Contribution of Central Nervous System Endothelial Nitric Oxide Synthase to Neurohumoral Activation in Heart Failure Rats
Neurohumoral activation, a hallmark in heart failure (HF), is linked to the progression and mortality of HF patients. Thus, elucidating its precise underlying mechanisms is of critical importance. Other than its classic peripheral vasodilatory actions, the gas NO is a pivotal neurotransmitter in the central nervous system control of the circulation. While accumulating evidence supports a contribution of blunted NO function to neurohumoral activation in HF, the precise cellular sources, and NO synthase (NOS) isoforms involved, remain unknown. Here, we used a multidisciplinary approach to study the expression, cellular distribution, and functional relevance of the endothelial NOS isoform within the hypothalamic paraventricular nucleus in sham and HF rats. Our results show high expression of endothelial NOS in the paraventricular nucleus (mostly confined to astroglial cells), which contributes to constitutive NO bioavailability, as well as tonic inhibition of presympathetic neuronal activity and sympathoexcitatory outflow from the paraventricular nucleus. A diminished endothelial NOS expression and endothelial NOS-derived NO availability were found in the paraventricular nucleus of HF rats, resulting, in turn, in blunted NO inhibitory actions on neuronal activity and sympathoexcitatory outflow. Taken together, our study supports blunted central nervous system endothelial NOS-derived NO as a pathophysiological mechanism underlying neurohumoral activation in HF.
In addition to its classic peripheral vasodilatory actions, the gas NO is a key neurotransmitter within the central nervous system (CNS), particularly in regions involved in the neurohumoral control of the circulation,1–3 including the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei.4 Constitutively produced NO tonically inhibits neurosecretory and preautonomic neuronal activity,5,6 restraining, in turn, sympathohumoral outflow to the circulation.7,8 Importantly, blunted CNS NO function is linked to neurohumoral activation in heart failure (HF).8,9 Despite this evidence, the precise cellular sources and NO synthase (NOS) isoforms involved remain unknown. Given the abundance of NO-producing neurons within the SON and PVN,10,11 it is generally implicit that constitutive NO arises from a neuronal (nNOS) source. This, however, has not been compellingly demonstrated, given that most studies were based on the use of nonselective NOS blockers.8,9,12 An alternative source of constitutive NO is the endothelial NOS (eNOS), shown recently to influence the CNS control of the circulation.13–15 Different from nNOS, eNOS is localized within endothelial cells in brain capillaries,16 although recent evidence supports eNOS expression in astrocytes as well.17 Because eNOS can synthesize NO in a sustained manner,18,19 it is a likely source of tonic ambient NO levels within the CNS.20 Still, whether eNOS contributes to tonic PVN NO levels and what its functional role is in the regulation of neuronal activity and neurohumoral outflow in physiological and pathological conditions, remain unknown. Here, we used a multidisciplinary approach to study eNOS cellular distribution and functional significance in the PVN of control and HF rats. We found eNOS to contribute to constitutive NO levels and to tonic inhibition of PVN neuronal activity and sympathetic outflow. Our results also support a role for eNOS in blunted NO availability and elevated sympathoexcitation during HF.
An expanded Methods section is available in the online Data Supplement at http://hyper.ahajournals.org.
Male Wistar rats (180 to 220 g) were used. All of the procedures were approved by the institutional animal care and use committees at Georgia Health Sciences University and the University of Nebraska Medical Center.
Induction of HF
HF was induced by coronary artery ligation.12,21 Sham animals underwent the same procedure, but the coronary artery was not occluded. All of the animals were used 6 to 7 weeks after surgery. Transthoracic echocardiography (Vevo 770, Visual Sonics) was performed to evaluate cardiac parameters.
Retrograde Labeling of Rostral Ventrolateral Medulla-Projecting PVN Neurons
PVN neurons innervating the rostral ventrolateral medulla (RVLM; PVN-RVLM) were retrogradely labeled with rhodamine microspheres (Lumaflor) injected unilaterally (400 nL) within the RVLM, and the location and extension of the injections sites were confirmed histologically.5,22
Conventional immunofluorescence22 was used to characterize eNOS, phospho-eNOS (Ser1177 and Thr495), and its colocalization with nNOS, astroglial cells, microvessels, and oxytocin neurons. Confocal images were obtained and a densitometry analysis was used to compare eNOS immunoreactivity (ir) between sham and HF groups.22
Measurements of NO Availability
NO was visualized in living hypothalamic slices using the NO-sensitive dye 4,5 diaminofluorescein diacetate (DAF-2, Calbiochem).10 Slices were loaded with DAF-2 (2.5 μmol/L) in the presence or absence of relatively selective and nonselective eNOS and nNOS blockers. Confocal images were obtained, and DAF-2 was quantified in identified neurons and astrocytes.
Whole-Animal DAF-2 Infusion
Rats were intravenously injected with DAF-2 following modified methods described previously.23 Brains were then dissected, 20-μm hypothalamic sections were collected in a microscope slide, and images of DAF-2 were taken.
In Vitro Electrophysiological Recordings From PVN-RVLM Neurons
Whole-cell patch-clamp recordings of PVN-RVLM neurons were obtained from hypothalamic sections in HF and sham rats.5 The mean firing rate recorded during a 2 minutes period, before and after bath drug applications, was calculated and compared between groups.
Hemodynamic and Renal Sympathetic Nerve Activity Measurements
Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and heart rate (HR) were monitored.9 The peak response of RSNA to the administration of drugs into the PVN was expressed as a percentage of change from baseline and values compared with those evoked by a microinjection of the same volume of artificial cerebrospinal fluid.
eNOS Antisense Delivery into the PVN
The eNOS antisense oligonucleotide (ODN) sequence used in this study, 5′-ATGGGCAACTTGAAGAG-3′, was designed according to the rat eNOS mRNA sequence (GenBank accession No. NM021838). The ODNs were administered into the PVN by unilateral microinjections (100 nL). This dose and protocol were based on our previous successful use of antisense ODN against nNOS within the PVN.24
Data are presented as mean±SEM. Unpaired or paired t tests, as well as 1- or 2-way ANOVA, followed by Bonferroni post hoc tests, were used as indicated. Values of P<0.05 were considered statistically significant.
Representative echocardiography images and mean cardiac functional data for sham and ligated rats (n=31 per group) are shown in Figure S1 and Table S1 (available in the online Data Supplement). Compared with sham rats, ligated rats showed an increased left ventricle internal diameter throughout the cardiac cycle and a decreased percentage in posterior wall thickening, ejection fraction, and fractional shortening (all P<0.05). Moreover, a macroanatomical examination of hearts in ligated rats revealed a dense scar and thinning of the anterior left ventricular wall (data not shown).
eNOS Expression in the PVN Localizes Primarily Within Astrocytes and Perivascular Elements
A dense eNOSir, with a noticeable contrasting pattern to that of nNOS, was observed in the PVN (Figure 1A through 1C; n=4 rats). Although nNOS was almost exclusively localized in principal neurons,5,11 eNOS was more diffusely distributed. No overlap between eNOSir and nNOSir (Figure 1D through 1F) or eNOS and oxytocin (Figure S2) was detected in the PVN and the SON. The specificity of the eNOS antibody is supported by the lack of staining in the absence of primary antibody (data not shown) and in brain tissue obtained from eNOS knockout mice (Figure S3).
Dual immunohistochemical studies for eNOS and a microvasculature (anti-endothelial cell antigen RECA-1) or astrocyte markers (glial fibrilary acidic protein [GFAP] and S100β for processes and cell bodies, respectively) were performed. Images were also obtained from the SON, in which astrocytes have a distinct anatomic distribution, with cell bodies congregated in the ventral glial laminae.25 Although eNOS was found closely associated with the PVN and SON microvasculature (Figure 1G through 1I), it did not overlap with RECA-1 but rather appeared to be present in perivascular profiles abutting on the vessel wall. Conversely, a high degree of colocalization was found between eNOS and perivascular astrocyte processes (GFAP; Figure 1J through 1L) and cell bodies (S100β; Figure 1M through 1O). Similar results were observed in larger microvessels (arterioles) of the cortex (Figure S4).
eNOS Contributes to Constitutive NO Availability and Influences Basal Sympathoexcitatory Outflow From the PVN
Hypothalamic brain sections were loaded with the NO-sensitive indicator DAF-2 in the presence or absence of the relatively eNOS-selective inhibitors (l-N5-(1-Iminoethyl)-ornithine.2HCI [l-NIO]; 10 μmol/L)26 or cavtratin (10 μmol/L; Figure 2A through 2C).27 Preincubation of sections with l-NIO significantly decreased PVN DAF-2 staining (≈19% inhibition; P<0.05; Figure 2G). These effects were absent in eNOS knockout mice (Figure 2H). Similar results were obtained with cavtratin (≈18% inhibition; P<0.05). The nNOS selective inhibitor 1-(2-trifluoromethylphenyl) imidazole (TRIM; 100 μmol/L; Figure 2D) reduced DAF-2 fluorescence to a similar extent (≈21% inhibition; P<0.05), whereas the nonselective NOS blocker NG-nitro-l-arginine methyl ester (l-NAME 200 μmol/L; Figure 2E) resulted in an ≈2-fold reduction in DAF-2 (≈54% inhibition; P<0.05), when compared with the eNOS or nNOS blockers. An almost complete blockade was observed when l-NAME was coapplied with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l1-oxyl-3-oxide (C-PTIO; 100 μmol/L, Figure 2F; ≈90% inhibition, P<0.05, Figure 2G).
In vivo microinjections of l-NIO (50 to 200 pmol) into the PVN of sham rats (n=6) increased RSNA, MAP, and HR in a dose-dependent manner (Figure 2I; P<0.05 for all of the variables, 1-way ANOVA). No changes were observed when a similar volume of vehicle was administered (data not shown). In separate experiments, eNOS antisense was delivered directly within the PVN of sham rats (n=3), and the effects of intra-PVN injection of the nonselective NOS blocker NG-monomethyl-l-arginine (l-NMMA; 200 pmol/200 nL) were assessed before and 2 hours after eNOS antisense delivery. We reasoned that if both eNOS and nNOS isoforms contributed to basal constitutive NO levels, a proportion of l-NMMA effect should be blocked by eNOS antisense pretreatment. As summarized in Figure 2J, the increases in RSNA and MAP evoked by l-NMMA under control conditions were significantly diminished 2 hours after eNOS antisense delivery (P<0.05 for RSNA and MAP, respectively; paired t test). Taken together, our studies support a contribution of eNOS to constitutive NO production and tonic regulation of sympathoexcitatory outflow from the PVN.
Diminished eNOS Expression in the PVN of HF Rats
We assessed then whether eNOSir, as well as its activation/inhibition phosphorylation sites, the phospho-eNOS-Ser1177 and eNOS-Thr495, respectively28 (n=3 per group), were altered in HF rats, in PVN subnuclei containing presympathetic (ventromedial and posterior parvocellular) and magnocellular neurosecretory neurons (lateral magnocellular).4 We found a lower eNOSir in the ventromedial parvocellular and lateral magnocellular PVN subnuclei (P<0.05). A strong tendency, while not reaching statistical significance, was observed in the posterior parvocellular subnucleus. The eNOS-Ser1177ir was lower in the ventromedial parvocellular and posterior parvocellular (P<0.05) but not in the lateral magnocellular subnuclei. Conversely, eNOS-Thr495ir was unaffected in all of the subnuclei (Figure 3).
Blunted eNOS-Derived NO in Presympathetic Regions of the PVN in HF Rats
Basal DAF-2 staining and changes evoked by the eNOS blocker l-NIO were measured in 3 different PVN subnuclei of sham and HF rats (n=6 per group; Figure 4A through 4C). In the presympathetic ventromedial and posterior parvocellular subnuclei of sham rats, we found a higher basal DAF-2 staining (compared with HF rats), which was diminished by l-NIO (P<0.05 in both cases, Figure 4D, 4E, 4G and 4H). Conversely, l-NIO failed to affect DAF-2 in HF rats (Figure 4F and 4I). In the lateral magnocellular subnucleus, we found no differences in basal DAF-2 between sham (Figure 4J and 4K) and HF rats, which was similarly diminished by l-NIO in both groups (P<0.05; Figure 4L). Similar results were obtained with cavtratin in a subset of sham and HF rats (n=2 per group, 697 cells sampled; data not shown).
Diminished In Vivo Parenchymal Perivascular PVN NO Production in HF Rats
Given the lack of evident DAF-2 staining in parenchymal vessels in brain sections, we used an alternative in vivo approach, which efficiently detects perivascular DAF-2 staining in the CNS.23 Systemic intravenous infusions of DAF-2 (100 μL of 5 mmol/L solution, 15 minutes) readily stained parenchymal perivascular NO-induced fluorescence without staining other neuropile elements (Figure 5), as described previously in the cortex.23 We found PVN perivascular DAF-2 to be diminished in HF rats (≈34%; P<0.05 versus sham rats; n=3 per group).
Blunted eNOS Contribution to Tonic NO Inhibition of PVN Neuronal Activity and RSNA in HF Rats
Last, we assessed whether the blunted eNOS-derived NO resulted in blunted NO inhibition of presympathetic PVN neuronal activity and, consequently, enhanced sympathoexcitatory drive in HF rats.8,9 In vitro patch-clamp recordings from retrogradely labeled PVN-RVLM neurons showed that blockade of eNOS (l-NIO, 10 μmol/L) increased neuronal activity in sham (≈55.0%; n=10 cells; P<0.05) but not in HF rats (≈0.5%; n=12 cells; Figure 6A and 6B). Similarly, in vivo studies showed that the increase in RSNA (expressed as percentage of change from baseline), MAP, and HR evoked by microinjections of l-NIO into the PVN in sham rats was diminished in HF rats (n=5 per group; P<0.05, sham versus HF, 2-way ANOVA; Figure 6C).
A large body of evidence supports an integral role for CNS NO in the control of the circulation,1–3 as well as a contribution of blunted NO function to neurohumoral activation in HF.8,9 Most of these studies, however, focused on the targets and outcomes of NO actions, whereas only a few specifically addressed cellular sources and isoforms mediating NO actions.10,14,24 Given that the strength and specificity of NO actions are influenced by the spatial distribution and efficacy of NOS isoforms and their sensitivity and proximity to their targets,2 elucidating the cellular sources and isoforms contributing to NO availability is of critical importance. Results from the present studies show the following: (1) in addition to nNOS, eNOS is abundantly expressed in the SON and PVN; (2) nNOS and eNOS display a segregated although spatially interrelated cellular distribution (neuronal and astroglial, respectively); (3) eNOS contributes to constitutive NO production and tonic NO-dependent inhibition of neuronal activity and sympathetic outflow from the PVN; and (4) eNOS is involved in blunted NO availability and actions in HF rats. Taken together, our studies support eNOS-derived NO as a critical neuromodulator of presympathetic PVN neuronal activity and sympathoexcitatory outflow from the PVN and indicate that blunted CNS eNOS function contributes to sympathoexcitation in HF.
eNOS of a Likely Glial Location Contributes to Basal NO Bioavailability
We found eNOS immunoreactivity in the PVN to be largely localized in astrocyte cell bodies and processes. Given that eNOS did not colocalize with nNOS, which is exclusively expressed in neurons,5,11 our studies support a cellular segregation between these 2 isoforms. Still, the precise cellular distribution of eNOS in the CNS remains controversial. Although eNOS was reported both in astrocytic cultures29 and brain tissue,30,31 including recently in the nucleus tractus solitarius,17 others failed to detect eNOS in astrocytes.16 In addition, although we found eNOS staining in close association with the local microvasculature, it did not overlap with endothelial cells but rather with processes in contact with the abluminal side of the microvessels. These processes were immunoreactive for the glial-specific marker GFAP, likely representing astrocytic endfeet.32 This result is somewhat inconsistent with previous studies showing eNOS in brain endothelium.16 Thus, methodological dissimilarities, including antibodies used,17 fixation procedures, and overall sensitivity, could explain such reported differences.
To determine whether eNOS contributed to constitutive NO availability, we monitored NO using DAF-2, a well-established NO-sensitive fluorescent indicator.33 Our results showing a diminished basal DAF-2 in slices pretreated with l-NIO or cavtratin, 2 different eNOS selective blockers26,27 support a tonic contribution of eNOS to PVN NO levels. The lack of l-NIO effects in eNOS knockout mice support its eNOS selectivity in our experimental conditions. eNOS-dependent changes in DAF-2 were observed in areas enriched with magnocellular neurosecretory and presympathetic neurons,4 supporting the contribution of eNOS to NO availability within these distinct hypothalamic systems. Post hoc tissue processing for immunohistochemistry affected our ability to reliably quantify DAF-2 staining (unpublished observations), preventing us from identifying astrocytes. Moreover, as shown in similar studies,34 we failed to detect microvascular DAF-2 in vitro. Rather than reflecting the lack of NO in the microvasculature, we believe this to be a sensitivity limitation because of the small size of the endothelium and/or limited endothelium dye loading under our experimental conditions. In fact, using an in vivo approach shown previously to efficiently load vascular structures,23 we showed perivascular DAF-2 staining in the PVN. Thus, quantification of DAF-2 in vitro was restricted to neurons, in which we found a diminished DAF-2 fluorescence after eNOS blockade. Our results indicate that, despite eNOS segregated cellular distribution, the close proximity among neurons, astrocytes, and the local microvasculature in these nuclei,25 along with the ability of NO to freely diffuse from its site of production, ensues that eNOS-derived NO from either astrocytes or microvessels contributes to NO availability and actions in nearby neurons. This is important given previous controversial reports, showing that, whereas NO efficiently diminished the activity of most presympathetic PVN neurons,5 only a limited proportion of them expressed detectable levels of nNOS.5,35 Taken together, these studies suggest that NO produced by and diffusing from an alternative isoform (ie, eNOS) contributes to NO actions on PVN neuronal activity and sympathetic outflow.
eNOS-Derived NO Regulates Neuronal Activity and PVN Sympathetic Outflow
Our combined in vitro patch-clamp experiments, whole animal nerve recordings, and cardiovascular hemodynamic studies support eNOS-derived NO within the PVN as functionally relevant to the CNS control of the circulation. eNOS blockade increased PVN-RVLM firing discharge, indicating that their activity is tonically restrained by eNOS-derived NO. The RVLM is a critical target of sympathoexcitatory PVN descending projections,36,37 and overactivation of this pathway contributes to sympathoexcitation and increased MAP in hypertension38 and dehydration.39 In agreement with our in vitro studies, we found that microinjection of l-NIO directly into the PVN increased RSNA and MAP. Moreover, we found ≈50% of the excitatory effect evoked by the nonselective NOS inhibitor l-NMMA to be blocked by previous PVN microinjection of an eNOS antisense. Taken together, these studies support a contribution of eNOS to the regulation of PVN neuronal activity and sympathoexcitatory outflow to the circulation.
Differential Contribution of eNOS and nNOS to NO Bioavailability?
By comparing results obtained with NOS selective and nonselective blockers (in vitro DAF-2), along with in vivo eNOS antisense studies, we could speculate that eNOS and nNOS contribute to a similar degree to basal PVN NO bioavailability. However, when comparing the functional significance of these 2 isoforms, it is important to consider other properties as well. Based on their distinct cellular sources and bioactive properties,40 it is likely that NO originating from these alternative sources mediates distinct functions. For example, antagonistic effects were reported in the medulla, where eNOS and nNOS mediated inhibition and excitation of baroreflex function, respectively.14,41 Conversely, results from our laboratories indicate that both isoforms inhibit the firing activity of hypothalamic neurosecretory and presympathetic neurons, as well as sympathoexcitatory outflow to the circulation.6,7,10 This raises questions about the functional significance of the presence of, in principle, 2 similar NO sources. One possibility is that each NO source is activated by different signaling mechanisms and/or conditions. This is supported by the presence of at least two NO-mediated signaling modalities, phasic (rapid and transient) and tonic (sustained), mediated likely by nNOS- and eNOS-derived NO, respectively. Thus, activation of nNOS via associated N-methyl-d-aspartate (NMDA) receptors in dendritic spines results in a brief, low-amplitude NO transient, which is spatially and temporally restricted to the site of production.19,42 Therefore, this NMDA-nNOS phasic modality is better suited to act in a synapse-specific manner.20 Conversely, eNOS is able to synthesize NO in a sustained manner, even at resting cytosolic calcium concentrations,18,19 supporting eNOS as the likely primary source of tonic ambient NO levels, mediating more widespread effects of NO within CNS networks.20,43 Taken together, our results further support the notion that eNOS contributes to sustained NO bioavailability and actions within the PVN. However, whether PVN eNOS and nNOS are activated under different conditions or by different signals remains to be determined.
Diminished eNOS Expression and Function Contribute to Blunted NO Actions in HF Rats
Previous studies showed elevated PVN neuronal activity44 and blunted PVN NO actions as major contributing factors to increased sympathoexcitatory outflow in HF.9,12 However, whether eNOS contributes to blunted CNS NO function in HF remained unexplored. This is supported by several lines of evidence in this work. First, we found a diminished PVN eNOS immunoreactivity in HF rats. Given that most eNOSir was localized in perivascular structures and that a diminished in vivo perivascular DAF-2 was observed in HF rats, it is likely that a diminished perivascular eNOS expression occurred in HF rats. Second, eNOS activity can be efficiently regulated by phosphorylation of various sites, particularly the Ser1177 and the Thr495, resulting in increased and decreased activity, respectively.28 Our results showing diminished staining for Ser1177 in HF rats suggest that, in addition to changes in eNOS expression, dysfunctional phosphorylation at Ser1177 also contributes to blunted eNOS-derived NO in HF rats. Interestingly, a diminished Sert1177 is commonly observed in the vasculature of hypertensive rats.45 Lastly, we found a blunted effect of eNOS blockade on NO bioavailability, PVN-RVLM firing activity, RSNA, MAP, and HR in HF rats. Thus, in addition to previously reported diminished eNOS function in the peripheral vasculature during HF,46 our study supports a contribution of blunted CNS eNOS function to elevated neuronal activity and sympathoexcitation in this condition.
Increased neurohumoral drive, characterized by sympathoexcitation, and elevated circulating neurohormones constitute a common finding in humans and experimental animal models of HF.47,48 Given that sympathoexcitation increases the progression and mortality in HF,47 there is a great deal of interest in elucidating mechanisms underlying sympathoexcitation in HF. Our results showing the involvement of eNOS in blunted NO availability and actions during HF support eNOS as an important underlying pathophysiological mechanism in neurohumoral activation in HF, as well as a promising therapeutic target for the treatment of this disease.
Sources of Funding
This work was supported by American Heart Association grant 0640092N and National Institutes of Health grant HL085767 to J.E.S.; and National Heart, Lung, and Blood Institute grant HL-62222 to K.P.P.
We thank Dr Jessica A. Filosa, Georgia Health Sciences University, for critical reading of the article and Dr Thomas A. Kent, Baylor College of Medicine, for help with experimental in vivo protocols for perivascular NO measurements.
- Received May 5, 2011.
- Revision received May 25, 2011.
- Accepted July 7, 2011.
- © 2011 American Heart Association, Inc.
- Stern JE,
- Ludwig M
- Zheng H,
- Li YF,
- Cornish KG,
- Zucker IH,
- Patel KP
- Zhang K,
- Li YF,
- Patel KP
- Paton JF,
- Deuchars J,
- Ahmad Z,
- Wong LF,
- Murphy D,
- Kasparov S
- Gingerich S,
- Krukoff TL
- Hopper RA,
- Garthwaite J
- Francis J,
- Weiss RM,
- Wei SG,
- Johnson AK,
- Felder RB
- Biancardi VC,
- Campos RR,
- Stern JE
- Fabian RH,
- Perez-Polo JR,
- Kent TA
- Wang Y,
- Liu XF,
- Cornish KG,
- Zucker IH,
- Patel KP
- Theodosis DT,
- Poulain DA,
- Oliet SH
- Bauser-Heaton HD,
- Song J,
- Bohlen HG
- Fleming I,
- Busse R
- Sporbert A,
- Mertsch K,
- Smolenski A,
- Haseloff RF,
- Schonfelder G,
- Paul M,
- Ruth P,
- Walter U,
- Blasig IE
- Tagawa T,
- Dampney RA
- Allen AM
- Stocker SD,
- Keith KJ,
- Toney GM
- Knowles RG,
- Moncada S
- Smith CJ,
- Sun D,
- Hoegler C,
- Roth BS,
- Zhang X,
- Zhao G,
- Xu XB,
- Kobari Y,
- Pritchard K Jr.,
- Sessa WC,
- Hintze TH
- Cohn JN