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. Author manuscript; available in PMC: 2012 Sep 1.
Published in final edited form as: Hypertension. 2011 Aug 8;58(3):454–463. doi: 10.1161/HYPERTENSIONAHA.111.175810

Contribution of Central Nervous System eNOS to Neurohumoral Activation in Heart Failure Rats

Vinicia Campana Biancardi 1, Sook Jin Son 1, Patrick M Sonner 2, Hong Zheng 3, Kaushik P Patel 3, Javier E Stern 1
PMCID: PMC3189581  NIHMSID: NIHMS317196  PMID: 21825233

Abstract

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. Besides its classical peripheral vasodilatory actions, the gas nitric oxide (NO) is a pivotal neurotransmitter in the central nervous system (CNS) 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 (eNOS) within the hypothalamic paraventricular (PVN) nucleus in Sham and HF rats. Our results show high expression of eNOS in the PVN (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 PVN. A diminished eNOS expression and eNOS-derived NO availability was found in the PVN of HF rats, resulting in turn in blunted NO inhibitory actions on neuronal activity and sympathoexcitatory outflow. Taken together, our study supports blunted CNS eNOS-derived NO as a pathophysiological mechanism underlying neurohumoral activation in HF.

Keywords: heart failure, nitric oxide, hypothalamus, astrocyte, sympathetic

Introduction

In addition to its classical peripheral vasodilatory actions, the gas nitric oxide (NO) is a key neurotransmitter within the central nervous system (CNS), particularly in regions involved in the neurohumoral control of the circulation1-3, including the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei4. Constitutively produced NO tonically inhibits neurosecretory and preautonomic neuronal activity5, 6, restraining in turn sympathohumoral outflow to the circulation7, 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 PVN10, 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 non-selective NOS blockers8, 9, 12. An alternative source of constitutive NO is the endothelial eNOS, recently shown to influence the CNS control of the circulation13-15. Differently from nNOS, eNOS is localized within endothelial cells in brain capillaries16, although recent evidence supports eNOS expression in astrocytes as well17. Since eNOS can synthesize NO in a sustained manner18, 19, it is a likely source of tonic ambient NO levels within the CNS20. 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, remains 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.

Methods

An expanded Methods section is available in the Online Data Supplement at http://hyper.ahajournals.org

Animals

Male Wistar rats (180-220g) were used. All procedures were approved by the Institutional Animal Care and Use Committee at Georgia Health Sciences University and the University of Nebraska Medical Center.

Induction of heart failure

HF was induced by coronary artery ligation12, 21. Sham animals underwent the same procedure but the coronary artery was not occluded. All animals were used 6 to 7 weeks after surgery. Trans-thoracic echocardiography (Vevo 770, Visual Sonics) was performed to evaluate cardiac parameters.

Retrograde labeling of rostral ventrolateral medulla (RVLM)-projecting PVN neurons

PVN neurons innervating the RVLM (PVN-RVLM) were retrogradely labeled with rhodamine microspheres (Lumaflor) injected unilaterally (400nL) within the RVLM, and the location and extension of the injections sites were confirmed histologically5, 22.

Immunohistochemistry

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 groups22.

Measurements of NO availability

A-hypothalamic slices: 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 (2.5μmol/L) in the presence or absence of a relatively selective and non-selective eNOS and nNOS blockers. Confocal images were obtained and DAF-2 was quantified in identified neurons and astrocytes. B- Whole-animal DAF infusion: Rats were intravenously injected with DAF-2 following modified methods previously described23. Brains were then dissected, 20μm 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 slices in HF and Sham rats5. The mean firing rate recorded during a 2min period, before and after bath drug applications was calculated and compared between groups.

Hemodynamic and renal sympathetic nerve activity (RSNA) measurements

RSNA, mean arterial pressure (MAP) and heart rate (HR) were monitored9. The peak response of RSNA to the administration of drugs into the PVN was expressed as a percent change from baseline, and values compared to those evoked by a microinjection of the same volume of artificial cerebrospinal fluid.

eNOS antisense delivery into the PVN

The eNOS antisense oligonucleotides (AS-ODN) sequence used in this study, 5′-ATGGGCAACTTGAAGAG-3′, was designed according to the rat eNOS mRNA sequence (GenBank accession n°.NM021838). The ODNs were administered into the PVN by unilateral microinjections (100nL). This dose and protocol were based on our previous successful use of AS-ODN against nNOS within the PVN24.

Statistical Analysis

Data are presented as mean ± SEM. Unpaired or paired t-tests, as well as one or two-way analysis of variance (ANOVA), followed by Bonferroni posthoc tests, were used as indicated. Values of P<0.05 were considered statistically significant.

Results

Representative echocardiography images and mean cardiac functional data for Sham and ligated rats (n=31/group) are shown in Fig.S1 and Table S1. Compared to 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 (not shown).

eNOS expression in the PVN localizes primarily within astrocytes and perivascular elements

A dense eNOS immunoreactivity (ir), with a noticeable contrasting pattern to that of nNOS, was observed in the PVN (Fig.1A-C, n=4 rats). While nNOS was almost exclusively localized in principal neurons5, 11, eNOS was more diffusely distributed. No overlap between eNOSir and nNOSir (Fig.1D-F) or eNOS and oxytocin (Fig.S2). was detected in the PVN and the SON. The specificity of the eNOS antibody is supported by the lack of staining (a) in the absence of primary antibody (not shown), and (b) in brain tissue obtained from eNOS knockout mice (Fig.S3). Dual immunohistochemical studies for eNOS and a microvasculature (RECA for endothelial cells) or astrocyte markers (GFAP and S100β for processes and cell bodies, respectively) were performed. Images were also obtained from the SON, in which astrocytes have a distinct anatomical distribution, with cell bodies congregated in the ventral glial laminae25. While eNOS was found closely associated with the PVN and SON microvasculature (Fig.1G-I), it did not overlap with RECA, 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, Fig.1J-L) and cell bodies (S100β, Fig.1M-O). Similar results were observed in larger microvessels (arterioles) of the cortex (Fig.S4).

Figure 1. eNOS is densely expressed in segregated cell populations in the PVN and SON.

Figure 1

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A and D: Immunostaining of eNOS (green) within the PVN and SON, respectively. B and E: nNOS staining (red) was confined to main neurons in both nuclei. C and F: Merged higher magnification images of the areas outlined by squares in B (PVN) and E (SON), showing lack of colocalization between eNOS (green) and nNOS (red) in both nuclei. G-I: eNOS (green) and the endothelial cell marker RECA-1 (red) immunoreactivities within the PVN (G) and SON (I). J-L: eNOS (green) and the glial marker GFAP (red) immunoreactivities within the PVN (J) and the SON (L). M-O: eNOS (green) and the glial marker S100β (red) immunoreactivities within the PVN (M) and SON (O). Details of PVN staining are shown in H, K and N, higher magnification images of the PVN area outlined in G, J and M respectively. Note the dense eNOS staining in the SON ventral glial laminae (I, L, O, arrows), and the colocalization (yellow color: green + red) with glial markers, but not with endothelial, RECA-1 positive cells. Scale bars: 50μm. 3V: third ventricle; OT: optic tract.

eNOS contributes to constitutive NO availability and influences basal sympathoexcitatory outflow from the PVN

Hypothalamic brain slices were loaded with the NO-sensitive indicator DAF-2 in the presence or absence of the relatively eNOS selective inhibitors L-NIO (10μmol/L)26 or Cavtratin (10μmol/L)27 (Fig.2A-C). Preincubation of slices with L-NIO significantly decreased PVN DAF-2 staining (∼19% P<0.05, Fig.2G). These effects were absent in eNOS KO mice (Fig.2H). Similar results were obtained with Cavtratin (∼18% inhibition, P<0.05). The nNOS selective inhibitor TRIM (100μmol/L, Fig.2D) reduced DAF-2 fluorescence to a similar extent (∼21%, P<0.05), while the non-selective NOS blocker L-NAME (200μmol/L, Fig.2E) resulted in a ∼2 fold reduction in DAF-2 (∼54%, P<0.05), when compared to the eNOS or nNOS blockers. An almost complete blockade was observed when L-NAME was co-applied with the NO scavenger C-PTIO (100μmol/L, Fig.2F) (∼90%, P<0.05, Fig.2G).

Figure 2. eNOS contributes to constitutive NO availability and actions in the PVN.

Figure 2

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Photomicrographs of PVN DAF-2 staining in control rats in (A) control ACSF, (B) eNOS inhibitor L-NIO (10μmol/L), (C) eNOS inhibitor Cavtratin (10μmol/L), (D) nNOS inhibitor TRIM (100μmol/L), (E) non selective NOS inhibitor L-NAME (200μmol/L) and (F) combined L-NAME + NO scavenger C-PTIO (200μmol/L). G: Summary data showing mean DAF-2 intensity in each group.*†‡P<0.05 vs.: *ACSF; †-L-NIO, CAVTRATIN and TRIM; ‡L-NAME; n=809 neurons from 18 rats. H: Summary data showing the effects of L-NIO (10μmol/L) in wild-type and eNOS-KO mice.*P<0.05 vs. ACSF; n=195 neurons from 6 rats. I: Summary data showing dose-dependent increases in RSNA, MAP and HR after microinjection of L-NIO (50, 100 and 200pmol) into the PVN of control rats (n=6, P<0.05). J: RSNA, MAP and HR responses to L-NMMA (200pmol/200nL) before and 2h after microinjection of eNOS antisense (AS-ODN, 100pmol/100nL) into the PVN in a sham rat. The L-NMMA-mediated increases in RSNA and MAP were significantly reduced when L-MMMA was re-applied 2h after the AS-ODN microinjection.*P<0.05, vs. before AS-ODN; n=3/group. Scale bars: 50μm. AU: arbitrary units.

In vivo microinjections of L-NIO (50 - 200pmol) into the PVN of sham rats (n=6/group) increased RSNA, MAP and HR, in a dose-dependent manner (Fig.2I, P<0.05 for all variables, one-way ANOVA). No changes were observed when a similar volume of vehicle was administered (not shown). In separate experiments, eNOS antisense was delivered directly within the PVN of sham rats (n=3/group), and the effects of intra-PVN injection of the non-selective NOS blocker N(G)-monomethyl-L-arginine (L-NMMA, 200pmol/200nL) were assessed before and 2h 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 Fig.2J, the increases in RSNA and MAP evoked by L-NMMA under control conditions were significantly diminished 2h 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/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, though 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 subnuclei (Fig.3A-G).

Figure 3. PVN eNOS and eNOS-Ser1177 are decreased in HF rats.

Figure 3

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A-C: Examples of eNOSir (A), eNOS-Ser 1177ir (B) and eNOS-Thr496ir (C) in the PVN of a Sham rat. The inset in C shows the same section at lower magnification, displaying also oxytocin immunoreactive neurons (white) as landmark. Asterisk: center of the lateral magnocellular subdivision. D-F: Examples of eNOSir (D), eNOS-Ser 1177ir (E) and eNOS-Thr496ir (F) in the PVN of a HF rat. G: Summary data showing mean differences between Sham and HF rats in different PVN subnuclei.*P<0.05 vs. Sham; n=3/group. Scale bars: 50μm. 3V: third ventricle.

Blunted eNOS-derived NO in presympathetic regions of the PVN in HF rats

Basal DAF-2 staining, as well as changes evoked by the eNOS blocker L-NIO, were measured in three different PVN subnuclei of Sham and HF rats (n=6/group) (Fig.4A-C). In the presympathetic ventromedial and posterior parvocellular subnuclei of Sham rats, we found a higher basal DAF-2 staining (compared to HF rats), which was diminished by L-NIO (P<0.05 in both cases, Fig.4D-E, G-H). Conversely, L-NIO failed to affect DAF-2 in HF rats (Fig.4F,I). In the lateral magnocellular subnucleus, we found no differences in basal DAF-2 between Sham (Fig.4J-K) and HF rats, which was similarly diminished by L-NIO in both groups (P<0.05, Fig.4L). Similar results were obtained with Cavtratin in a subset of Sham and HF rats (n=2/group, 697 cells sampled, not shown).

Figure 4. Blunted eNOS contribution to constitutive NO in the PVN of HF rats.

Figure 4

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A: Diagram depicting PVN subnuclei and projection targets (modified from4). B and C: Toto-3 counterstaining (nuclear marker, white) depicting the Lateral Magnocellular (1), Ventromedial Parvocellular (2), and Posterior Parvocellular (3) subnuclei in the PVN. Images were taken at ∼1.8–2.1 (B) and 2.1–2.3mm (C) caudal to bregma. Representative confocal images of DAF-2 fluorescence in the Ventromedial Parvocellular (D and E), Posterior Parvocellular (G and H) and Lateral Magnocellular (J and K) PVN subnuclei in the absence or presence of 10μmol/L L-NIO, respectively, in a Sham rat. F, I and L: summary data of DAF-2 intensity in neurons for each subnucleus in Sham and HF rats.*P<0.05 vs. respective ACSF. †P<0.05 Sham ACSF vs. HF ACSF; n=6/group. Scale bar: 50μm.

Diminished in vivo parenchymal perivascular PVN NO production in HF rats

Given the lack of evident DAF-2 staining in parenchymal vessels in brain slices, we used an alternative in vivo approach, which efficiently detects perivascular DAF-2 staining in the CNS23. Systemic intravenous infusions of DAF-2 (100μl of 5mmol/L solution, 15min), readily stained parenchymal perivascular NO-induced fluorescence, without staining other neuropile elements (Fig.5), as previously described in the cortex23. We found PVN perivascular DAF-2 to be diminished in HF rats (∼34%, P<0.05 vs. Sham rats, n=3/group).

Figure 5. – Diminished PVN perivascular NO availability in HF rats following in vivo infusions of DAF-2.

Figure 5

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A-B: Epifluorescence images of DAF-2 fluorescence in the Posterior Parvocellular subnucleus in a Sham (A) and HF (B) rat. C-D: higher magnification image of the PVN area outlined in A and B, respectively. Arrows point to typical examples of staining in rounded, coronally cut microvessels. E: Summary data showing a diminished perivascular DAF-2 staining in the PVN of HF rats.*P<0.05, n=3/group. Scale bar: 50μm. 3V: third ventricle.

Blunted eNOS contribution to tonic NO inhibition of PVN neuronal activity and RSNA in HF rats

Lastly, 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 rats8, 9. In vitro patch-clamp recordings from retrogradely-labeled PVN-RVLM neurons showed that blockade of eNOS (L-NIO, 10μmol/L) increased neuronal activity in Sham (∼55%, n=10 cells, P<0.05), but not in HF rats (∼0.5%, n=12 cells Fig.6A-B). Similarly, in vivo studies showed that the increase in RSNA (expressed as percent 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/group, P<0.05, Sham vs. HF, two-way ANOVA, Fig.6C).

Figure 6. eNOS blockade-mediated increases in PVN-RVLM firing activity and RSNA are blunted in HF rats.

Figure 6

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A: in vitro recordings of firing activity in a PVN-RVLM neuron before (top), during (middle) and after (bottom) bath application of L-NIO (10μmol/L). B: Summary data for mean firing frequency in PVN-RVLM neurons from Sham and HF rats. *P<0.05, vs. Sham ACSF; n=10 cells from Sham and 12 cells from HF rats. C: Summary data showing mean percent changes in RSNA, and changes in MAP and HR following microinjection of L-NIO (50, 100 and 200pmol) into the PVN in Sham and HF rats. *P<0.05 vs. respective dose in Sham rats; n=5/group.

Discussion

A large body of evidence supports an integral role for CNS NO in the control of the circulation1-3, as well as a contribution of blunted NO function to neurohumoral activation in HF8, 9. Most of these studies however, focused on the targets and outcomes of NO actions, while only a few specifically addressed cellular sources and isoforms mediating NO actions10, 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 targets2, elucidating the cellular sources and isoforms contributing to NO availability is of critical importance. Results from the present studies show that (a) in addition to nNOS, eNOS is abundantly expressed in the SON and PVN; (b) nNOS and eNOS display a segregated, though spatially interrelated cellular distribution (neuronal and astroglial, respectively); (c) eNOS contributes to constitutive NO production and tonic NO-dependent inhibition of neuronal activity and sympathetic outflow from the PVN, and (d) 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 neurons5, 11, our studies support a cellular segregation between these two isoforms. Still, the precise cellular distribution of eNOS in the CNS remains controversial. While eNOS was reported both in astrocytic cultures29 and brain tissue30, 31, including recently in the NTS17, others failed to detect eNOS in astrocytes16. In addition, while 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 endfeet32. This result is somewhat inconsistent with previous studies showing eNOS in brain endothelium16. Thus, methodological dissimilarities, including antibodies used17, 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 indicator33. Our results showing a diminished basal DAF-2 in slices pre-treated with L-NIO or Cavtratin, two different eNOS selective blockers26, 27 support a tonic contribution of eNOS to PVN NO levels. The lack of L-NIO effects in eNOS KO 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 neurons4, supporting its contribution 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 studies34, 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, due to the small size of the endothelium and/or limited endothelium dye loading under our experimental conditions. In fact, using an in vivo approach previously shown to efficiently load vascular structures23, 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 following eNOS blockade. Our results indicate that despite eNOS segregated cellular distribution, the close proximity among neurons, astrocytes and the local microvasculature in these nuclei25, 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 while NO efficiently diminished the activity of most presympathetic PVN neurons5, only a limited proportion of them expressed detectable levels of nNOS5, 35. Taken together, these studies suggest that NO produced by, and diffusing from an alternative isoform (i.e., 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 heir activity is tonically restrained by eNOS-derived NO. The RVLM is a critical target of sympathoexcitatory PVN descending projections36, 37, and over-activation of this pathway contributes to sympathoexcitation and increased MAP in hypertension38 and dehydration39. 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 non-selective NOS inhibitor L-NMMA to be blocked by prior 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 non-selective blockers (in vitro DAF-2), along with in vivo eNOS antisense studies (Figs.2G,J), 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 two isoforms, it is important to consider other properties as well. Based on their distinct cellular sources and bioactive properties40, 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, respectively14, 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 circulation6, 7, 10. This raise questions about the functional significance of the presence of, in principle, two 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 NMDA receptors in dendritic spines results in a brief, low-amplitude NO transient, which is spatially and temporally restricted to the site of production19, 42. Therefore, this NMDA-nNOS phasic modality is better suited to act in a synapse-specific manner20. Conversely, eNOS is able to synthesize NO in a sustained manner, even at resting cytosolic calcium concentrations18, 19, supporting eNOS as the likely primary source of tonic ambient NO levels, mediating more widespread effects of NO within CNS networks20, 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 contributes 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 HF9, 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. Firstly, 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. Secondly, eNOS activity can be efficiently regulated by phosphorylation of various sites, particularly the Ser1177 and the Thr495, resulting in increased and decreased activity, respectively28. 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 rats45. 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 HF46, our study supports a contribution of blunted CNS eNOS function to elevated neuronal activity and sympathoexcitation in this condition.

Perspectives

Increased neurohumoral drive, characterized by sympathoexcitation, and elevated circulating neurohormones constitute a common finding in humans and experimental animal models of HF47, 48. Given that sympathoexcitation increases the progression and mortality in HF47, 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.

Supplementary Material

1

Acknowledgments

We thank Dr. Jessica A. Filosa, Georgia Health Sciences University, for critical reading of the manuscript and Dr. Thomas A. Kent, Baylor College of Medicine, for help with experimental in vivo protocols for perivascular NO measurements.

Sources of funding: This work was supported by AHA 0640092N and NIH HL085767 to Stern JE; and National Heart, Lung, and Blood Institute Grant HL-62222 to Patel KP.

Footnotes

Conflict of Interest/ Disclosure: None.

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