10 (2008) 268e275. [10] J.M. Beaulieu, R.R. Gainetdinov, The physiology, signaling, and pharmacology of dopamine receptors, Pharmacol. Rev. 63 (2011) 182e217. [11] M.O. Kim, P.O. Koh, J.H. Kim, J.S. Kim, S.S. Kang, G.J. Cho, K. Kim, W.S. Choi, Localization of dopamine D1 and D2 receptor mRNAs in the rat systemic and pulmonary vasculatures, Mol. Cells 9 (1999) 417e421. [12] J.E. de Leeuw van Weenen, E.T. Parlevliet, P. Maechler, L.M. Havekes, J.A. Romijn, D.M. Ouwens, H. Pijl, B. Guigas, The dopamine receptor D2 agonist bromocriptine inhibits glucose-stimulated insulin secretion by direct activation of the alpha2-adrenergic receptors in beta cells, Biochem. Pharmacol. 79 (2010) 1827e1836. [13] R.A. Defronzo, Bromocriptine: a sympatholytic, d2-dopamine agonist for the treatment of type 2 diabetes, Diabetes Care 34 (2011) 789e794. [14] I. Garcia-Tornadu, A.M. Ornstein, A. Chamson-Reig, M.B. Wheeler, D.J. Hill, E. Arany, M. Rubinstein, D. Becu-Villalobos, Disruption of the dopamine d2 receptor impairs insulin secretion and causes glucose intolerance, Endocrinology 151 (2010) 1441e1450. [15] F. Lopez Vicchi, G.M. Luque, B. Brie, J.P. Nogueira, I. Garcia Tornadu, D. BecuVillalobos, Dopaminergic drugs in type 2 diabetes and glucose homeostasis, Pharmacol. Res. 109 (2016) 74e80. [16] C. Yu, Z. Wang, Y. Han, Y. Liu, W.E. Wang, C. Chen, H. Wang, P.A. Jose, C. Zeng, Dopamine D(4) receptors inhibit proliferation and migration of vascular smooth muscle cells induced by insulin via down-regulation of insulin receptor expression, Cardiovasc. Diabetol. 13 (2014) 97. [17] S. Zarei, M. Frieden, B. Rubi, P. Villemin, B.R. Gauthier, P. Maechler, U.M. Vischer, Dopamine modulates von Willebrand factor secretion in endothelial cells via D2-D4 receptors, J. Thromb. Haemost. 4 (2006) 1588e1595. [18] K. Ishige, Q. Chen, Y. Sagara, D. Schubert, The activation of dopamine D4 receptors inhibits oxidative stress-induced nerve cell death, J. Neurosci. 21 (2001) 6069e6076. [19] X. Zhen, J. Zhang, G.P. Johnson, E. Friedman, D(4) dopamine receptor differentially regulates Akt/nuclear factor-kappa b and extracellular signalregulated kinase pathways in D(4)MN9D cells, Mol. Pharmacol. 60 (2001) 857e864. [20] V. Altabas, Diabetes, endothelial dysfunction, and vascular repair: what should a diabetologist keep his eye on? Internet J. Endocrinol. 2015 (2015) 848272. [21] C.G. Schalkwijk, C.D. Stehouwer, Vascular complications in diabetes mellitus: the role of endothelial dysfunction, Clin. Sci. (Lond.) 109 (2005) 143e159. [22] M.I. Uusitupa, Early lifestyle intervention in patients with non-insulindependent diabetes mellitus and impaired glucose tolerance, Ann. Med. 28 (1996) 445e449. [23] P.Y. Zhang, Cardiovascular disease in diabetes, Eur. Rev. Med. Pharmacol. Sci. 18 (2014) 2205e2214. [24] C. Gragnoli, G.M. Reeves, J. Reazer, T.T. Postolache, Dopamine-prolactin pathway potentially contributes to the schizophrenia and type 2 diabetes comorbidity, Transl. Psychiatry 6 (2016) e785. [25] G.M. Luque, F. Lopez-Vicchi, A.M. Ornstein, B. Brie, C. De Winne, E. Fiore,
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[38]
[39] [40]
[41]
559
M.I. Perez-Millan, G. Mazzolini, M. Rubinstein, D. Becu-Villalobos, Chronic hyperprolactinemia evoked by disruption of lactotrope dopamine D2 receptors impacts on liver and adipocyte genes related to glucose and insulin balance, Am. J. Physiol. Endocrinol. Metab. 311 (2016) E974eE988. F. Contreras, C. Foullioux, B. Pacheco, C. Maroun, H. Bolivar, M. Lares, E. Leal, R. Cano, V. Bermudez, M. Velasco, Effect of drugs interacting with the dopaminergic receptors on glucose levels and insulin release in healthy and type 2 diabetic subjects, Am. J. Therapeut. 15 (2008) 397e402. A. Marwaha, M.F. Lokhandwala, Tempol reduces oxidative stress and restores renal dopamine D1-like receptor- G protein coupling and function in hyperglycemic rats, Am. J. Physiol. Renal. Physiol. 291 (2006) F58eF66. B. Rubi, S. Ljubicic, S. Pournourmohammadi, S. Carobbio, M. Armanet, C. Bartley, P. Maechler, Dopamine D2-like receptors are expressed in pancreatic beta cells and mediate inhibition of insulin secretion, J. Biol. Chem. 280 (2005) 36824e36832. B.R. Lawford, M. Barnes, C.P. Morris, E.P. Noble, P. Nyst, K. Heslop, R.M. Young, J. Voisey, J.P. Connor, Dopamine 2 receptor genes are associated with raised blood glucose in schizophrenia, Can. J. Psychiatr. 61 (2016) 291e297. V.S.H. Kumar, B.V. M, N.P. A, S. Aithal, S.R. Baleed, U.N. Patil, Bromocriptine, a dopamine (d2) receptor agonist, used alone and in combination with glipizide in sub-therapeutic doses to ameliorate hyperglycaemia, J. Clin. Diagn. Res. 7 (2013) 1904e1907. J. Fu, Y. Han, H. Wang, Z. Wang, Y. Liu, X. Chen, Y. Cai, W. Guan, D. Yang, L.D. Asico, L. Zhou, P.A. Jose, C. Zeng, Impaired dopamine D1 receptormediated vasorelaxation of mesenteric arteries in obese Zucker rats, Cardiovasc. Diabetol. 13 (2014) 50. D. Detaille, B. Guigas, C. Chauvin, C. Batandier, E. Fontaine, N. Wiernsperger, X. Leverve, Metformin prevents high-glucose-induced endothelial cell death through a mitochondrial permeability transition-dependent process, Diabetes 54 (2005) 2179e2187. R. Gomez, M. Gonzalez-Izquierdo, R.C. Zimmermann, E. Novella-Maestre, I. Alonso-Muriel, J. Sanchez-Criado, J. Remohi, C. Simon, A. Pellicer, Low-dose dopamine agonist administration blocks vascular endothelial growth factor (VEGF)-mediated vascular hyperpermeability without altering VEGF receptor 2-dependent luteal angiogenesis in a rat ovarian hyperstimulation model, Endocrinology 147 (2006) 5400e5411. G.J. Pyne-Geithman, D.N. Caudell, M. Cooper, J.F. Clark, L.A. Shutter, Dopamine D2-receptor-mediated increase in vascular and endothelial NOS activity ameliorates cerebral vasospasm after subarachnoid hemorrhage in vitro, Neurocritical Care 10 (2009) 225e231. G.N. Shah, T.O. Price, W.A. Banks, Y. Morofuji, A. Kovac, N. Ercal, C.M. Sorenson, E.S. Shin, N. Sheibani, Pharmacological inhibition of mitochondrial carbonic anhydrases protects mouse cerebral pericytes from high glucose-induced oxidative stress and apoptosis, J. Pharmacol. Exp. Ther. 344 (2013) 637e645. M. Silambarasan, J.R. Tan, D.S. Karolina, A. Armugam, C. Kaur, K. Jeyaseelan, MicroRNAs in hyperglycemia induced endothelial cell dysfunction, Int. J. Mol. Sci. 17 (2016) 518. A. Sandoo, J.J. van Zanten, G.S. Metsios, D. Carroll, G.D. Kitas, The endothelium and its role in regulating vascular tone, Open Cardiovasc. Med. J. 4 (2010) 302e312. U. Forstermann, T. Munzel, Endothelial nitric oxide synthase in vascular disease: from marvel to menace, Circulation 113 (2006) 1708e1714. X. Shu, T.C.t. Keller, D. Begandt, J.T. Butcher, L. Biwer, A.S. Keller, L. Columbus, B.E. Isakson, Endothelial nitric oxide synthase in the microcirculation, Cell. Mol. Life Sci. 72 (2015) 4561e4575. D.A. Yuen, B.E. Stead, Y. Zhang, K.E. White, M.G. Kabir, K. Thai, S.L. Advani, K.A. Connelly, T. Takano, L. Zhu, A.J. Cox, D.J. Kelly, I.W. Gibson, T. Takahashi, R.C. Harris, A. Advani, eNOS deficiency predisposes podocytes to injury in diabetes, J. Am. Soc. Nephrol. 23 (2012) 1810e1823.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Dopamine D4 receptor protected against hyperglycemia-induced endothelial dysfunction via PI3K /eNOS pathway He Wang a, b, 1, Yonggang Yao c, 1, Juncheng Liu b, 1, Yingjie Cao b, Chunying Si b, Rongfei Zheng b, Chunyu Zeng d, e, Huaimin Guan b, **, Ling Li a, * a
Department of Cardiology, The First Affiliated Hospital of Zhengzhou University, Henan, PR China Department of Cardiology, The First Affiliated Hospital of Henan University of Chinese Medicine, Henan, PR China Department of Critical Care Medicine, Chongqing Hospital of Traditional Chinese Medicine, Chongqing, PR China d Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing, PR China e Chongqing Institute of Cardiology, Chongqing Key Laboratory for Hypertension Research, Chongqing, PR China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 August 2019 Accepted 13 August 2019 Available online 22 August 2019
Hyperglycemia-induced endothelial dysfunction is generally believed to be the basis of diabetic vascular complications. Dopamine receptors is known to play an important protective role in diabetes. However, the protective effect of dopamine receptors against hyperglycemia-induced endothelial damage in diabetic rats is still unknown. In the present study, we established a cell model of hyperglycemia-induced endothelial dysfunction by treating human umbilical vein endothelial cells (HUVEC) with high glucose. MTT and lactate dehydrogenase assays results showed that high glucose treatment significantly reduced the cell viability and down-regulated dopamine D4 receptor. Pre-treatment with PD168077, a specific D4 receptor agonist, greatly improved endothelial cell viability and decreased apoptosis. Furthermore, pharmacological inhibition of phosphoinositide 3-kinase (PI3K) and endothelial nitric oxide synthase (eNOS) eliminated the protective effect of D4 receptor against endothelial injury. More importantly, the expression level of D4 receptor was also dramatically down-regulated in the arterial endothelium of rats with streptozotocin-(STZ)-induced diabetes, and the STZ-induced impairment of acetylcholine-induced vasodilation was reversed by activation of D4 receptor. In conclusion, our results indicated that dopamine D4 receptor protected against hyperglycemia-induced endothelial dysfunction via the PI3K/eNOS pathway, which may provide a novel strategy in the treatment of diabetes. © 2019 Elsevier Inc. All rights reserved.
Keywords: Dopamine D4 receptor Endothelial dysfunction HUVEC PI3K eNOS
1. Introduction Diabetes mellitus has become a serious threat to human health and causes the rapidly increasing cardiovascular events worldwide [1e3]. Cardiovascular complications are the leading causes of morbidity and mortality in diabetes mellitus patients [2]. In addition to being a physical barrier between the blood and tissues, the endothelium also serves other functions, including the regulation and maintenance of vascular tone, coagulation, fibrinolysis, cell
* Corresponding author. NO.1, Jianshe East Road, 2.7 District, Zhengzhou city, Henan Province, PR China. ** Corresponding author. No.19, Renmin Road, Jinshui District, Zhengzhou city, Henan Province, PR China. E-mail addresses: [email protected] (H. Guan), liling20181010@ 163.com (L. Li). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bbrc.2019.08.080 0006-291X/© 2019 Elsevier Inc. All rights reserved.
growth and platelet and leukocyte adherence [4,5]. Endothelial dysfunction is the earliest pathologic event and contributes significantly to the initiation and progression of diabetic vascular complications [6]. Therefore, improving endothelial function is an attractive therapeutic intervention in the prevention and treatment of diabetic vascular complications [7]. Dopamine, a neurotransmitter in the central nervous system, is also an important modulator of renal and adrenal function, sodium and fluid balance, and blood pressure [8]. Dopamine receptors are classified into two subfamilies depending on their ability to activate or inhibit adenylyl cyclase: the D1-like receptor subfamily includes D1 and D5 receptors, while D2, D3 and D4 receptors belong to the D2like receptor subfamily [9,10]. Kim MO et al. found that D1- like receptor mRNAs localized mainly in the smooth muscle cells and D2-like receptor mRNAs were mainly found in the arterial endothelium [11]. The D2-like receptors have been showed to play an important role in diabetes. Dopamine D2 receptor agonist, such as
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bromocriptine, could inhibit insulin secretion in isolated islets of rodents [12] and has recently been approved for the treatment of type 2 diabetes [13]. Mutant mice lacking the D2 receptor are glucose intolerant and have abnormal insulin secretion [14]. In humans, administration of neuroleptic drugs, which block dopamine receptors, can cause hyperinsulinemia, increase weight gain and glucose intolerance [15]. Our previous work found that D4 receptor agonists reduced the vascular smooth muscle cell proliferation and migration by down-regulating insulin receptors [16]. However, although expressed on endothelial cells [17], the effect of D4 receptor on endothelial dysfunction in diabetes is still unknown. In the present study, we found that dopamine D4 receptor was down-regulated on a cell model of hyperglycemia-induced endothelial injury and on the arterial endothelium of STZ-induced diabetic rats. However, whether this down-regulation of D4 receptor expression is of pathological importance still needs to be elucidated. Therefore, the purpose of this study is to clarify the role of the D4 receptor on hyperglycemia-induced endothelial dysfunction, and its underlying mechanisms.
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assay kit (Zhongshan Company, Beijing, China). 2.6. Detection of LDH releasing in HUVEC Lactate dehydrogenase (LDH) concentration of HUVEC was determined using the LDH releasing assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. The level of LDH was expressed as micromoles per liter supernatant. The absorbance of LDH was detected at wavelength of 490 nm. 2.7. Western blot The total proteins were extracted from HUVEC or aortic endothelium of rats using RIPA lysis buffer at 4 C. Protein concentration was determined using a bicinchoninic acid (BCA) protein Assay. Primary antibody: D4 receptor (1:500, Abcam), eNOS (1:800, Cell Signaling Technology, CST), phosphor (p)-eNOS (1:800, CST), protein kinase B (Akt; 1:1000, CST), phosphorylated-Akt (1:1000, CST), casepase-3 (1:800, CST) and b-actin (1:10000, CST).
2. Material and methods 2.8. Apoptosis in HUVECs 2.1. Animal All the procedures on rats were conformed to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publication No.85-23), and was approved at Daping Hospital. 30 healthy male Sprague-Dawley (SD) were randomly divided into three groups (10 animals/group): control group, STZ group and STZ þ PD168077 group. See supplemental material. 2.2. Blood sample and aorta preparation SD rats were fasted for 12 h before surgery and were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg). Blood samples were collected from the heart in ethylene diamine tetra acetic acid (EDTA)-coated tubes for measurement of plasma glucose and insulin at the end of the experiment. In the endothelium-denuded aorta group, the endothelial layer was removed by abrading the lumen using a rough-surfaced needle. The dissected endothelium was collected and used for immunoblotting.
A fluorescein in situ cell death detection kit was used according to the manufacturer's instructions for terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay (Roche Applied Science, Mannheim, Germany). 2.9. Evaluation of intracellular NO levels in HUVEC with DAF-2DA The concentration of NO in cells was measured using a DAF-2 DA fluorescence assay. Fluorescence was measured with the excitation wavelength set at 495 nm and the emission wave length at 515 nm, using fluorescence microscopy (Olympus America, Inc., Melville, NY). 2.10. Evaluation of vasorelaxation The third-order branches of the mesenteric arteries were dissected and cut in segments of 2 mm in length and mounted on 40 mm stainless-steel wires in an isometric Mulvany-Halpern smallvessel myograph (model 91 M610; J.P. Trading, Aarhus, Denmark). See supplemental material.
2.3. Blood glucose and plasma insulin concentration analysis 2.11. Statistical analysis Blood (5 mL) was centrifugated at 1800g for 10min and the serum was extracted and collected. Blood glucose values were determined by using the Accu-Chek Advantage glucose monitoring system (Roche Diagnostics GmbH, Sandhofer, Germany). Plasma insulin levels were measured by a rat insulin 96 well plate assay from Millipore Corporation (Billerica, MA). 2.4. Cell culture of human umbilical vein endothelial cell Human umbilical vein endothelial cell (HUVEC) line was purchased from ATCC Company (Manassas, VA, USA). HUVEC were cultured in DMEM or DMEM high glucose medium (Gibco) supplemented with 10% fetal bovine serum, in a humidified incubator at 37 C with 95% air/5% CO2. The cells were serum-starved overnight before the experiment. 2.5. MTT assay of cell viability HUVEC viability was measured by 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2-H-tetrazolium bromide (MTT) cell proliferation
Data are expressed as mean ± SEM. Relaxation in each arterial segment is expressed as the percentage of the contraction induced by PHE (10 mmol/L). Comparison within groups was made by repeated-measures ANOVA (or paired t-test when only 2 groups were compared), and comparison among groups was made by factorial ANOVA with the Holm-Sidak ad hoc test (or t-test when only 2 groups were compared). A value of P < 0.05 was considered significant. 3. Results 3.1. Hyperglycemia-induced endothelial injury and decreased expression of dopamine D4 receptor in HUVEC To establish a cell model of hyperglycemia-induced endothelial injury, HUVEC was treated with different concentration of Dglucose (10, 25, 30 mM) in basal HUVEC media with 5% fetal calf serum for 24 h. As showed in Fig. 1A, the cell viability determined by MTT was significantly decreased in a does-dependent manner
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L745870. As shown in Supplemental Fig. 1B and 1C, the protective effects of PD168077 were completely eliminated by L745870 treatment. 3.3. Activation of the D4 receptor protected against high-glucoseinduced cell apoptosis To determine whether the protective effect of D4 receptor on high-glucose-induced cytotoxicity is via the effect on cell apoptosis, TUNEL staining was performed to measure the rate of cell apoptosis. As shown in Fig. 2AeB, incubation with high glucose induced a significant increase in the number of TUNEL-positive cells. However, the number of apoptotic cells was greatly decreased by pre-treatment with PD168077 (107M,48 h). Here, the activity of pro-apoptotic caspase-3 was also measured using a caspase 3 colorimetric assay Kit. We found that the caspase 3 activity was significantly increased by high glucose treatment, but was inhibited by PD168077 treatment (Fig. 2C). Consistent with this result, high-glucose-induced up-regulation of cleaved caspase 3 was prevented by PD168077 treatment (Fig. 2D and E). The specificity of PD168077 was also confirmed by L745870 treatment, in
Fig. 1. Activation of D4 receptor protected against high-glucose-induced injury on endothelial cells. (A) and (B) Cell viability and LDH release were determined by MTT and LDH release assays. (C) and (D) D4 receptor expression level in HUVEC was estimated by Western blot. (E) The specificity of D-glucose (30 mM) on D4 receptor expression in endothelial cells. (F) MTT assay showing activation of D4 receptor by PD168077 increased the cell viability. *P < 0.05 vs. Control, #P < 0.05 vs. HG groups. HG: high glucose. PD: PD168077. L: L745870.
after D-glucose treatment, consistent with the increased level of LDH levels (Fig. 1B), suggesting a gyperglycemia-induced endothelial injury in HUVEC. Moreover, we found that D-glucose treatment dramatically down-regulated D4 receptor expression in timedependent and does-dependent manners (Fig. 1CeD). However, 30 mM L-glucose and 30 mM mannitol both can not alter the expression level of D4 receptor (Fig. 1E), showing the specificity of D-glucose on D4 receptor expression. 3.2. Activation of D4 receptor protected against high-glucoseinduced injury on endothelial cells To investigate the effect of D4 receptor on high-glucose-induced endothelial cells injury, we treated the HUVEC with a selective dopamine D4 receptor agonist, PD168077, for 48 h after 24 h high glucose treatment. MTT assay showed that in the presence of PD168077 (108-106 M), high-glucose-induced endothelial cells injury was partly prevented (Fig. 1F). Similar result was observed in LDH release assay (Supplemental figure 1A), indicating that activation of D4 receptor protected HUVEC against high-glucoseinduced injury. To rule out the side effect of PD168077, we treated HUVEC simultaneously with D4 receptor antagonist,
Fig. 2. Activation of the D4 receptor protected against high-glucose-induced cell apoptosis. (A) Apoptotic cells were evaluated by TUNEL-staining. Representative TUNEL staining images ( 200). DAPI: blue. TUNEL staining: green. (B) The percentage of apoptotic cells (C) Caspase-3 enzyme activity (D) and (E) Protein levels of cleaved caspase-3 (17kd and 19kd) were assessed by Western blot. *P < 0.05 vs. Control, # P < 0.05 vs. HG group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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which pretreatment with L745870 (107M) abrogated the antiapoptotic effect of PD168077 in high-glucose-treated endothelial cells (Fig. 2BeE).
3.4. PI3K/Akt/eNOS/NO pathway involved in the protective effects of D4 receptor against high-glucose-induced endothelial cells injury To explore whether PI3K/Akt/eNOS pathway involve in the protective effects of D4 receptor, we first detected the phosphorylation of Akt and eNOS (Ser1177) after activating D4 receptor in high-glucose-treated HUVEC. We found that treatment with 107M PD168077 dramatically increased the phosphorylation of Akt and eNOS (Fig. 3A and B), indicating activation of D4 receptor by PD168077 activated the downstream PI3K/Akt/eNOS pathway. To confirm the involvement of PI3K/Akt/eNOS pathway in the protective effects of D4 receptor, Akt inhibitor, LY294002, and eNOS inhibitor, L-NAME, was pre-treated with HUVEC prior to PD168077 treatment. MTT and LDH assays showed that the protective effect of D4 receptor on high-glucose-induced endothelial cell injury was eliminated by 105M LY294002 or 104M L-NAME pre-treatment (Fig. 3C and D). Moreover, the effect of D4 receptor activation on
Fig. 3. Role of PI3K/Akt/eNOS/NO signal pathway in the protective effect of D4 receptor on HUVEC after high-glucose-induced injury. (A) and (B) Western blot results showing Akt and p-Akt, eNOS and p-eNOS expression levels. *P < 0.05 vs. HG, #P < 0.05 vs. HG þ PD group. (C) and (D) MTT assay and LDH release assay. *P < 0.05 vs. Control, # P < 0.05 vs. HG, & P < 0.05 vs. HG þ PD group. (E) Intracellular NO production was measured by using DAF-2DA. Representative images of NO detection ( 10). (F) The summary of fluorescence intensity data. *P < 0.05 vs. HG groups, #P < 0.05 vs. HG þ PD (n ¼ 6). HG: high glucose. PD: PD168077. L: L745870. *P < 0.05 vs. HG groups, #P < 0.05 vs. HG þ PD.
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NO production was also determined by DAF-2DA fluorescence. As shown in Fig. 4EeF, PD168977 treatment greatly elevated the NO production. However, the NO production was completely returned to control level by Akt inhibitor, LY294002, indicating that activation of D4 receptor induces NO production via PI3K/Akt/eNOS pathway in endothelial cells.
3.5. Down-regulation of D4 receptor in arterial endothelium of STZinduced diabetic rats To investigate the protective effects of dopamine D4 receptor against hyperglycemia-induced endothelial dysfunction in vivo, we established a diabetic rat model by injecting intraperitoneally with STZ. Three days after 60 mg/kg STZ injection [18], these rats showed a significant increase in their plasma glucose (Fig. 4A), accompanied with decreased plasma insulin (Fig. 4B), indicating the successful establishment of a type I diabetic rat model. To evaluate the expression level of D4 receptor in the arterial endothelium, endothelia was denuded from the thoracic aorta. Western bolt result showed that the protein level of D4 receptor was significantly down-regulated in the arterial endothelium of STZ-induced diabetic rats (Fig. 4C), consistent with the above cell model of hyperglycemia-induced endothelial injury.
Fig. 4. Activation of D4 receptor improved endothelium-dependent vasodilation of mesenteric arteries in STZ-induced diabetic rats. (A) and (B). The concentrations of blood glucose (A) and plasma insulin (B) in STZ-induced diabetic rats. (C) The protein expression of D4 receptors in the aortic endothelium of STZ-induced diabetic and control rats. (D) and (E) The vascular endothelium cumulative concentration-response curves for Ach (A) or SNP (B) in aortic rings. Relaxation of aortic rings is expressed as a percentage of contraction induced by PHE (10-5 M). *P < 0.05 vs. control rats, #P < 0.05 vs. STZ.
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3.6. Activation of D4 receptor improved endothelium-dependent vasodilation of mesenteric arteries in STZ-induced diabetic rats To determine the effect of the D4 receptor on endothelial dysfunction in STZ-induced diabetic rats, PD168077 (6 mg/kg/day) were intraperitoneally injected for 2 weeks. Compared to control, the weight in STZ-induced diabetic rats was greatly reduced, accompanied with significantly increased plasma glucose level and decreased insulin level. However, treatment with PD168077 in the STZ-induced diabetic rats did not improve the body weight, plasma glucose and insulin levels (Table 1). Consistent with other reports [19], the acetylcholine (Ach, 109105M)-induced vasodilation was impaired in the mesenteric arteries of STZ-induced diabetic rats. This impairment was partially reversed by PD168077 treatment in the STZ-induced diabetic rats (Fig. 4D). However, sodium nitroprusside (SNP)-induced vasodilation did not change in STZ-induced diabetic rats with or without PD168077 treatment (Fig. 4E), indicating that activation of D4 receptor improved endothelium-dependent vasodilation of mesenteric arteries in STZ-induced diabetic rats. 4. Discussion Endothelial dysfunction is the earliest pathologic event and contributes significantly to the initiation and progression of diabetic vascular complications [20,21]. Glucose intolerance and hyperglycemia are the main metabolic disorders found in diabetes mellitus and are important risk factors for cardiovascular diseases [22,23]. Reducing the injury of glucose on endothelial cells may provide new insights in the prevention of diabetic vascular complications. The salient feature in the present study is that the dopamine D4 receptor agonist was proven to be a remarkable endothelial protective agent in the diabetic animal model. Accumulating evidence indicate an interaction between glucose and dopamine receptors in various tissues and cells [12,14,17,24,25]. The renal dopaminergic system is impaired in hyperglycemic rats and type-2 diabetes patients [26,27]. In pancreatic beta cells, the D2-like receptor agonist, quinpirole, inhibits glucose-stimulated insulin secretion [28]. Lawford et al. have found that the D2 receptor gene is associated with raised blood glucose in cases of schizophrenia [29]. KumarV et al. have found that the D2 receptor agonist, bromocriptine, could ameliorate hyperglycaemia [30]. Our previous studies have found that hyperglycemia decreases arterial D1 receptor expression and increases D1 receptor phosphorylation in obese Zucker rats [31], thereby causing D1 receptor dysfunction. Zarei et al. found that endothelial cells only express dopamine D2like receptors, and dopamine modulates von Willebrand factor secretion in endothelial cells [17]. Our present study showed that the expression of the D4 receptor was reduced in the endothelium of rats with STZ-induced diabetes and high-glucose-treated HUVEC. The decreases D4 receptor expression is due to high Dglucose, but not the hyperosmolarity, since L-glucose and mannitol both did not change the expression level of D4 receptor. High glucose, apoptosis, and endothelial dysfunction were reported to play an important role in the pathophysiology of diabetic disease. Accelerated endothelial cell apoptosis induced by
Table 1 The basal index of rats (mean ± SEM, n ¼ 6).
Body weight(g) Blood glucose(mmol/L) Insulin(ng/mL) *P < 0.05 vs. Control.
Control
STZ
PD þ STZ
284 ± 9.0 5.50 ± 0.3 0.799 ± 0.056
235 ± 6.0* 26.20 ± 0.8* 0.421 ± 0.086*
248 ± 7.0* 24.8 ± 0.7* 0.478 ± 0.032*
hyperglycemia is an important event in the process of diabetesassociated vascular complications [32e34]. It is well established that endothelial cells cultured under the high glucose environment led to a significant increase in apoptosis [35,36]. Similar to those studies, our results showed that high glucose induced a significant decrease in cell viability and a significant increase in the number of apoptotic cells. Interestingly, we found that stimulation of the D4 receptor by PD168077 could alleviate high-glucose-induced endothelial cell injury by promoting endothelial cell survival and inhibiting cell apoptosis. Endothelial cells regulate vascular homeostasis by generating paracrine factors that regulate vascular tone, inhibit platelet function, prevent adhesion of leukocytes, and limit proliferation of vascular smooth muscle [38]. The most important weapon of endothelial cells to fight vascular diseases is eNOS, an enzyme that generates the vasoprotective molecule NO [39]. Endothelial dysfunction characterized by inactivation or reduced synthesis of NO, alone or in combination, is implicated in the effect of diabetes induced cardiovascular injury [40]. Evidence has shown that the PI3K/Akt/eNOS pathway plays an important role in preventing high glucose-induced endothelial cell injury [41]. Previous study has also shown that D4 dopamine receptor agonist, PD168077, activates Akt in time-an dose-dependent manner in D4MN9D cells [19]. Similarly, our study showed that activation of the D4 receptor by PD168077 could increase PI3K, Akt and eNOS phosphorylation and NO production. Moreover, the protective effect of PD168077 on high-glucose-induced endothelial cell injury was blocked by the PI3K inhibitor LY294002 and eNOS inhibitor L-NAME, indicating that PI3K/Akt-dependent eNOS pathway is involved in the D4-receptor-mediated protective effect on endothelial cells. Based on our findings, we concluded that dopamine D4 receptor has protective effects on endothelial cells of STZ-induced diabetic rats, partially by reducing apoptosis via the PI3K/Akt/eNOS/NO pathway. The protective effect is of pathological significance, as pretreatment with activators of the D4 receptor could reverse the impaired acetylcholine-induced vasodilation, which may lead to a novel strategy in the treatment of diabetic vascular complications. Declaration of interest No conflicts of interest, financial or otherwise, are declared by the authors. Acknowledgment These studies were supported in part by grants from the National Natural Science Foundation of China (81603432,81603431). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.08.080. References [1] S.D. de Ferranti, I.H. de Boer, V. Fonseca, C.S. Fox, S.H. Golden, C.J. Lavie, S.N. Magge, N. Marx, D.K. McGuire, T.J. Orchard, B. Zinman, R.H. Eckel, Type 1 diabetes mellitus and cardiovascular disease: a scientific statement from the American Heart Association and American Diabetes Association, Circulation 130 (2014) 1110e1130. [2] B.M. Leon, T.M. Maddox, Diabetes and cardiovascular disease: epidemiology, biological mechanisms, treatment recommendations and future research, World J. Diabetes 6 (2015) 1246e1258. [3] A.M. Schmidt, Diabetes mellitus and cardiovascular disease, Arterioscler. Thromb. Vasc. Biol. 39 (2019) 558e568. [4] P.E. Szmitko, C.H. Wang, R.D. Weisel, J.R. de Almeida, T.J. Anderson, S. Verma, New markers of inflammation and endothelial cell activation: Part I, Circulation 108 (2003) 1917e1923.
H. Wang et al. / Biochemical and Biophysical Research Communications 518 (2019) 554e559 [5] P.E. Szmitko, C.H. Wang, R.D. Weisel, G.A. Jeffries, T.J. Anderson, S. Verma, Biomarkers of vascular disease linking inflammation to endothelial activation: Part II, Circulation 108 (2003) 2041e2048. [6] M. Brownlee, Biochemistry and molecular cell biology of diabetic complications, Nature 414 (2001) 813e820. [7] M.A. Potenza, S. Gagliardi, C. Nacci, M.R. Carratu, M. Montagnani, Endothelial dysfunction in diabetes: from mechanisms to therapeutic targets, Curr. Med. Chem. 16 (2009) 94e112. [8] I. Armando, V.A. Villar, P.A. Jose, Dopamine and renal function and blood pressure regulation, Comp. Physiol. 1 (2011) 1075e1117. [9] A.A. Banday, M.F. Lokhandwala, Dopamine receptors and hypertension, Curr. Hypertens. Rep. 10 (2008) 268e275. [10] J.M. Beaulieu, R.R. Gainetdinov, The physiology, signaling, and pharmacology of dopamine receptors, Pharmacol. Rev. 63 (2011) 182e217. [11] M.O. Kim, P.O. Koh, J.H. Kim, J.S. Kim, S.S. Kang, G.J. Cho, K. Kim, W.S. Choi, Localization of dopamine D1 and D2 receptor mRNAs in the rat systemic and pulmonary vasculatures, Mol. Cells 9 (1999) 417e421. [12] J.E. de Leeuw van Weenen, E.T. Parlevliet, P. Maechler, L.M. Havekes, J.A. Romijn, D.M. Ouwens, H. Pijl, B. Guigas, The dopamine receptor D2 agonist bromocriptine inhibits glucose-stimulated insulin secretion by direct activation of the alpha2-adrenergic receptors in beta cells, Biochem. Pharmacol. 79 (2010) 1827e1836. [13] R.A. Defronzo, Bromocriptine: a sympatholytic, d2-dopamine agonist for the treatment of type 2 diabetes, Diabetes Care 34 (2011) 789e794. [14] I. Garcia-Tornadu, A.M. Ornstein, A. Chamson-Reig, M.B. Wheeler, D.J. Hill, E. Arany, M. Rubinstein, D. Becu-Villalobos, Disruption of the dopamine d2 receptor impairs insulin secretion and causes glucose intolerance, Endocrinology 151 (2010) 1441e1450. [15] F. Lopez Vicchi, G.M. Luque, B. Brie, J.P. Nogueira, I. Garcia Tornadu, D. BecuVillalobos, Dopaminergic drugs in type 2 diabetes and glucose homeostasis, Pharmacol. Res. 109 (2016) 74e80. [16] C. Yu, Z. Wang, Y. Han, Y. Liu, W.E. Wang, C. Chen, H. Wang, P.A. Jose, C. Zeng, Dopamine D(4) receptors inhibit proliferation and migration of vascular smooth muscle cells induced by insulin via down-regulation of insulin receptor expression, Cardiovasc. Diabetol. 13 (2014) 97. [17] S. Zarei, M. Frieden, B. Rubi, P. Villemin, B.R. Gauthier, P. Maechler, U.M. Vischer, Dopamine modulates von Willebrand factor secretion in endothelial cells via D2-D4 receptors, J. Thromb. Haemost. 4 (2006) 1588e1595. [18] K. Ishige, Q. Chen, Y. Sagara, D. Schubert, The activation of dopamine D4 receptors inhibits oxidative stress-induced nerve cell death, J. Neurosci. 21 (2001) 6069e6076. [19] X. Zhen, J. Zhang, G.P. Johnson, E. Friedman, D(4) dopamine receptor differentially regulates Akt/nuclear factor-kappa b and extracellular signalregulated kinase pathways in D(4)MN9D cells, Mol. Pharmacol. 60 (2001) 857e864. [20] V. Altabas, Diabetes, endothelial dysfunction, and vascular repair: what should a diabetologist keep his eye on? Internet J. Endocrinol. 2015 (2015) 848272. [21] C.G. Schalkwijk, C.D. Stehouwer, Vascular complications in diabetes mellitus: the role of endothelial dysfunction, Clin. Sci. (Lond.) 109 (2005) 143e159. [22] M.I. Uusitupa, Early lifestyle intervention in patients with non-insulindependent diabetes mellitus and impaired glucose tolerance, Ann. Med. 28 (1996) 445e449. [23] P.Y. Zhang, Cardiovascular disease in diabetes, Eur. Rev. Med. Pharmacol. Sci. 18 (2014) 2205e2214. [24] C. Gragnoli, G.M. Reeves, J. Reazer, T.T. Postolache, Dopamine-prolactin pathway potentially contributes to the schizophrenia and type 2 diabetes comorbidity, Transl. Psychiatry 6 (2016) e785. [25] G.M. Luque, F. Lopez-Vicchi, A.M. Ornstein, B. Brie, C. De Winne, E. Fiore,
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[38]
[39] [40]
[41]
559
M.I. Perez-Millan, G. Mazzolini, M. Rubinstein, D. Becu-Villalobos, Chronic hyperprolactinemia evoked by disruption of lactotrope dopamine D2 receptors impacts on liver and adipocyte genes related to glucose and insulin balance, Am. J. Physiol. Endocrinol. Metab. 311 (2016) E974eE988. F. Contreras, C. Foullioux, B. Pacheco, C. Maroun, H. Bolivar, M. Lares, E. Leal, R. Cano, V. Bermudez, M. Velasco, Effect of drugs interacting with the dopaminergic receptors on glucose levels and insulin release in healthy and type 2 diabetic subjects, Am. J. Therapeut. 15 (2008) 397e402. A. Marwaha, M.F. Lokhandwala, Tempol reduces oxidative stress and restores renal dopamine D1-like receptor- G protein coupling and function in hyperglycemic rats, Am. J. Physiol. Renal. Physiol. 291 (2006) F58eF66. B. Rubi, S. Ljubicic, S. Pournourmohammadi, S. Carobbio, M. Armanet, C. Bartley, P. Maechler, Dopamine D2-like receptors are expressed in pancreatic beta cells and mediate inhibition of insulin secretion, J. Biol. Chem. 280 (2005) 36824e36832. B.R. Lawford, M. Barnes, C.P. Morris, E.P. Noble, P. Nyst, K. Heslop, R.M. Young, J. Voisey, J.P. Connor, Dopamine 2 receptor genes are associated with raised blood glucose in schizophrenia, Can. J. Psychiatr. 61 (2016) 291e297. V.S.H. Kumar, B.V. M, N.P. A, S. Aithal, S.R. Baleed, U.N. Patil, Bromocriptine, a dopamine (d2) receptor agonist, used alone and in combination with glipizide in sub-therapeutic doses to ameliorate hyperglycaemia, J. Clin. Diagn. Res. 7 (2013) 1904e1907. J. Fu, Y. Han, H. Wang, Z. Wang, Y. Liu, X. Chen, Y. Cai, W. Guan, D. Yang, L.D. Asico, L. Zhou, P.A. Jose, C. Zeng, Impaired dopamine D1 receptormediated vasorelaxation of mesenteric arteries in obese Zucker rats, Cardiovasc. Diabetol. 13 (2014) 50. D. Detaille, B. Guigas, C. Chauvin, C. Batandier, E. Fontaine, N. Wiernsperger, X. Leverve, Metformin prevents high-glucose-induced endothelial cell death through a mitochondrial permeability transition-dependent process, Diabetes 54 (2005) 2179e2187. R. Gomez, M. Gonzalez-Izquierdo, R.C. Zimmermann, E. Novella-Maestre, I. Alonso-Muriel, J. Sanchez-Criado, J. Remohi, C. Simon, A. Pellicer, Low-dose dopamine agonist administration blocks vascular endothelial growth factor (VEGF)-mediated vascular hyperpermeability without altering VEGF receptor 2-dependent luteal angiogenesis in a rat ovarian hyperstimulation model, Endocrinology 147 (2006) 5400e5411. G.J. Pyne-Geithman, D.N. Caudell, M. Cooper, J.F. Clark, L.A. Shutter, Dopamine D2-receptor-mediated increase in vascular and endothelial NOS activity ameliorates cerebral vasospasm after subarachnoid hemorrhage in vitro, Neurocritical Care 10 (2009) 225e231. G.N. Shah, T.O. Price, W.A. Banks, Y. Morofuji, A. Kovac, N. Ercal, C.M. Sorenson, E.S. Shin, N. Sheibani, Pharmacological inhibition of mitochondrial carbonic anhydrases protects mouse cerebral pericytes from high glucose-induced oxidative stress and apoptosis, J. Pharmacol. Exp. Ther. 344 (2013) 637e645. M. Silambarasan, J.R. Tan, D.S. Karolina, A. Armugam, C. Kaur, K. Jeyaseelan, MicroRNAs in hyperglycemia induced endothelial cell dysfunction, Int. J. Mol. Sci. 17 (2016) 518. A. Sandoo, J.J. van Zanten, G.S. Metsios, D. Carroll, G.D. Kitas, The endothelium and its role in regulating vascular tone, Open Cardiovasc. Med. J. 4 (2010) 302e312. U. Forstermann, T. Munzel, Endothelial nitric oxide synthase in vascular disease: from marvel to menace, Circulation 113 (2006) 1708e1714. X. Shu, T.C.t. Keller, D. Begandt, J.T. Butcher, L. Biwer, A.S. Keller, L. Columbus, B.E. Isakson, Endothelial nitric oxide synthase in the microcirculation, Cell. Mol. Life Sci. 72 (2015) 4561e4575. D.A. Yuen, B.E. Stead, Y. Zhang, K.E. White, M.G. Kabir, K. Thai, S.L. Advani, K.A. Connelly, T. Takano, L. Zhu, A.J. Cox, D.J. Kelly, I.W. Gibson, T. Takahashi, R.C. Harris, A. Advani, eNOS deficiency predisposes podocytes to injury in diabetes, J. Am. Soc. Nephrol. 23 (2012) 1810e1823.