612 Integrative systems
Cardiovascular effects of endothelin-11–31 microinjected into the nucleus tractus solitarius of anesthetized rats Peiwu Lia, Xu Fub, Yan Luc, Wen Yinb, Li Mab and Zhongxue Fua Endothelin-11–31 (ET-11–31) is a 31-amino-acid vasoactive peptide that plays an important role in the regulation of cardiovascular function. However, the cardiovascular effects of central ET-11–31 are still not fully understood. In this study, we assess the effects of ET-11–31 within the nucleus tractus solitarius (NTS) of anesthetized rats and explore the underlying mechanisms of these effects. Bilateral microinjections of ET-11–31 into the NTS produced dose-dependent hypotension and bradycardia, very similar to the effects of a unilateral microinjection of ET-11–31 into the NTS. Bilateral microinjections of ET-11–31 into the NTS significantly decreased baroreflex function in a timedependent manner. The hypotensive and bradycardic effects induced by the microinjection of ET-11–31 into the NTS were significantly decreased by the ETA receptor antagonist BQ123 and by kynurenic acid, but not by the ETB receptor antagonist BQ788. These results show that
ET-11–31 injected into the NTS produces hypotension and bradycardia, mediated by ETA receptors and, at least partly, by the glutamate receptor. NeuroReport c 2014 Wolters Kluwer Health | Lippincott 25:612–617 Williams & Wilkins.
Introduction
of baroreceptor afferents activates excitatory amino acid receptors within the NTS. NTS neurons then send excitatory amino acid projections to the caudal ventrolateral medulla (CVLM), which in turn inhibits RVLM neurons through a GABAergic inhibitory pathway. Salusin-b is a vasoactive peptide similar to ET-1 that is relevant to the cardiovascular system, which causes hypertension and bradycardia in a dose-dependent manner by activating its receptors within the medulla [9]. However, the cardiovascular functions of ET-11–31 have not yet been determined [10,11]. Thus, this study was designed to determine the effects of exogenous ET-11–31 on cardiovascular activity at the NTS level.
Endothelin-1 (ET-1), the dominant isoform of the ET family, is derived from vascular endothelial cells and plays an important role in the regulation of cardiovascular function by activating the ETA or ETB receptors [1]. A large amount of evidence indicates that the ET receptors (ETA and/or ETB) can be activated directly by ET-11–31 in vitro [2,3]. The systemic administration of ET-11–31 increases the mean arterial pressure (MAP) in mice and rabbits and induces vasoconstriction in vivo [4,5]. However, the cardiovascular effects of ET-11–31 in the central nervous system are still not fully understood. Mast cell-derived chymase is important not only in degradation, but also in the synthesis of ET-11–31 from its precursor, big ET-1, in vitro. There are reports that human chymase exists in the pineal and pituitary glands and is observably upregulated under certain pathological states, such as in a human brain infested with cysticerci [6]. Although no evidence has yet been found of ET-11–31 in the brain, it is hypothesized that ET-11–31 may be involved in central cardiovascular regulation. In the central nervous system of mice, intracerebroventricular and rostral ventrolateral medullary (RVLM) injections of ET-11–31 produce similar cardiovascular effects: a typical biphasic response of a rapid increase and then a protracted decrease in MAP. These effects are thought to be mediated by the ETA receptors, but not the ETB receptors [7,8]. The nucleus tractus solitaries (NTS) is the primary site of termination of afferent fibers from the arterial baroreceptors and chemoreceptors. Stimulation c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4965
NeuroReport 2014, 25:612–617 Keywords: baroreflex sensitivity, cardiovascular, endothelin-11–31, endothelin receptors, nucleus tract solitarius a Department of General Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing, bDepartment of Emergency, Lanzhou University Second Hospital and cDepartment of Clinical Laboratory, San Aitang Hospital, Lanzhou, China
Correspondence to Zhongxue Fu, PhD, Department of General Surgery, First Affiliated Hospital, Chongqing Medical University, 1 College Road, Yuzhong District, Chongqing 400010, China Tel/fax: + 86 0931 8942031; e-mail: [email protected] Received 31 December 2013 accepted 19 February 2014
Materials and methods Animals and drugs
Ethical approval for the study was obtained from the Ethics Committee of Chongqing Medical University. Male Sprague–Dawley rats (weighing between 250 and 300 g, CQLA-2009-010) were obtained from the Animal Experimental Centre of Chongqing Medical University (Chongqing, China). ET-11–31 was obtained from the Peptide Institute (Tokyo, Japan) and kynurenine (KYN), BQ123, and BQ788 were obtained from Sigma-Aldrich (St Louis, Missouri, USA). Organic reagents and phenylephrine were obtained from Shanghai Biotechnology Ltd. (Shanghai, China). General procedure
Male Sprague–Dawley rats (weighing between 300 and 350 g) were used in this study. Animal preparation, DOI: 10.1097/WNR.0000000000000149
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Cardiovascular effects of endothelin-11–31 in NTS Li et al. 613
histologic procedures, and microinjections were executed according to institutional guidelines [12]. To test the cardiovascular effects of injection of ET-11–31 (0.5–2.0 pmol) at different dosage levels into the NTS and compare unilateral and bilateral doses, 50 male Sprague–Dawley rats were divided into five groups. Two groups were set aside as the bilateral (n = 8) and unilateral (n = 4) artificial cerebrospinal fluid (aCSF) control groups. Another two groups were administered bilateral (n = 21) and unilateral (n = 21) microinjections of ET-11–31 in a dose-dependent manner. The remaining group (n = 7) was bilaterally microinjected with ET-11–31 to observe the effects of ET-11–31 on the arterial baroreflex (BRS) function within the NTS. A further 29 rats were pretreated with the ETA receptor antagonist BQ123, the ETB receptor antagonist BQ788, or the nonselective glutamate receptor antagonist KYN to explore the mechanisms of action of ET-11–31 in the NTS. A catheter, inserted into the right femoral artery, was connected to a pressure transducer (PT400; Omega, Washington, District of Columbia, USA) to directly measure blood pressure (BP). The heart rate (HR) was computed from the BP waveforms and displayed on a channel of the recording system.
Microinjection procedure
NTS microinjections were administered using a method referred to in the relevant literature [13]. The injections were made into the NTS using stereotaxic coordinates from the rat brain atlas (Paxinos and Watson, 1986): 0.5–0.8 mm lateral to the medial line, 0.5–0.8 mm caudal to the bregma, and 0.5 mm deep from the bone surface. ET-11–31 was initially dissolved in 0.1% acetic acid and diluted with aCSF (in mM: 3.4 KCl, 133.3 NaCl, 1.3 CaCl2, 0.6 NaH2PO4, 32.0 NaHCO3, 1.2 MgCl2, and 3.4 glucose, pH 7.4). The final concentration of dimethyl sulfoxide in aCSF was not more than 1%. BQ788 (20 pmol) and KYN (20 pmol) were initially dissolved in dimethyl sulfoxide and then diluted in aCSF, and BQ123 (20 pmol) was dissolved in double distilled water. All drugs were slowly (over 30 s) injected into the NTS in a volume of 100 nl.
BRS measurement
BRS measurements were made on the basis of procedures described in the literature [14]. Once a rat’s BP was stable, a rapid intravenous infusion of 0.4 g/l phenylephrine raised the BP by 30–40 mmHg. Systolic BP was regarded as the independent variable and cardiac cycle (heart period, HP) as the dependent variable in the linear regression analysis (the correlation coefficient R2 > 0.8). The slope of the line of the resulting regression is the BRS (ms/mmHg). The data were recorded using a computer at three time points (preadministration, and 5 and 10 min after medication).
Statistical analysis
All of the values are expressed as mean±SE. BP is reported as the MAP, which was calculated as diastolic + [(systolic – diastolic)/3]. The magnitudes of the changes in MAP and HR at 5 and 10 min after injection of the agents were compared with that on injection of the vehicle by a one-way analysis of variance. If the difference was significant, further pairwise comparisons were performed using analysis of covariance (Dunnett’s test) and a significance level of 0.05.
Results Changes in MAP and HR
Bilaterally (n = 7) or unilaterally (n = 7) microinjected ET-11–31 produced dose-dependent hypotension and bradycardia. A unilateral NTS microinjection of ET-11–31 generated hypotension (from – 10±2 to – 22±3 mmHg, P < 0.05) and bradycardia (from – 11±4 to – 21±3 bpm, P < 0.05). The effects of unilateral injections were similar to those of bilateral injections, and were not shorter or less intense. In control tests, bilateral and unilateral microinjections of the same volume of aCSF had no effect on BP or HR. With both bilateral and unilateral microinjections, the cardiovascular effects began within 30 s of microinjection, lasted 1–2 min, and then returned to normal. The changes in MAP and HR (DMAP and DHR) in response to microinjection of ET-11–31 into the NTS are summarized in Tables 1 and 2. Effects on BRS function
Bilateral administration of ET-11–31 into the NTS timedependently attenuated BRS function. Typically, BRS function significantly decreased in the 5 min following
Table 1 The effects of bilateral microinjection of ET-11–31 into the NTS on DMAP and DHR aCSF
ET-11–31
Groups
100 nl
0.5 pmol
1.0 pmol
2.0 pmol
DMAP (mmHg) DHR (bpm)
– 1±1 – 3±2
– 10±2* – 11±4*
– 16±3* – 16±4*
– 22±3* – 21±3*
Values are means±SE. aCSF, artificial cerebrospinal fluid; HR, heart rate; ET-1, endothelin-1; MAP, mean arterial pressure; NTS, nucleus tractus solitarius. *P < 0.05 compared with the control group.
Table 2 The effects of unilateral microinjection of ET-11–31 into the NTS on the DMAP and DHR aCSF Groups DMAP (mmHg) DHR (bpm)
ET-11–31
100 nl
0.5 pmol
1.0 pmol
2.0 pmol
– 0.5±1 – 2±2
– 7±3* – 5±4*
– 12±3* – 11±6*
– 18±3* – 16±5*
Values are means±SE. aCSF, artificial cerebrospinal fluid; HR, heart rate; ET-1, endothelin-1; MAP, mean arterial pressure; NTS, nucleus tractus solitarius. *P < 0.05 compared with the control group.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
614 NeuroReport 2014, Vol 25 No 8
Fig. 1
(a)
HR (bpm)
BP (mmHg)
Baseline
5 min
Baseline
30 min
5 min
30 min
150
50 500
400
(b) 1.0
BRS (ms/mmHg)
BRS (ms/mmHg)
1.0
0.5
0.0
Baseline
5 min aCSF
30 min
n=7
∗
∗
5 min
30 min
0.5
0.0
Baseline
ET-11–31 n=7
(a) Representative tracings showing the BP and HR responses to microinjections of aCSF and ET-11–31 into the NTS. (b) Bar graphs showing the effects of bilateral microinjection of ET-11–31 (1.0 pmol) or aCSF into the NTS on the BRS response. *P < 0.05 compared with baseline. aCSF, artificial cerebrospinal fluid; BP, blood pressure; BRS, baroreflex; HR, heart rate; ET-1, endothelin-1; NTS, nucleus tractus solitarius.
intra-NTS application of ET-11–31 and the changes increased over the period of action of the drug (30 min after the treatment). The effects of intra-NTS administration of ET-11–31 or aCSF on BP and HR are summarized in Fig. 1.
microinjection of ET-11–31 into the NTS. The effects of the ET receptor antagonists and aCSF on BP and HR following a unilateral microinjection of ET-11–31 into the NTS are summarized in Fig. 2.
Effects of blockade of ETA and ETB receptors
Effects of the nonspecific glutamate receptor antagonist KYN
Pretreatment with BQ123 completely prevented the lowering of BP and significantly reduced the slowing of HR caused by the microinjection of ET-11–31 (1.0 pmol) into the NTS (P < 0.05). In contrast, prior microinjection of the ETB receptor antagonist BQ788 (20 pmol) into the NTS did not alter the ET-11–31-induced reduction of BP and HR (P > 0.05). Microinjection of the same volume of aCSF into the NTS had no effect on the basal BP and HR or on the hypotension and bradycardia responses to
Unilateral pretreatment with KYN (20 pmol) significantly inhibited the hypotension and bradycardia (P < 0.05) arising from the intra-NTS microinjection of ET-11–31 (1.0 pmol). Unilateral microinjection of the same volume of aCSF did not affect MAP and HR responses to ET-11–31 within the NTS. The changes in MAP and HR in response to the microinjection of ET-11–31 into the NTS after pretreatment with KYN are illustrated in Fig. 3.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Cardiovascular effects of endothelin-11–31 in NTS Li et al. 615
Fig. 2
Fig. 3
aCSF+ET-11–31
(a)
n=4
BQ123+ET-11–31
n=7
BQ788+ET-11–31
(a)
n=7
0
0
aCSF+ET-11–31
KYN+ET-11–31
n=4
n=7
−10
Change in MAP (mmHg)
Change in MAP (mmHg)
∗
−20
∗
−10
−20
−30 aCSF+ET-11–31
(b) 0
n=4
BQ123+ET-11–31
n=7
−30
BQ788+ET-11–31
n=7
(b) 0
KYN+ET-11–31
n=4
n=7
−10
∗
−20
Change in HR (bmp)
Change in HR (bpm)
∗
aCSF+ET-11–31
−10
−20
−30
Bar graphs showing the effects of pretreatment with BQ123 (20 pmol), BQ788 (20 pmol), and aCSF on (a) hypotension (BP) and (b) bradycardia (HR) responses to microinjection of ET-11–31 (1.0 pmol) into the NTS. *P < 0.05 compared with aCSF. aCSF, artificial cerebrospinal fluid; BP, blood pressure; HR, heart rate; ET-1, endothelin-1; NTS, nucleus tractus solitarius.
Discussion Within the low brain stem, cerebellum, and spinal cord, it is well known that ET-1 binding sites are widely distributed in the area postrema, the spinal nucleus of the trigeminal nerve, the dorsal motor nucleus of the vagus nerve, the hypoglossal nuclei, and the NTS, suggesting that ET-1 may play an important role in central cardiovascular regulation [15]. In addition, the NTS, the first level of the central baroreflex, accepts projections from arterial baroreceptors, chemoreceptors, and other cardiovascular receptors and integrates the information projected in the CVLM and RVLM to maintain the relative stability of BP [16]. Our previous studies have suggested that ET-11–31 regulates cardiovascular activity in anesthetized rats through the lateral
−30 Bar graphs showing the effects of pretreatment with KYN on (a) hypotension (BP) and (b) bradycardia (HR) responses to microinjection of ET-11–31 into the NTS. *P < 0.05 compared with aCSF. aCSF, artificial cerebrospinal fluid; BP, blood pressure; HR, heart rate; ET-1, endothelin-1; KYN, kynurenic acid; NTS, nucleus tractus solitarius.
ventricle or RVLM [7,8]. However, the physiological role of ET-11–31 in the regulation of cardiovascular function is still not fully understood. Further, the cardiovascular responses caused by intra-NTS ET-11–31 administration have, until now, not been reported. Therefore, the main objective of this study was to obtain evidence that intraNTS ET-11–31 plays a key role in cardiovascular responses. It has been proven that ET-11–21 participates in the regulation of cardiovascular activity in the central nervous system [17]. Early data have revealed that the
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616 NeuroReport 2014, Vol 25 No 8
cardiovascular effects of exogenous ET-11–21 are relied upon to activate the autonomic nervous system at the level of the NTS [18]. Previous research has also indicated that the microinjection of ET-11–21 into the NTS not only decreases MAP but also lowers HR in vivo [19]. However, the exact mechanisms of these depressor effects are controversial. Some argue that, accompanied by a reduction in blood flow to the brain, the sustained hypotension by centrally administered ET-1 is due largely to intense vasoconstriction caused by medullary ischemia [20]. Others hold that the hypotensive action of centrally administered ET-1 may be a consequence of bradycardia [19]. Given that bradycardia can be completely eliminated by the intravenous injection of hexamethonium, it is possible that bradycardia produced by the microinjection of ET-11–21 into the NTS may depend on the activation of the vagal nerve [18]. In this study, we increased local ET-11–31 concentrations through intra-NTS microinjection of ET-11–31 in anesthetized rats to investigate the cardiovascular effects of ET-11–31 in the NTS. Our results suggest that bilateral and unilateral NTS microinjections of ET-11–31 (0.5–2.0 pmol) produce dose-dependent hypotension and bradycardia responses. Bilateral microinjection of ET-11–31 into the NTS (1.0 pmol) also appeared to significantly inhibit the BRS function in anesthetized rats. The effects of microinjection of ET-11–31 into the NTS were similar to those produced by ET-11–21 and big endothelin (big ET-1), suggesting that the cardiovascular effects produced by ET-11–31 in the NTS are likely to be consistent with those produced by ET-11–21. Further experiments have shown that the cardiovascular effects produced by ET-11–31, such as lowered BP, slower HR, and suppressed BRS, are mediated by the ETA receptor rather than by the ETB receptor. Glutamate receptors, which play an important role in cardiovascular regulation in the medulla oblongata, are widely distributed in the NTS and other regions of the medulla oblongata [21]. Therefore, we hypothesized that the cardiovascular effects produced by ET-11–31 in the NTS may be mediated by a glutamate receptor. To test this hypothesis, the glutamate receptor antagonist KYN was used. The results show that microinjection of KYN into the NTS partially blocks the hypotension and bradycardia responses induced by a subsequent microinjection of ET11–31 into the NTS, which suggests that the cardiovascular effects caused by ET-11–31 in the NTS are, at least in part, produced through the activation of glutamate receptors that then activate the relevant neurons.
Conclusion ET-11–31 within the NTS caused hypotension and bradycardia and significantly decreased the BRS function in rats. Further study indicated that the cardiovascular
effects of ET-11–31 within the NTS are mediated by the ETA receptor and, at least in part, by the glutamate receptor. These findings suggest that ET-11–31 plays a significant role in the physiological alteration of cardiovascular effects in rats.
Acknowledgements The authors thank Li Ma and Zi-Li Li for their helpful discussions and expert statistical assistance, respectively. Conflicts of interest
There are no conflicts of interest.
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Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Cardiovascular effects of endothelin-11–31 microinjected into the nucleus tractus solitarius of anesthetized rats Peiwu Lia, Xu Fub, Yan Luc, Wen Yinb, Li Mab and Zhongxue Fua Endothelin-11–31 (ET-11–31) is a 31-amino-acid vasoactive peptide that plays an important role in the regulation of cardiovascular function. However, the cardiovascular effects of central ET-11–31 are still not fully understood. In this study, we assess the effects of ET-11–31 within the nucleus tractus solitarius (NTS) of anesthetized rats and explore the underlying mechanisms of these effects. Bilateral microinjections of ET-11–31 into the NTS produced dose-dependent hypotension and bradycardia, very similar to the effects of a unilateral microinjection of ET-11–31 into the NTS. Bilateral microinjections of ET-11–31 into the NTS significantly decreased baroreflex function in a timedependent manner. The hypotensive and bradycardic effects induced by the microinjection of ET-11–31 into the NTS were significantly decreased by the ETA receptor antagonist BQ123 and by kynurenic acid, but not by the ETB receptor antagonist BQ788. These results show that
ET-11–31 injected into the NTS produces hypotension and bradycardia, mediated by ETA receptors and, at least partly, by the glutamate receptor. NeuroReport c 2014 Wolters Kluwer Health | Lippincott 25:612–617 Williams & Wilkins.
Introduction
of baroreceptor afferents activates excitatory amino acid receptors within the NTS. NTS neurons then send excitatory amino acid projections to the caudal ventrolateral medulla (CVLM), which in turn inhibits RVLM neurons through a GABAergic inhibitory pathway. Salusin-b is a vasoactive peptide similar to ET-1 that is relevant to the cardiovascular system, which causes hypertension and bradycardia in a dose-dependent manner by activating its receptors within the medulla [9]. However, the cardiovascular functions of ET-11–31 have not yet been determined [10,11]. Thus, this study was designed to determine the effects of exogenous ET-11–31 on cardiovascular activity at the NTS level.
Endothelin-1 (ET-1), the dominant isoform of the ET family, is derived from vascular endothelial cells and plays an important role in the regulation of cardiovascular function by activating the ETA or ETB receptors [1]. A large amount of evidence indicates that the ET receptors (ETA and/or ETB) can be activated directly by ET-11–31 in vitro [2,3]. The systemic administration of ET-11–31 increases the mean arterial pressure (MAP) in mice and rabbits and induces vasoconstriction in vivo [4,5]. However, the cardiovascular effects of ET-11–31 in the central nervous system are still not fully understood. Mast cell-derived chymase is important not only in degradation, but also in the synthesis of ET-11–31 from its precursor, big ET-1, in vitro. There are reports that human chymase exists in the pineal and pituitary glands and is observably upregulated under certain pathological states, such as in a human brain infested with cysticerci [6]. Although no evidence has yet been found of ET-11–31 in the brain, it is hypothesized that ET-11–31 may be involved in central cardiovascular regulation. In the central nervous system of mice, intracerebroventricular and rostral ventrolateral medullary (RVLM) injections of ET-11–31 produce similar cardiovascular effects: a typical biphasic response of a rapid increase and then a protracted decrease in MAP. These effects are thought to be mediated by the ETA receptors, but not the ETB receptors [7,8]. The nucleus tractus solitaries (NTS) is the primary site of termination of afferent fibers from the arterial baroreceptors and chemoreceptors. Stimulation c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4965
NeuroReport 2014, 25:612–617 Keywords: baroreflex sensitivity, cardiovascular, endothelin-11–31, endothelin receptors, nucleus tract solitarius a Department of General Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing, bDepartment of Emergency, Lanzhou University Second Hospital and cDepartment of Clinical Laboratory, San Aitang Hospital, Lanzhou, China
Correspondence to Zhongxue Fu, PhD, Department of General Surgery, First Affiliated Hospital, Chongqing Medical University, 1 College Road, Yuzhong District, Chongqing 400010, China Tel/fax: + 86 0931 8942031; e-mail: [email protected] Received 31 December 2013 accepted 19 February 2014
Materials and methods Animals and drugs
Ethical approval for the study was obtained from the Ethics Committee of Chongqing Medical University. Male Sprague–Dawley rats (weighing between 250 and 300 g, CQLA-2009-010) were obtained from the Animal Experimental Centre of Chongqing Medical University (Chongqing, China). ET-11–31 was obtained from the Peptide Institute (Tokyo, Japan) and kynurenine (KYN), BQ123, and BQ788 were obtained from Sigma-Aldrich (St Louis, Missouri, USA). Organic reagents and phenylephrine were obtained from Shanghai Biotechnology Ltd. (Shanghai, China). General procedure
Male Sprague–Dawley rats (weighing between 300 and 350 g) were used in this study. Animal preparation, DOI: 10.1097/WNR.0000000000000149
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Cardiovascular effects of endothelin-11–31 in NTS Li et al. 613
histologic procedures, and microinjections were executed according to institutional guidelines [12]. To test the cardiovascular effects of injection of ET-11–31 (0.5–2.0 pmol) at different dosage levels into the NTS and compare unilateral and bilateral doses, 50 male Sprague–Dawley rats were divided into five groups. Two groups were set aside as the bilateral (n = 8) and unilateral (n = 4) artificial cerebrospinal fluid (aCSF) control groups. Another two groups were administered bilateral (n = 21) and unilateral (n = 21) microinjections of ET-11–31 in a dose-dependent manner. The remaining group (n = 7) was bilaterally microinjected with ET-11–31 to observe the effects of ET-11–31 on the arterial baroreflex (BRS) function within the NTS. A further 29 rats were pretreated with the ETA receptor antagonist BQ123, the ETB receptor antagonist BQ788, or the nonselective glutamate receptor antagonist KYN to explore the mechanisms of action of ET-11–31 in the NTS. A catheter, inserted into the right femoral artery, was connected to a pressure transducer (PT400; Omega, Washington, District of Columbia, USA) to directly measure blood pressure (BP). The heart rate (HR) was computed from the BP waveforms and displayed on a channel of the recording system.
Microinjection procedure
NTS microinjections were administered using a method referred to in the relevant literature [13]. The injections were made into the NTS using stereotaxic coordinates from the rat brain atlas (Paxinos and Watson, 1986): 0.5–0.8 mm lateral to the medial line, 0.5–0.8 mm caudal to the bregma, and 0.5 mm deep from the bone surface. ET-11–31 was initially dissolved in 0.1% acetic acid and diluted with aCSF (in mM: 3.4 KCl, 133.3 NaCl, 1.3 CaCl2, 0.6 NaH2PO4, 32.0 NaHCO3, 1.2 MgCl2, and 3.4 glucose, pH 7.4). The final concentration of dimethyl sulfoxide in aCSF was not more than 1%. BQ788 (20 pmol) and KYN (20 pmol) were initially dissolved in dimethyl sulfoxide and then diluted in aCSF, and BQ123 (20 pmol) was dissolved in double distilled water. All drugs were slowly (over 30 s) injected into the NTS in a volume of 100 nl.
BRS measurement
BRS measurements were made on the basis of procedures described in the literature [14]. Once a rat’s BP was stable, a rapid intravenous infusion of 0.4 g/l phenylephrine raised the BP by 30–40 mmHg. Systolic BP was regarded as the independent variable and cardiac cycle (heart period, HP) as the dependent variable in the linear regression analysis (the correlation coefficient R2 > 0.8). The slope of the line of the resulting regression is the BRS (ms/mmHg). The data were recorded using a computer at three time points (preadministration, and 5 and 10 min after medication).
Statistical analysis
All of the values are expressed as mean±SE. BP is reported as the MAP, which was calculated as diastolic + [(systolic – diastolic)/3]. The magnitudes of the changes in MAP and HR at 5 and 10 min after injection of the agents were compared with that on injection of the vehicle by a one-way analysis of variance. If the difference was significant, further pairwise comparisons were performed using analysis of covariance (Dunnett’s test) and a significance level of 0.05.
Results Changes in MAP and HR
Bilaterally (n = 7) or unilaterally (n = 7) microinjected ET-11–31 produced dose-dependent hypotension and bradycardia. A unilateral NTS microinjection of ET-11–31 generated hypotension (from – 10±2 to – 22±3 mmHg, P < 0.05) and bradycardia (from – 11±4 to – 21±3 bpm, P < 0.05). The effects of unilateral injections were similar to those of bilateral injections, and were not shorter or less intense. In control tests, bilateral and unilateral microinjections of the same volume of aCSF had no effect on BP or HR. With both bilateral and unilateral microinjections, the cardiovascular effects began within 30 s of microinjection, lasted 1–2 min, and then returned to normal. The changes in MAP and HR (DMAP and DHR) in response to microinjection of ET-11–31 into the NTS are summarized in Tables 1 and 2. Effects on BRS function
Bilateral administration of ET-11–31 into the NTS timedependently attenuated BRS function. Typically, BRS function significantly decreased in the 5 min following
Table 1 The effects of bilateral microinjection of ET-11–31 into the NTS on DMAP and DHR aCSF
ET-11–31
Groups
100 nl
0.5 pmol
1.0 pmol
2.0 pmol
DMAP (mmHg) DHR (bpm)
– 1±1 – 3±2
– 10±2* – 11±4*
– 16±3* – 16±4*
– 22±3* – 21±3*
Values are means±SE. aCSF, artificial cerebrospinal fluid; HR, heart rate; ET-1, endothelin-1; MAP, mean arterial pressure; NTS, nucleus tractus solitarius. *P < 0.05 compared with the control group.
Table 2 The effects of unilateral microinjection of ET-11–31 into the NTS on the DMAP and DHR aCSF Groups DMAP (mmHg) DHR (bpm)
ET-11–31
100 nl
0.5 pmol
1.0 pmol
2.0 pmol
– 0.5±1 – 2±2
– 7±3* – 5±4*
– 12±3* – 11±6*
– 18±3* – 16±5*
Values are means±SE. aCSF, artificial cerebrospinal fluid; HR, heart rate; ET-1, endothelin-1; MAP, mean arterial pressure; NTS, nucleus tractus solitarius. *P < 0.05 compared with the control group.
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614 NeuroReport 2014, Vol 25 No 8
Fig. 1
(a)
HR (bpm)
BP (mmHg)
Baseline
5 min
Baseline
30 min
5 min
30 min
150
50 500
400
(b) 1.0
BRS (ms/mmHg)
BRS (ms/mmHg)
1.0
0.5
0.0
Baseline
5 min aCSF
30 min
n=7
∗
∗
5 min
30 min
0.5
0.0
Baseline
ET-11–31 n=7
(a) Representative tracings showing the BP and HR responses to microinjections of aCSF and ET-11–31 into the NTS. (b) Bar graphs showing the effects of bilateral microinjection of ET-11–31 (1.0 pmol) or aCSF into the NTS on the BRS response. *P < 0.05 compared with baseline. aCSF, artificial cerebrospinal fluid; BP, blood pressure; BRS, baroreflex; HR, heart rate; ET-1, endothelin-1; NTS, nucleus tractus solitarius.
intra-NTS application of ET-11–31 and the changes increased over the period of action of the drug (30 min after the treatment). The effects of intra-NTS administration of ET-11–31 or aCSF on BP and HR are summarized in Fig. 1.
microinjection of ET-11–31 into the NTS. The effects of the ET receptor antagonists and aCSF on BP and HR following a unilateral microinjection of ET-11–31 into the NTS are summarized in Fig. 2.
Effects of blockade of ETA and ETB receptors
Effects of the nonspecific glutamate receptor antagonist KYN
Pretreatment with BQ123 completely prevented the lowering of BP and significantly reduced the slowing of HR caused by the microinjection of ET-11–31 (1.0 pmol) into the NTS (P < 0.05). In contrast, prior microinjection of the ETB receptor antagonist BQ788 (20 pmol) into the NTS did not alter the ET-11–31-induced reduction of BP and HR (P > 0.05). Microinjection of the same volume of aCSF into the NTS had no effect on the basal BP and HR or on the hypotension and bradycardia responses to
Unilateral pretreatment with KYN (20 pmol) significantly inhibited the hypotension and bradycardia (P < 0.05) arising from the intra-NTS microinjection of ET-11–31 (1.0 pmol). Unilateral microinjection of the same volume of aCSF did not affect MAP and HR responses to ET-11–31 within the NTS. The changes in MAP and HR in response to the microinjection of ET-11–31 into the NTS after pretreatment with KYN are illustrated in Fig. 3.
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Cardiovascular effects of endothelin-11–31 in NTS Li et al. 615
Fig. 2
Fig. 3
aCSF+ET-11–31
(a)
n=4
BQ123+ET-11–31
n=7
BQ788+ET-11–31
(a)
n=7
0
0
aCSF+ET-11–31
KYN+ET-11–31
n=4
n=7
−10
Change in MAP (mmHg)
Change in MAP (mmHg)
∗
−20
∗
−10
−20
−30 aCSF+ET-11–31
(b) 0
n=4
BQ123+ET-11–31
n=7
−30
BQ788+ET-11–31
n=7
(b) 0
KYN+ET-11–31
n=4
n=7
−10
∗
−20
Change in HR (bmp)
Change in HR (bpm)
∗
aCSF+ET-11–31
−10
−20
−30
Bar graphs showing the effects of pretreatment with BQ123 (20 pmol), BQ788 (20 pmol), and aCSF on (a) hypotension (BP) and (b) bradycardia (HR) responses to microinjection of ET-11–31 (1.0 pmol) into the NTS. *P < 0.05 compared with aCSF. aCSF, artificial cerebrospinal fluid; BP, blood pressure; HR, heart rate; ET-1, endothelin-1; NTS, nucleus tractus solitarius.
Discussion Within the low brain stem, cerebellum, and spinal cord, it is well known that ET-1 binding sites are widely distributed in the area postrema, the spinal nucleus of the trigeminal nerve, the dorsal motor nucleus of the vagus nerve, the hypoglossal nuclei, and the NTS, suggesting that ET-1 may play an important role in central cardiovascular regulation [15]. In addition, the NTS, the first level of the central baroreflex, accepts projections from arterial baroreceptors, chemoreceptors, and other cardiovascular receptors and integrates the information projected in the CVLM and RVLM to maintain the relative stability of BP [16]. Our previous studies have suggested that ET-11–31 regulates cardiovascular activity in anesthetized rats through the lateral
−30 Bar graphs showing the effects of pretreatment with KYN on (a) hypotension (BP) and (b) bradycardia (HR) responses to microinjection of ET-11–31 into the NTS. *P < 0.05 compared with aCSF. aCSF, artificial cerebrospinal fluid; BP, blood pressure; HR, heart rate; ET-1, endothelin-1; KYN, kynurenic acid; NTS, nucleus tractus solitarius.
ventricle or RVLM [7,8]. However, the physiological role of ET-11–31 in the regulation of cardiovascular function is still not fully understood. Further, the cardiovascular responses caused by intra-NTS ET-11–31 administration have, until now, not been reported. Therefore, the main objective of this study was to obtain evidence that intraNTS ET-11–31 plays a key role in cardiovascular responses. It has been proven that ET-11–21 participates in the regulation of cardiovascular activity in the central nervous system [17]. Early data have revealed that the
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616 NeuroReport 2014, Vol 25 No 8
cardiovascular effects of exogenous ET-11–21 are relied upon to activate the autonomic nervous system at the level of the NTS [18]. Previous research has also indicated that the microinjection of ET-11–21 into the NTS not only decreases MAP but also lowers HR in vivo [19]. However, the exact mechanisms of these depressor effects are controversial. Some argue that, accompanied by a reduction in blood flow to the brain, the sustained hypotension by centrally administered ET-1 is due largely to intense vasoconstriction caused by medullary ischemia [20]. Others hold that the hypotensive action of centrally administered ET-1 may be a consequence of bradycardia [19]. Given that bradycardia can be completely eliminated by the intravenous injection of hexamethonium, it is possible that bradycardia produced by the microinjection of ET-11–21 into the NTS may depend on the activation of the vagal nerve [18]. In this study, we increased local ET-11–31 concentrations through intra-NTS microinjection of ET-11–31 in anesthetized rats to investigate the cardiovascular effects of ET-11–31 in the NTS. Our results suggest that bilateral and unilateral NTS microinjections of ET-11–31 (0.5–2.0 pmol) produce dose-dependent hypotension and bradycardia responses. Bilateral microinjection of ET-11–31 into the NTS (1.0 pmol) also appeared to significantly inhibit the BRS function in anesthetized rats. The effects of microinjection of ET-11–31 into the NTS were similar to those produced by ET-11–21 and big endothelin (big ET-1), suggesting that the cardiovascular effects produced by ET-11–31 in the NTS are likely to be consistent with those produced by ET-11–21. Further experiments have shown that the cardiovascular effects produced by ET-11–31, such as lowered BP, slower HR, and suppressed BRS, are mediated by the ETA receptor rather than by the ETB receptor. Glutamate receptors, which play an important role in cardiovascular regulation in the medulla oblongata, are widely distributed in the NTS and other regions of the medulla oblongata [21]. Therefore, we hypothesized that the cardiovascular effects produced by ET-11–31 in the NTS may be mediated by a glutamate receptor. To test this hypothesis, the glutamate receptor antagonist KYN was used. The results show that microinjection of KYN into the NTS partially blocks the hypotension and bradycardia responses induced by a subsequent microinjection of ET11–31 into the NTS, which suggests that the cardiovascular effects caused by ET-11–31 in the NTS are, at least in part, produced through the activation of glutamate receptors that then activate the relevant neurons.
Conclusion ET-11–31 within the NTS caused hypotension and bradycardia and significantly decreased the BRS function in rats. Further study indicated that the cardiovascular
effects of ET-11–31 within the NTS are mediated by the ETA receptor and, at least in part, by the glutamate receptor. These findings suggest that ET-11–31 plays a significant role in the physiological alteration of cardiovascular effects in rats.
Acknowledgements The authors thank Li Ma and Zi-Li Li for their helpful discussions and expert statistical assistance, respectively. Conflicts of interest
There are no conflicts of interest.
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