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. Author manuscript; available in PMC: 2019 Oct 1.
Published in final edited form as: Hypertension. 2018 Oct;72(4):994–1001. doi: 10.1161/HYPERTENSIONAHA.118.11497

Corticotropin-Releasing Hormone Receptor 2 in the Nucleus of the Solitary Tract Contributes to Intermittent Hypoxia-Induced Hypertension

Lei A Wang 1, Dianna H Nguyen 1, Steve W Mifflin 1
PMCID: PMC6205726  NIHMSID: NIHMS1500961  PMID: 30354709

Abstract

This study tested the hypothesis that corticotropin-releasing hormone (CRH) receptors in the nucleus of the solitary tract (NTS) contribute to the hypertension induced by intermittent hypoxia (IH) exposure in rats. Initial studies using in situ hybridization revealed low mRNA level of CRH type 1 receptor (CRHR1) but high mRNA level of CRH type 2 receptor (CRHR2) in the NTS. Calcium imaging studies on NTS slice preparations using Fura-2-acetoxymethyl ester demonstrated that CRH induced a transient increase of intracellular calcium level. The CRH-induced calcium response was reproduced in the presence of TTX but was abolished by depletion of extracellular calcium or by the L-type calcium channel blocker nifedipine. The CRH-induced calcium influx was attenuated by the CRHR2 antagonist K41498 but not by the CRHR1 antagonist NBI-35965. Calcium influx can be induced by the CRHR2 agonist Urocortin II but not by the CRHR1 agonist Stressin I. IH exposure did not affect CRHR1 mRNA level but significantly decreased CRHR2 mRNA level and the CRH-induced calcium influx in the NTS. Further in vivo studies showed that intra-4th ventricle infusion of K41498 did not affect the basal blood pressure but significantly attenuated the IH-induced hypertension; intra-4th ventricle infusion of Urocortin II significantly increased basal blood pressure and exacerbated the IH-induced hypertension. Collectively, these results suggest that CRHR2 in the NTS contributes to the IH-induced hypertension; down-regulation of CRHR2 and CRHR2-mediated calcium influx in the NTS may serve as an adaptive response to protect against the IH-induced hypertension.

Keywords: intermittent hypoxia, CRH, CRF, CRHR2, CRFR2, NTS, hypertension

Introduction

Sleep apnea is a major risk factor for hypertension and cardiovascular diseases1. It is estimated that 26% of US population between 30 and 70 years old have sleep apnea2. More importantly, the prevalence of sleep apnea in the US has increased 28% in men and 32% in women during the last three decades2. Patients with sleep apnea exhibit increases in peripheral chemoreflex sensitivity, sympathetic nerve activity, and blood pressure3,4. The sympathetic activation and hypertension occur not only during sleep but also when awake3. Treatments that reduce sleep apnea induced hypertension also decrease sympathetic activity3. This evidence indicates that sympathetic activation contributes to sleep apnea induced hypertension.

Exposing rats to intermittent hypoxia (IH) mimics the arterial hypoxemias that occur in patients with sleep apnea, resulting in sympathetic activation and hypertension5–7. IH exposure activates neurons in the paraventricular nucleus of the hypothalamus (PVN)8. The PVN contains a large population of neurons synthesizing corticotropin releasing hormone (CRH), indicating that IH may activate CRH-producing neurons in the PVN. Indeed, one study has reported that IH exposure increased the CRH mRNA levels in the hypothalamus9. CRH is well known to act in the pituitary gland to activate the hypothalamic-pituitary-adrenal axis, a key pathway in regulating the responses to stress. Interestingly, CRH-producing neurons in the PVN also project to the rostral ventrolateral medulla (RVLM)10,11 and the nucleus of the solitary tract (NTS)12, where they may regulate sympathetic activity and blood pressure10,13–16. There are two types of CRH receptors: CRH type 1 and type 2 receptors (CRHR1 and CRHR2)17. Both CRHR1 and CRHR2 are expressed in the NTS18, a key nucleus in integrating the peripheral chemoreflex and baroreflex inputs and regulating the sympathetic outflow. Neurons in the NTS have been shown to contribute to sustained increase of blood pressure during IH exposure19,20, but the role of NTS CRH receptors in IH-induced hypertension remains unknown.

The present study tested the hypothesis that CRH receptors in the NTS contribute to IH-induced hypertension. We first used in situ hybridization to study how IH exposure influences the anatomical distribution of CRH receptors in the NTS. We then conducted calcium imaging on brain slices to study how CRH regulates intracellular calcium dynamics of NTS neurons and how IH exposure affects the CRH-induced changes of intracellular calcium levels in NTS neurons. Based on the findings from the in situ hybridization and calcium imaging experiments, we chronically infused agonist and antagonist for CRHR2 into the 4th ventricle of rats and studied the cardiovascular responses to IH exposure.

Methods

The data that support the findings of this study are available from the corresponding author upon reasonable request. For detailed methods, please see the Data Supplement at http://hyper.ahajournals.org.

Animals

Male Sprague-Dawley rats (8 weeks old, 200–250 g), purchased from Charles River Laboratories (Wilmington, MA), were individually housed in a temperature-controlled room on a 12 h light:12 h dark cycle (on at 0700, off at 1900) and given ad libitum access to food and water. Rats that did not recover from surgeries or lost telemetry signals were excluded from the study. The study used only male rats because female rats (similar age compared to the male rats used in the present study) are protected from the hypertension induced by IH exposure21. All procedures were approved by the University of North Texas Health Science Center Institutional Animal Care and Use Committee.

Intermittent Hypoxia

The IH protocol has been previously described20. Briefly, individually housed rats were placed in custom-built plexiglass chambers and were exposed to cycles of hypoxia. Each cycle consisted of 3 mins of 10% O2 followed by 3 mins of 21% O2. Rats were exposed to IH for 80 cycles (8 h, 0800 to 1600) daily for 7 consecutive days. Rats were euthanized the day after the 7 days IH exposure.

Data Analysis

Group Assignment, data collection, and data analysis were conducted by the same people. Data were analyzed using RStudio22 and were visualized using the ‘ggplot2’ package. Figures were assembled using the ‘magick’ package. In situ hybridization results were analyzed using two-way mixed model ANOVA with ‘receptor type’ (CRHR1 vs. CRHR2) or ‘treatment’ (CON vs. IH) as a between subject factor and ‘location’ (Distance caudal to Bregma) as a within subject factor. Calcium imaging results were analyzed using one way ANOVA with post hoc Tukey’s honest significance test; the results were also analyzed using Chi-Square test to determine whether different treatments affected the percentage of cells responded. The 24-hour average mean arterial pressure (MAP) and heart rate (HR) values were calculated to represent daily levels for each treatment group; the results were then analyzed using two-way mixed model ANOVA with ‘treatment’ as a between subject factor and ‘day’ as a within subject factor. Post hoc comparisons for two-way mixed model ANOVA were conducted using the least squares mean method with p values adjusted using Tukey method and degrees of freedom calculated using satterthwaite method; Post hoc comparisons were conducted using the ‘lsmeans’ package. Significance level was set at P < 0.05.

Results

NTS had higher mRNA levels of CRHR2 than CRHR1

Using RNAscope in situ hybridization, we detected both CRHR1 and CRHR2 mRNAs in the caudal NTS (13.68 – 14.76 mm caudal to Bregma; Figure 1, Figure S2, and Figure S4). Two-way mixed model ANOVA analysis revealed significant effects of the factor ‘receptor type’ (P < 0.001; F(1,30) = 101.39), the factor ‘location’ (P < 0.001, F(9,27) = 13.29), and the interaction between the factors ‘receptor type’ and ‘location’ (P < 0.001, F(9,30) = 16.57). Post hoc multiple comparisons of the mRNA levels between the factor ‘receptor type’ within the factor ‘location’ revealed that CRHR2 mRNA levels were significantly higher than CRHR1 mRNA levels in the segment of NTS that is 13.92–14.28 mm caudal to Bregma (P <= 0.0007 in all 4 locations, t values ranged from 3.39 to 10.35; Figure 1 E & F).

Figure 1.

Figure 1.

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IH exposure reduces CRHR2 but not CRHR1 mRNA levels in the NTS. (A & C) CRHR1 mRNA (punctate white dots) and (B & D) CRHR2 mRNA (punctate white dots) in the NTS (14.04 mm caudal to Bregma) of (A-B) CON rats (n = 4) and (C-D) IH-exposed rats (n = 3). Images illustrating a larger area of the NTS are presented in Figure S2–S5. Scale bars = 20 μm. (E-F) Summary of the effects of IH exposure on mRNA levels of CRHR1 and CRHR2 in the NTS. #, P < 0.05 CON CRHR1 vs. CON CRHR2; *, P < 0.05 CON CRHR2 vs. IH CRHR2.

IH decreased CRHR2 but not CRHR1 mRNA levels in the NTS

Two-way mixed model ANOVA analysis revealed that IH did not affect the CRHR1 mRNA level (P = 0.91, F(1,5) = 0.01; CON, n = 4; IH, n = 3; Figure 1, Figure S2, and Figure S3) but significantly decreased the CRHR2 mRNA level (P = 0.0001, F(1,5) = 108.2; CON, n = 4; IH, n = 3; Figure 1, Figure S3, and Figure S4) in the NTS. Post hoc multiple comparisons revealed that the decrease of CRHR2 mRNA level was mainly localized in the segment of NTS that is 13.92 – 14.28 mm caudal to Bregma (13.92 mm - 14.16 mm, P <= 0.0004, t values ranged from 3.52 to 8.69; 14.28 mm, P = 0.01, t = 2.53; Figure 1F).

CRH induced calcium influx in NTS neurons

Perfusion of 100 nM CRH elicited an increase of intracellular calcium level in NTS neurons (Figure 2A). This response was reproduced in the presence of TTX (Figure 2B). TTX did not affect the percentage of cells that responded to CRH (without TTX 21.9% (18 out of 82 cells from 10 brain slices) vs. with TTX 19.0% (4 out of 21 cells from 3 brain slices), Chi-square test: P = 0.38) or the magnitude of calcium response induced by CRH (Normalized calcium response to CRH: without TTX, 57.1% ± 5.17% vs. with TTX, 42.1% ± 8.52%; P = 0.67), indicating that the CRH-induced calcium response was independent of action potentials. The CRH-induced increase of intracellular calcium level was abolished by depletion of extracellular calcium (0 out of 79 cells responded from 9 brain slices, Figure 2C) or by the L-type calcium channel blocker nifedipine (0 out of 49 cells responded from 7 brain slices, Figure 2D), indicating that CRH induced calcium influx.

Figure 2.

Figure 2.

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CRH induces calcium influx in NTS neurons. (A-B) CRH induces increase of intracellular calcium level (indicated by the ratio of 550 nm fluorescent intensity excited by 340nm vs. 380nm) in NTS neurons in the absence of TTX (A, 18 out of 82 cells showed response) or in the presence of TTX (B, 4 out of 21 cells showed response). (C-D) Perfusion of CRH causes no change of intracellular calcium levels when (C) extracellular calcium was depleted (0 out of 79 cells showed response) or when (D) CRH was co-perfused with Nifedipine (0 out of 49 cells showed response). Horizontal scale bars = 100 sec; Vertical scale bars = 0.05 (Δ 340nm/380nm Ratio).

CRH induced calcium influx in NTS neurons by activating CRHR2

Co-perfusion of CRH and CRHR1 antagonist NBI-35965 induced calcium influx (13 out of 79 cells responded from 7 brain slices, Figure 3A) with similar magnitude compared to that induced by CRH alone (P ≈ 1, Figure 3E). Co-perfusion of CRH and CRHR2 antagonist K41498 induced calcium influx (11 out of 124 cells responded from 8 brain slices, Figure 3B) but the magnitude of the response was significantly smaller than that induced by CRH alone (P = 0.003, Figure 3E). CRHR1 agonist Stressin-1 did not affect intracellular calcium level (0 out of 121 cells responded from 14 brain slices, Figure 3C) but CRHR2 agonist Urocortin-II induced calcium influx (9 out of 69 cells responded from 7 brain slices, Figure 3D) similar to that induced by CRH (P = 0.58, Figure 3E).

Figure 3.

Figure 3.

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CRH induces calcium influx in NTS neurons by activating CRHR2. (A-D) Responses of NTS intracellular calcium levels to (A) co-perfusion of CRH and CRHR1 antagonist NBI35963 (13 out of 79 cells responded), (B) co-perfusion of CRH and CRHR2 antagonist K41498 (11 out of 124 cells responded), (C) perfusion of CRHR1 agonist Stressin 1 (0 out of 121 cells responded), and (D) perfusion of CRHR2 agonist Urocortin II (9 out of 69 cells responded). Horizontal scale bars = 100 sec; Vertical scale bars = 0.05 (Δ 340nm/380nm Ratio). (E) Summary of the magnitude of calcium influx induced by different drugs. * P < 0.05.

IH attenuated the CRH-induced calcium influx in NTS neurons

To study whether IH-induced down-regulation of CRHR2 mRNA leads to reduction of the CRH-induced calcium influx, we compared the CRH-induced calcium influx in IH-exposed rats to that in control rats. The magnitude of the CRH-induced calcium influx in IH-exposed rats was 44.4% less compared to that in control rats (P = 0.001; Figure 4 A-C). IH exposure also significantly decreased the percentage of cells that responded to CRH (CON 21.9% (18 out of 82 cells from 10 brain slices) vs. IH 11.1% (19 out of 171 cells from 15 brain slices), Chi-square test: P = 0.036).

Figure 4.

Figure 4.

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IH exposure reduces the CRH-induced calcium influx in NTS neurons. (A-B) CRH-induced calcium influx in NTS neurons of (A) a CON rat (18 out of 82 cells responded) and (B) a rat exposed to IH (19 out of 171 cells responded). Horizontal scale bars = 100 sec; Vertical scale bars = 0.05 (Δ 340nm/380nm Ratio). (C) The effect of IH exposure on the magnitude of CRH-induced calcium influx. * P < 0.05.

Intra-4th ventricle infusion of CRHR2 antagonist attenuated IH-induced hypertension during the initiation phase

Rats were randomly assigned to aCSF treatment group (n = 7) or K41498 treatment group (n = 6). At the beginning of the study, two treatment groups had no difference in baseline MAP (aCSF, 104 ± 1 mmHg vs. K41498, 106 ± 1 mmHg; P = 0.217, t (11) = 1.31) or HR (aCSF, 371 ± 28 BPM vs. K41498, 391 ± 8 BPM; P = 0.53, t (11) = 0.65).

Intra-4th ventricle infusion of K41498 (Dose: 688 pmol/h) did not affect the basal MAP but significantly attenuated IH-induced increase of MAP (aCSF, n = 7; K41498, n = 6; Figure 5A). Two-way mixed model ANOVA analysis revealed significant effects of the factor ‘day’ (P < 0.0001, F(11,121) = 3.16) and the interaction between the factors ‘day’ and ‘treatment’ (aCSF or K41498) (P = 0.01, F(11, 121) = 2.36). Post hoc multiple comparisons of the MAP between the factor ‘day’ within the factor ‘treatment’ revealed that, in rats infused with aCSF, the MAP was significantly increased from day 6 to day 12 (day 6, P = 0.005, t(121) = 4.07; day 7–12 P <= 0.0001, t(121) ranged from 5.20 – 7.12; Figure 5A) compared to day 5 (the day before IH started), suggesting that IH significantly increased MAP in rats infused with aCSF. Post hoc multiple comparisons of the MAP between the factor ‘treatment’ within the factor ‘day’ revealed that the MAP was significantly decreased in rats infused with K41498 compared to rats infused with aCSF from day 7 to day 9 (day 7, P = 0.03, t(36.61) = 2.24; day 8, P = 0.003, t(36.61) = 3.20; day 9, P = 0.03, t(36.61) = 2.23; Figure 5A). The MAP was not different between rats infused with K41498 and rats infused with aCSF from day 10 to day 12 (P values ranged from 0.07 to 0.1; t values ranged from 1.67 to 1.86). We also found that the average MAP during the 7-day IH exposure period was significantly decreased by 40% in rats infused with K41498 compared to rats infused with aCSF (aCSF, 5 ± 1 mmHg vs. K41498, 3 ± 1 mmHg; P = 0.03, t(11) = 2.47). These results combined suggest that K41498 significantly attenuated IH-induced hypertension during the initiation phase. A lower dose of K41498 (100 pmol/h) was initially tested and did not affect the MAP at basal condition (average Delta MAP of day 2–5; aCSF, 1 ± 1 mmHg vs. low dose K41498, 1 ± 2 mmHg; P = 0.90) or during IH exposure (average Delta MAP of day 6–12; aCSF, 5 ± 1 mmHg vs. low dose K41498, 5 ± 1 mmHg; p = 0.92).

Figure 5.

Figure 5.

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Effects of activating/blocking hindbrain CRHR2 on MAP and HR. (A-B) Intra-4th ventricle infusion of CRHR2 antagonist K41498 did not affect the basal levels of (A) MAP or (B) HR but significantly attenuated the increase of MAP and HR induced by IH exposure. aCSF, n = 7; K41498, n = 6. (C) Intra-4th ventricle infusion of CRHR2 agonist Urocortin II increased MAP at basal condition and during IH exposure but (D) did not affect HR. Vehicle, n = 5; Urocortin II, n = 5. * P < 0.05 aCSF vs. K41498 or Vehicle vs. Urocortin II; @ P < 0.05 vs. day 5 in control rats infused with aCSF or Vehicle.

Intra-4th ventricle infusion of K41498 (Dose: 688 pmol/h) did not affect the basal HR but significantly attenuated IH-induced increase of HR (aCSF, n = 7; K41498, n = 6; Figure 5B). Two-way mixed model ANOVA analysis revealed significant effects of the factor ‘day’ (P < 0.0001, F(11,121) = 4.31) and the interaction between the factors ‘day’ and ‘treatment’ (aCSF or K41498) (P = 0.0006, F(11, 121) = 3.26). Post hoc multiple comparisons of the HR between the factor ‘day’ within the factor ‘treatment’ revealed that, in rats infused with aCSF, the HR was significantly increased on day 7–9 and day 11 (day 7, P = 0.02, t(121) = 3.53; day 8, P = 0.001, t(121) = 4.42; day 9, P = 0.0001, t(121) = 5.19; day 11, P = 0.003, t(121) = 4.18; Figure 5B) compared to day 5 (the day before IH started), suggesting that IH significantly increased HR in rats infused with aCSF. Post hoc multiple comparisons of the HR between the factor ‘treatment’ within the factor ‘day’ revealed that the HR was significantly decreased in rats infused with K41498 compared to rats infused with aCSF on day 8–9 and day 11–12 (day 8, P = 0.025, t(62.3) = 2.30; day 9, P = 0.015, t(62.3) = 2.50; day 11, P = 0.02, t(62.3) = 2.38; day 12, P = 0.048, t(62.3) = 2.02; Figure 5B).

Intra-4th ventricle infusion of CRHR2 agonist exacerbated IH-induced hypertension

Rats were randomly assigned to vehicle treatment group (n = 5) or Urocortin II treatment group (n = 5). At the beginning of the study, two treatment groups had no difference in baseline MAP (vehicle, 102 ± 2 mmHg vs. Urocortin II, 99 ± 3 mmHg; P = 0.52, t (8) = 0.67) or HR (vehicle, 315 ± 50 BPM vs. Urocortin II, 243 ± 31 BPM; P = 0.24, t (8) = 1.28).

Intra-4th ventricle infusion of Urocortin II (Dose: 100 pmol/h) significantly increased the basal MAP and the MAP during IH exposure (Vehicle, n = 5; Urocortin II, n = 5; Figure 5C). Two-way mixed model ANOVA analysis revealed significant effects of the factor ‘day’ (P < 0.0001, F(11,88) = 34.2), the factor ‘treatment’ (Vehicle or Urocortin II) (P = 0.001, F(1,8) = 25.4), and the interaction between the factors ‘day’ and ‘treatment’ (P < 0.0001, F(11, 88) = 6.68). Post hoc multiple comparisons of the MAP between the factor ‘day’ within the factor ‘treatment’ revealed that, in rats infused with aCSF, the MAP was significantly increased from day 7 to day 12 (day 7, P = 0.01, t(88) = 1.42; day 8 – 12, P <= 0.0003, t(88) ranged from 4.88 to 5.96; Figure 5C) compared to day 5 (the day before IH started), suggesting that IH significantly increased MAP in rats infused with Vehicle (10% DMSO in aCSF). Post hoc multiple comparisons of the MAP between the factor ‘treatment’ within the factor ‘day’ revealed that the MAP was significantly increased in rats infused with Urocortin II compared to rats infused with vehicle from day 3 to day 12 (P < 0.001 in all comparisons, t(20.6) ranges from 3.90 to 5.38, Figure 5C), suggesting that Urocortin II significantly increased baseline blood pressure and exacerbated IH-induced hypertension.

Intra-4th ventricle infusion of Urocortin II did not affect the HR at baseline condition or during IH exposure (Vehicle, n = 5; Urocortin II, n = 5). Two-way mixed model ANOVA analysis revealed no effects of the factor ‘day’ (P = 0.46, F(11,88) = 0.99), or the factor ‘treatment’ (P = 0.99, F(1,8) = 0), or the interaction between the factors ‘day’ and ‘treatment’ (P = 1, F(11, 88) = 0.087).

Discussion

CRH has been well documented to regulate blood pressure by acting in the central nervous system. A number of studies have shown that ICV injection of CRH increased blood pressure23–28. ICV injection of CRH elevated blood pressure in hypophysectomized rats and dexamethasone-treated rats but not in ganglionically blocked rats24. ICV injection of CRH increased sympathetic nerve activity and/or plasma catecholamine levels in rats and rabbits26,29,30. These studies suggest that sympathetic activation mediates the CRH-induced pressor response.

We found that the NTS, a key nucleus regulating sympathetic function, expresses high level of CRHR2 mRNA and relatively lower level of CRHR1 mRNA. This finding is consistent with a previous study by Van Pett and colleagues18. The study by Van Pett et al. also showed that among all the brain stem regions that regulate sympathetic outflow to the cardiovascular system, CRHR2 is highly expressed in the NTS18. Therefore, the effects of intra-4th ventricle infusion of CRHR2 agonist/antagonist on IH-induced hypertension can be mainly attributed to the CRHR2 located in the NTS. The current study also found that IH exposure attenuated the mRNA level of CRHR2 in the NTS but did not affect that of CRHR1, indicating that IH exposure down-regulates CRHR2 signaling pathways in the NTS.

The CRHR2 signaling pathways in the NTS have not been well established. The current study is the first to show that CRH increases intracellular calcium levels in NTS neurons by activating CRHR2 rather than CRHR1. This response was also observed in the presence of TTX, suggesting that the CRH-induced calcium response was independent of action potentials. The CRH-induced calcium response was abolished by depleting extracellular calcium or by perfusion of nifedipine, indicating that CRH induces calcium influx from the extracellular region. The effect of the CRH-induced calcium influx on neuronal activity remains unknown. Increasing intracellular calcium levels may activate calmodulin-dependent protein kinases, which can increase the single channel conductance of glutamate receptors31 and facilitate glutamate-mediated neuronal excitation. Increasing intracellular calcium levels may activate calcium gated potassium channels32, resulting in inhibition of neuronal activity. Increasing intracellular calcium levels may regulate gene transcription33, which can lead to either facilitation or inhibtion of neuronal activity.

We then asked the question whether inhibition of CRHR2 mRNA and CRHR2-mediated calcium influx contributes to or serves as an adaptive response to IH-induced hypertension. The finding that intra-4th ventricle infusion of CRHR2 antagonist attenuated IH-induced increase of MAP indicates that activation of CRHR2 contributes to IH-induced hypertension. Therefore, down-regulation of CRHR2 signaling pathway is likely an addptive response to protect against IH-induced hypertension. Activating CRHR2 in the NTS compromises this adaptive defensive mechanism, which explains why intra-4th ventricle infusion of CRHR2 agonist Urocortin II exacerbated IH-induced hypertension.

The current study observed that IH modestly increased MAP by ~ 5 mmHg, which is consistent with many previous studies5–7. Intra-4th ventricle infusion of CRHR2 antagonist attenuated IH-induced increase of MAP by 40%, indicating CRHR2 plays an important role in IH-induced hypertension. During the 7-day IH exposure, CRHR2 antagonist only attenuated the increase of MAP from day 2–4 but did not significantly affect the MAP from day 5–7, suggesting that CRHR2 mainly contributes to the initiation phase of IH-induced hypertension.

The cellular and molecular mechanisms how down-regulation of CRHR2 signaling pathways protects against IH-induced hypertension remain unestablished. It is well established that IH-induced hypertension is dependent on chemoreceptor inputs since carotid body denervation abolished IH-induced sympathetic activation and hypertension5,34,35. Increased chemoreceptor afferent activity can lead to sustained activation and increased reactivity of neurons in the NTS. Indeed, in vitro studies have reported that IH exposure not only increased the basal discharge rate of NTS neurons but also augmented the response of NTS neurons evoked by acute repetitive stimulation of the solitary tract at a rate similar to hypoxic sensory neuron discharge in vivo36; IH exposure also facilitated the response of NTS neurons to excitatory amino acid α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)37. Knocking down tyrosine hydroxylase or the transcription factor ΔFosB from NTS neurons attenuated IH-induced hypertension19,20, suggesting activation of NTS neurons contributes to IH-induced hypertension. Based on these studies, we propose that the decrease of CRHR2-mediated calcium influx observed in the present study may serve as an adaptive response to prevent over-excitation of NTS neurons caused by increased chemoreceptor afferent activity during IH exposure. Intra-4th ventricle infusion of CRHR2 antagonist possibly strengthens this adaptive defensive mechanism by further reducing the excitability of NTS neurons, resulting in attenuation of IH-induced hypertension.

The current study reveals a pressor effect of CRHR2 in the NTS, but it cannot determine whether CRHR2 in the NTS regulates chemoreflex or baroreflex. Contrary to the present study, some previous studies indicate that CRH in the NTS decreases blood pressure by activating baroreflex. Microinjection of CRH or CRHR agonists into the NTS rapidly (within 10 s) decreased basal levels of sympathetic nerve activity and blood pressure10,14–16; the depressor response was attenuated by intra-NTS microinjection of CRHR2 antagonist15. It must be noted that the injection sites of the previous microinjections studies were localized in the medial NTS14–16, where baroreflex afferents are mainly located38, whereas the 4th ventricle infusion conducted in the current study affects a broader area of the NTS. Additionally, the microinjection studies were conducted in anesthetized rats, whereas the current study was conducted in conscious rats. These differences in experimental conditions may explain the different effects of CRHR2 on regulating blood pressure in different studies.

The NTS receives CRH inputs from the PVN and the central nucleus of the amygdala (CeA)12,39. Which specific CRH pathway(s) is/are involved in IH-induced hypertension remains undetermined. To date, there are more studies indicating the involvement of PVN than those indicating the involvement of CeA. A major role of CeA is to regulate responses to psychological stress, but IH exposure did not affect the stress-induced neuronal activation (cFos) in the amygdala40. In comparison, after IH exposure, the transcription factor ΔFosB was elevated in the PVN to mediate neuronal activation8; unfortunately, the CeA wasn’t examined in that study. Functional studies have demonstrated that PVN neurons contribute to IH-induced sympathetic activation and hypertension6,41. Based on these studies, it is likely that the descending CRH pathway from the PVN to the NTS plays an important role in IH-induced hypertension.

Supplementary Material

Long In Vivo Checklist
Online Supplement

Novelty and Significance

What Is New?

  • Exposure to intermittent hypoxia (IH) decreases corticotropin releasing hormone (CRH) type 2 receptor (CRHR2) but not CRH type 1 receptor (CRHR1) mRNA levels in the nucleus of the solitary tract (NTS).

  • CRH induces calcium influx in NTS neurons by activating CRHR2. CRH-induced calcium influx in NTS neurons was reduced by IH exposure.

  • IH-induced hypertension was attenuated by 4th ventricle infusion of CRHR2 antagonist, but was exacerbated by 4th ventricle infusion of CRHR2 agonist.

What Is Relevant?

  • The current study provided the novel observation that CRHR2 in the NTS contributes to IH-induced hypertension.

Summary

This study demonstrates that CRHR2 in the NTS plays an important role in IH-induced hypertension. Down-regulation of CRHR2 and CRHR2-mediated calcium influx in the NTS may serve as an adaptive response to protect NTS neurons from being over-activated by increased chemoreceptor afferent activity; consequently, this adaptive mechanism may limit IH-induced hypertension.

Acknowledgments

The authors thank Sissy Cross for technical assistance.

Source(s) of Funding

This work was supported by American Heart Association Postdoctoral Fellowship 18POST34050013 and National Heart, Lung, and Blood Institute Grant HL-088052.

Footnotes

Perspectives

The present study provides the first evidence that CRHR2 in the NTS plays an important role in IH-induced hypertension. Down-regulation of CRHR2 and CRHR2-mediated calcium influx in the NTS during exposure to IH may serve as an adaptive response to protect NTS neurons from being over-activated by increased chemoreceptor afferent activity. Consequently, this adaptive mechanism may limit IH-induced hypertension.

Conflict(s) of Interest/Disclosure(s)

The authors declare no conflicts of interest.

References

  • 1.Somers VK, White DP, Amin R, Abraham WT, Costa F, Culebras A, Daniels S, Floras JS, Hunt CE, Olson LJ, others. Sleep apnea and cardiovascular disease: An american heart association/american college of cardiology foundation scientific statement from the american heart association council for high blood pressure research professional education committee, council on clinical cardiology, stroke council, and council on cardiovascular nursing in collaboration with the national heart, lung, and blood institute national center on sleep disorders research (national institutes of health). Circulation. 2008;118(10):1080–1111. [DOI] [PubMed] [Google Scholar]
  • 2.Peppard PE, Young T, Barnet JH, Palta M, Hagen EW, Hla KM. Increased prevalence of sleep-disordered breathing in adults. American journal of epidemiology. 2013;177(9):1006–1014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Somers VK, Dyken ME, Clary MP, Abboud FM. Sympathetic neural mechanisms in obstructive sleep apnea. The Journal of clinical investigation. 1995;96(4):1897–1904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Narkiewicz K, Van De Borne PJ, Pesek CA, Dyken ME, Montano N, Somers VK. Selective potentiation of peripheral chemoreflex sensitivity in obstructive sleep apnea. Circulation. 1999;99(9):1183–1189. [DOI] [PubMed] [Google Scholar]
  • 5.Fletcher EC, Lesske J, Behm R, Miller C 3rd, Stauss H, Unger T. Carotid chemoreceptors, systemic blood pressure, and chronic episodic hypoxia mimicking sleep apnea. Journal of applied physiology. 1992;72(5):1978–1984. [DOI] [PubMed] [Google Scholar]
  • 6.Sharpe AL, Calderon AS, Andrade MA, Cunningham JT, Mifflin SW, Toney GM. Chronic intermittent hypoxia increases sympathetic control of blood pressure: Role of neuronal activity in the hypothalamic paraventricular nucleus. American Journal of Physiology-Heart and Circulatory Physiology. 2013;305(12):H1772–H1780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Mifflin S, Cunningham JT, Toney GM. Neurogenic mechanisms underlying the rapid onset of sympathetic responses to intermittent hypoxia. Journal of applied physiology. 2015;119(12):1441–1448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Knight WD, Little JT, Carreno FR, Toney GM, Mifflin SW, Cunningham JT. Chronic intermittent hypoxia increases blood pressure and expression of fosb/Δfosb in central autonomic regions. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 2011;301(1):R131–R139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Feng X, Kang J, Wang Z, Wang W, Yu R. The effect of chronic intermittent hypoxia to hypothalamus-pituitary-adrenal axis and growth hormone level in rats during sleep. Zhong Guo Ying Yong Sheng Li Xue Za Zhi. 2005;21(4):414–417. [PubMed] [Google Scholar]
  • 10.Milner TA, Reis D, Pickel V, Aicher S, Giuliano R. Ultrastructural localization and afferent sources of corticotropin-releasing factor in the rat rostral ventrolateral medulla: Implications for central cardiovascular regulation. Journal of Comparative Neurology. 1993;333(2):151–167. [DOI] [PubMed] [Google Scholar]
  • 11.Coldren KM, Li D-P, Kline DD, Hasser EM, Heesch CM. Acute hypoxia activates neuroendocrine, but not presympathetic, neurons in the paraventricular nucleus of the hypothalamus: Differential role of nitric oxide. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 2017;312(6):R982–R995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Sawchenko P Evidence for differential regulation of corticotropin-releasing factor and vasopressin immunoreactivities in parvocellular neurosecretory and autonomic-related projections of the paraventricular nucleus. Brain research. 1987;437(2):253–263. [DOI] [PubMed] [Google Scholar]
  • 13.Bardgett ME, Sharpe AL, Toney GM. Activation of corticotropin-releasing factor receptors in the rostral ventrolateral medulla is required for glucose-induced sympathoexcitation. American Journal of Physiology-Endocrinology and Metabolism. 2014;307(10):E944–E953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Nakamura T, Kawabe K, Sapru HN. Cardiovascular responses to microinjections of urocortin 3 into the nucleus tractus solitarius of the rat. American Journal of Physiology-Heart and Circulatory Physiology. 2009;296(2):H325–H332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Nakamura T, Sapru HN. Cardiovascular responses to microinjections of urocortins into the nts: Role of inotropic glutamate receptors. American Journal of Physiology-Heart and Circulatory Physiology. 2009;296(6):H2022–H2029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Yamazaki T, Waki H, Kohsaka A, Nakamura T, Cui H, Yukawa K, Maeda M. Microinjection of urocortin into the rat nucleus tractus solitarii decreases arterial blood pressure. Autonomic Neuroscience: Basic and Clinical. 2008;142(1):51–54. [DOI] [PubMed] [Google Scholar]
  • 17.Dautzenberg FM, Dietrich K, Palchaudhuri MR, Spiess J. Identification of two corticotropin-releasing factor receptors from xenopus laevis with high ligand selectivity: Unusual pharmacology of the type 1 receptor. Journal of neurochemistry. 1997;69(4):1640–1649. [DOI] [PubMed] [Google Scholar]
  • 18.Van Pett K, Viau V, Bittencourt JC, Chan RK, Li H-Y, Arias C, Prins GS, Perrin M, Vale W, Sawchenko PE. Distribution of mRNAs encoding crf receptors in brain and pituitary of rat and mouse. Journal of Comparative Neurology. 2000;428(2):191–212. [DOI] [PubMed] [Google Scholar]
  • 19.Bathina CS, Rajulapati A, Franzke M, Yamamoto K, Cunningham JT, Mifflin S. Knockdown of tyrosine hydroxylase in the nucleus of the solitary tract reduces elevated blood pressure during chronic intermittent hypoxia. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 2013;305(9):R1031–R1039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Wu Q, Cunningham JT, Mifflin S. Transcription factor Δfosb acts within the nucleus of the solitary tract to increase mean arterial pressure during exposures to intermittent hypoxia. American Journal of Physiology-Heart and Circulatory Physiology. 2018;314(2):H270–H277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Hinojosa-Laborde C, Mifflin SW. Sex differences in blood pressure response to intermittent hypoxia in rats. Hypertension. 2005;46(4):1016–1021. [DOI] [PubMed] [Google Scholar]
  • 22.RStudio Team. RStudio: Integrated development environment for r. Boston, MA: RStudio, Inc.; 2015. [Google Scholar]
  • 23.Briscoe RJ, Cabrera CL, Baird TJ, Rice KC, Woods JH. Antalarmin blockade of corticotropin releasing hormone-induced hypertension in rats. Brain research. 2000;881(2):204–207. [DOI] [PubMed] [Google Scholar]
  • 24.Fisher LA, Jessen G, Brown MR. Corticotropin-releasing factor (crf): Mechanism to elevate mean arterial pressure and heart rate. Regulatory peptides. 1983;5(2):153–161. [DOI] [PubMed] [Google Scholar]
  • 25.Kalin NH, Shelton SE, Kraemer GW, McKinney WT. Corticotropin-releasing factor administered intraventricularly to rhesus monkeys. Peptides. 1983;4(2):217–220. [DOI] [PubMed] [Google Scholar]
  • 26.Korte S, Bouws G, Bohus B. Central actions of corticotropin-releasing hormone (crh) on behavioral, neuroendocrine, and cardiovascular regulation: Brain corticoid receptor involvement. Hormones and behavior. 1993;27(2):167–183. [DOI] [PubMed] [Google Scholar]
  • 27.Richter RM, Mulvany MJ. Comparison of hCRF and oCRF effects on cardiovascular responses after central, peripheral, and in vitro application. Peptides. 1995;16(5):843–849. [DOI] [PubMed] [Google Scholar]
  • 28.Scoggins BA, Coghlan JP, Denton DA, Fei DW, Nelson MA, Tregear GW, Tresham J, Wang X-M. Intracerebroventricular infusions of corticotrophin releasing factor (crf) and acth raise blood pressure in sheep. Clinical and Experimental Pharmacology and Physiology. 1984;11(4):365–367. [DOI] [PubMed] [Google Scholar]
  • 29.Brown MR, Fisher LA, Rivier J, Spiess J, Rivier C, Vale W. Corticotropin-releasing factor: Effects on the sympathetic nervous system and oxygen consumption. Life sciences. 1982;30(2):207–210. [DOI] [PubMed] [Google Scholar]
  • 30.Kurosawa M, Sato A, Swenson R, Takahashi Y. Sympatho-adrenal medullary functions in response to intracerebroventricularly injected corticotropin-releasing factor in anesthetized rats. Brain research. 1986;367(1–2):250–257. [DOI] [PubMed] [Google Scholar]
  • 31.Derkach V, Barria A, Soderling TR. Ca2+/calmodulin-kinase ii enhances channel conductance of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate type glutamate receptors. Proceedings of the National Academy of Sciences. 1999;96(6):3269–3274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Vergara C, Latorre R, Marrion NV, Adelman JP. Calcium-activated potassium channels. Current opinion in neurobiology. 1998;8(3):321–329. [DOI] [PubMed] [Google Scholar]
  • 33.Lyons MR, West AE. Mechanisms of specificity in neuronal activity-regulated gene transcription. Progress in neurobiology. 2011;94(3):259–295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Leke J, Fletcher EC, Bao G, Unger T. Hypertension caused by chronic intermittent hypoxia–influence of chemoreceptors and sympathetic nervous system. Journal of hypertension. 1997;15(12):1593–1603. [DOI] [PubMed] [Google Scholar]
  • 35.Prabhakar NR, Peng Y-J, Jacono FJ, Kumar GK, Dick TE. Cardiovascular alterations by chronic intermittent hypoxia: Importance of carotid body chemoreflexes. Clinical and experimental pharmacology and physiology. 2005;32(5–6):447–449. [DOI] [PubMed] [Google Scholar]
  • 36.Kline DD, Ramirez-Navarro A, Kunze DL. Adaptive depression in synaptic transmission in the nucleus of the solitary tract after in vivo chronic intermittent hypoxia: Evidence for homeostatic plasticity. Journal of Neuroscience. 2007;27(17):4663–4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.de Paula PM, Tolstykh G, Mifflin S. Chronic intermittent hypoxia alters nmda and ampa-evoked currents in nts neurons receiving carotid body chemoreceptor inputs. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 2007;292(6):R2259–R2265. [DOI] [PubMed] [Google Scholar]
  • 38.Andresen MC, Kunze DL. Nucleus tractus solitarius—gateway to neural circulatory control. Annual review of physiology. 1994;56(1):93–116. [DOI] [PubMed] [Google Scholar]
  • 39.Veening J, Swanson L, Sawchenko P. The organization of projections from the central nucleus of the amygdala to brainstem sites involved in central autonomic regulation: A combined retrograde transport-immunohistochemical study. Brain research. 1984;303(2):337–357. [DOI] [PubMed] [Google Scholar]
  • 40.Ma S, Mifflin SW, Cunningham JT, Morilak DA. Chronic intermittent hypoxia sensitizes acute hypothalamic–pituitary–adrenal stress reactivity and fos induction in the rat locus coeruleus in response to subsequent immobilization stress. Neuroscience. 2008;154(4):1639–1647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Blackburn MB, Andrade MA, Toney GM. Hypothalamic pvn contributes to acute intermittent hypoxia-induced sympathetic but not phrenic long-term facilitation. Journal of Applied Physiology. 2017;124(5):1233–1243. [DOI] [PMC free article] [PubMed] [Google Scholar]

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