HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Supplemental Figure All Versions of this Article: 293/6/C1797    most recent 00554.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (4) Citing Articles via Google Scholar Google Scholar Articles by Filosa, J. A. Articles by Gonzalez Bosc, L. V. Search for Related Content PubMed PubMed Citation Articles by Filosa, J. A. Articles by Gonzalez Bosc, L. V.

VASCULAR BIOLOGY

Activity-dependent NFATc3 nuclear accumulation in pericytes from cortical parenchymal microvessels

¹Department of Psychiatry, College of Medicine, University of Cincinnati, Cincinnati, Ohio; ²Department of Pharmacology, College of Medicine, University of Vermont, Burlington, Vermont; and ³Department of Cell Biology and Physiology, School of Medicine, University of New Mexico, Albuquerque, New Mexico

Submitted 30 October 2006 ; accepted in final form 12 September 2007

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The calcium-dependent transcription factor NFATc3, which is a member of the nuclear factor of activated T cells (NFAT) family of transcription factors, is critical for embryonic vascular development and differentiation. Despite its potential importance, nothing is known about NFATc3 regulation in the brain microcirculation. In the present study, we sought to investigate the role that glutamate, possibly through astrocytic communication, plays in the control of NFATc3 regulation in pericytes from parenchymal microvessels. Coronal cortical slices from neonatal rats were subjected to electrical field stimulation or were treated with the metabotropic glutamate receptor agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD). NFATc3, glial fibrillary acidic protein (an astrocyte-specific marker), and platelet-derived growth factor-β-receptor (a pericyte-specific marker) were detected by immunofluorescence. Electrical field stimulation induced NFATc3 nuclear accumulation in pericytes. This response was dependent on neuronal activity and group I metabotropic glutamate receptor (mGluR) activation. In addition, t-ACPD significantly increased NFATc3 nuclear accumulation in both astrocytes and pericytes. NFATc3 nuclear accumulation in pericytes was prevented when astrocytic function was abolished with the gliotoxin L--aminoadipate or by the inhibition of calcineurin, cyclooxygenase, and nitric oxide synthase. This is the first study to report NFATc3 expression in pericytes from parenchymal microvessels and in astrocytes from native tissue. Our results suggest a model by which glutamate, via mGluR activation, may regulate gene transcription in pluripotent vascular pericytes.

astrocytes; neuronal activity; brain cortex; rat; calcium

NFAT is a calcium-regulated transcription factor that was first associated with gene expression changes in T cells (9, 25, 46). It is now clear that NFAT plays an important role in the differentiation of a diverse array of cell types outside the immune system (5). To date, five members of the NFAT transcription factor family have been identified, including NFATc1 (NFAT2/c), NFATc2 (NFAT1/p), NFATc3 (NFAT4/x), NFATc4 (NFAT3), and NFAT5. NFAT has been implicated in vascular development and vessel maturation (18, 19). NFATc3-/NFATc4-null mice die in uterus at around the age of embryonic day 11 (E11), with defects in vascular pattern being attributed to endothelial cell dysfunction (18); however, single knockouts display no abnormalities. In the double-knockout mice, endothelial cells fail to respond to and give signals for vessel formation (5, 18). Along these lines, Hernandez et al. (23) showed that inhibition of the calcineurin/NFAT pathway prevents VEGF-mediated angiogenesis.

The subcellular localization of NFAT is an important regulatory event in the activation of this transcription factor. A rise in intracellular Ca²⁺ activates the Ca²⁺/calmodulin-dependent protein phosphatase, calcineurin, which dephosphorylates the NFAT molecule at specific NH₂-terminal serine residues (reviewed in references 5, 24, and 46). NFAT dephosphorylation causes a conformational change in the molecule that exposes nuclear localization signals (41), which allows NFAT nuclear import. NFAT nuclear accumulation is subject to further regulation by serine/threonine kinases, which promote the export of nuclear NFAT (3, 14).

We recently demonstrated activation of the NFATc3 isoform by G_q/11-coupled receptor agonists (uridine triphosphate, endothelin-1, and angiotensin II) and physiological intravascular pressure in VSMCs from extracerebral arteries (14, 15, 17, 53). Pressure-induced NFATc3 nuclear accumulation in VSMCs from cerebral arteries is dependent on Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channels, endothelium-derived nitric oxide (NO), and cGMP-dependent protein kinase (PKG) (17). Both PKG and NFAT have been associated with the maintenance of the contractile phenotype of VSMCs, which suggests that NFAT may be a linker in PKG-dependent gene transcription (17, 39, 54).

In the central nervous system, a rise in intracellular Ca²⁺ has been associated with increased NFAT transcriptional activity in both neurons and astrocytes through a glutamate-mediated pathway (20, 25, 28). The activation of metabotropic glutamate receptors (mGluR) in cortical astrocytes increases intracellular Ca²⁺ concentration ([Ca²⁺]_i) through an inositol 1,4,5-trisphosphate-mediated pathway (11). The rise in astrocytic [Ca²⁺]_i enhances the release of several vasoactive substances, some of which have been implicated in vessel maturation (i.e., prostanoids and NO) (33, 36, 40). Importantly, the activation of gene expression is necessary for the proper establishment of the cerebral microcirculation, and intercellular signaling is an essential step in this process. In the present study, we hypothesize that synaptically released glutamate signals pericytes, potentially via astrocytes, to increase NFATc3 nuclear accumulation. Nuclear translocation is a required step for NFATc3-mediated gene transcription and/or repression. Here we provide the first evidence that NFATc3 is expressed in pericytes from parenchymal microvessels. NFATc3 nuclear accumulation was induced by electrical field stimulation (EFS) and mGluR agonists, which suggests a mechanism by which activity-dependent glutamate release may regulate gene transcription in pluripotent vascular pericytes. Finally, we provide evidence that the flow of information for NFATc3 nuclear translocation in pericytes requires the participation of functional astrocytes.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Slice preparation. Ten-day-old neonatal Sprague-Dawley rats were used. Coronal cortical slices were prepared following procedures approved by the Office of Animal Care Management at the University of Vermont. The cortex was immediately removed and placed in ice-cold artificial cerebrospinal fluid (aCSF) equilibrated with 95% O₂-5% CO₂. Cortical slices of 150-µm thickness were cut by using a vibratome (Leica VT 1000S) and were immediately incubated in aCSF at room temperature (pH 7.45) until needed.

Experimental protocol. At the time of the experiment, cortical slices were incubated in aCSF equilibrated with 95% O₂-5% CO₂ at 37°C for 10 min for stabilization. The stabilization period was followed by 10 min of preincubation with or without antagonists and subsequently by 30 min of incubation with the mGluR agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD). The EFS protocol consisted of 3 min of stimulation (50 Hz, 0.3-ms pulses of 150 V). Brain slices were then fixed 15 min after the end of the stimulation to allow sufficient time for NFATc3 to translocate into the nucleus.

NFATc3 and nuclei immunofluorescence colocalization. At the end of the experimental protocol, cortical slices were immediately fixed in 4% formaldehyde/PBS overnight. After permeabilization and block of unspecific binding sites, primary antibody [rabbit anti-NFATc3 at 1:100 and goat anti-platelet-derived growth factor-β receptor (PDGFR-β) at 1:100 or goat anti-glial fibrillary acidic protein (GFAP) at 1:250 (Santa Cruz Biotechnology)] diluted in 0.2% gelatin-0.1% Triton-PBS was applied overnight at 4°C. Secondary antibodies (Cy5-anti-rabbit IgG and Cy3-anti-goat IgG at 1:500; Jackson ImmunoResearch) were applied for 2 h at room temperature. Nuclei were stained with the fluorescent nucleic acid dye Sytox (1:5,000; Molecular Probes). Individual cortical slices were then mounted (Aqua Polymount mounting medium; Polysciences) onto glass slides and were examined at x60 magnification by using a Bio-Rad 1000 laser-scanning confocal microscope. NFATc3, PDGFR-β or GFAP, and nuclei were detected by sequentially monitoring the Cy5, Cy3, and Sytox fluorescence by using an excitation wavelength of 650 nm, 550 nm, and 488 nm and an emission wavelength of 670 nm, 570 nm, and 520 nm, respectively. Before the generation of an overlay image, a threshold was applied to each individual channel to exclude pixels that corresponded to nonspecific staining. Nonspecific staining was originally determined in images collected from brain slices incubated with secondary antibodies alone. To avoid differences in fluorescence intensity between preparations, brain slices from all experimental groups were processed simultaneously; images were acquired with the same settings, and care was taken to obtain a similar level of pixel intensity between channels. Nuclei were optically sectioned when the majority of the nuclear surface was in focus. For scoring of NFATc3-positive nuclei, multiple fields for each slice were imaged and counted by two independent observers under double-blind conditions by using Metamorph software (Universal Imaging). The software was programmed so that individual pixels within a given image would appear white if colocalization of the green nucleic acid stain and the Cy5-NFATc3 stain occurred. The criteria for considering positive nuclei for quantification were as follows: a cell was considered positive if colocalization (white pixels) was uniformly distributed within the nucleus and if >10% of the nucleus surface area included white pixels. Nuclei were considered negative if the surface areas of the nucleus included <10% white pixels and if the white pixels were distributed in the perinuclear borders. The percentage of white pixels (255) over the entire number of pixels comprising the nucleus was calculated by tracing each individual nuclei and then obtaining a histogram distribution for all the pixels (0–255) in the nucleus by using ImageJ software (version 1.33u; W. Rasband, National Institutes of Health, Bethesda, MD) (Fig. 1).

View larger version (63K): [in this window] [in a new window]   Fig. 1. Quantification of NFATc3-positive nuclei. A: representative images of specific immunofluorescence staining for PDGF-β receptor (PDGFR-β), Sytox, NFATc3 [nuclear factor of activated T cells (NFAT)], and the corresponding overlay image of the three different channels. B: colocalization of NFATc3 with Sytox (left). White pixels represent the colocalization between the two channels. Sytox image showing nuclear NFATc3 accumulation (right). C: expanded image from B, right, showing traced nuclei and the corresponding colocalized (white) pixels. D: percentage of white pixels over total surface area pixels for each individual pericyte identified in A (PDGFR-β staining). Although cell no. 4 had >10% white pixel surface area, it was not included in the positive nuclei count; for details, see METHODS.

Calcium imaging. Ca²⁺ imaging was performed with the use of a confocal spinning unit (Yokogawa CSU 10) by using the imaging system and a Zeiss microscope (Axioscope 2FS) equipped with infrared differential interference contrast optics, a water immersion objective (Zeiss x63, numerical aperture 0.9), and an electron multiplying charge coupled device camera (iXon+885, Andor Tech). Cortical slices were incubated (1–3 h) at room temperature in aCSF containing 10 µM Fluo-4 AM (Invitrogen) and pluronic acid (2.5 µg/ml) in a custom chamber gassed with 95% O₂-5% CO₂. Using this loading protocol, we are able to visualize Ca²⁺ transients in both astrocytes and vascular cells. Fluo-4 was excited at 488 nm by using a diode-pumped solid-state laser (Melles Griot), and fluorescence emission was collected at >495 nm. Images were acquired at 3–16 frames/s. Fractional fluorescence (F/F_o) was determined by dividing the fluorescence intensity (F) within a region of interest (10 x 10 pixels, 2.5 x 2.5 µm) drawn on individual cells by a baseline fluorescence value (F_o) obtained from 50 images showing minimal fluorescence change. Data were analyzed with the use of custom software created by A. D. Bonev (Univ. of Vermont).

Solutions and drugs. The composition of the aCSF (in mM) was 5 KCl, 124 NaCl, 1.3 MgSO₄, 26 NaHCO₃, 1.24 KH₂PO₄, 10 glucose, and 2.4 CaCl_2, and it was equilibrated with 95% O₂-5% CO₂. The mGluR antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (Aida) and 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) were obtained from Tocris Cookson. FK-506 was kindly provided by Fujisawa. Cyclosporin A (CsA) was obtained from Calbiochem. All other drugs used were obtained from Sigma: mGluR agonist t-ACPD, tetrodotoxin, nifedipine, indomethacin (Indo), and N-nitro-L-arginine (L-NNA) and L--aminoadipate (LAA).

Data analysis. Results are expressed as means ± SE. Statistical significance was tested at the 95% (P < 0.05) confidence level by using one-way analysis of variance followed by Tukey's multiple-comparison test, where applicable.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Methodological considerations. To determine the number of NFATc3-positive nuclei accumulated in pericytes, brain slices were immunostained for NFATc3, the pericyte marker PDGFR-β, and the nuclear stain Sytox (Fig. 1A). PDGFR-β was used as a pericyte marker since it is selectively expressed in these cells throughout development, and the lack of these receptors, as in null-knockout mice, results in the absence of pericytes and in the formation of weak capillary walls (32). Nuclei in focus corresponding to a PDGFR-β-positive membrane staining were only considered for the analysis. Colocalization of NFATc3 and Sytox is shown in white (Fig. 1B), the nucleus of each pericyte was manually traced, and the corresponding histogram was obtained. Figure 1C shows representative nuclei corresponding to a cortical arteriole; nucleus no. 1 is negative (<10% white pixels), nuclei nos. 2, 3, and 7 are positive (>10% white pixels), and nucleus no. 3 is negative because it lacks a PDGFR-β-positive membrane staining and the white pixels are located in the perinuclear zone. Thus, from the vessel shown in Fig. 1, five out of eight nuclei (62.5%) were considered positive (Fig. 1D).

EFS induces NFATc3 nuclear accumulation in pericytes from cortical parenchymal microvessels. Figure 2A shows representative confocal images of control and EFS-stimulated cortical brain slices immunostained for NFATc3 and the pericyte marker PDGFR-β, as well as the nuclear stain Sytox. Colocalization of NFATc3 and the nuclear stain is shown by the white pixels. NFATc3 nuclear accumulation was significantly increased by EFS from a basal percent level of 13.4 ± 3.8% to 60.6 ± 9.0%, P < 0.001 (n = 8). This response was prevented by preincubation with the sodium channel blocker tetrodotoxin (1 µM), which suggests that increased neuronal activity activates the signaling pathway that mediates NFATc3 nuclear accumulation in pericytes (Fig. 2B).

View larger version (80K): [in this window] [in a new window]   Fig. 2. Electrical field stimulation (EFS) induces NFATc3 nuclear accumulation in parenchymal pericytes. A: representative immunofluorescence confocal microscopy images of control and EFS-stimulated cortical brain slices. NFATc3 immunostaining is shown in red, PDGFR-β (pericyte marker) in blue, and nuclei in green. White pixels represent colocalization of NFATc3 and nuclei. Scale bar represents 50 µm. Cortical brain slices were superfused with artificial cerebrospinal fluid (aCSF), EFS was applied for 3 min (50 Hz, 0.3 ms, 150 V), and slices were continuously superfused for an additional 15 min to allow sufficient time for NFAT to translocate into the nucleus. At the end of the experiments, slices were fixed with 4% formaldehyde in PBS, and the immunofluorescence was performed. A subgroup of slices was preincubated for 10 min with tetrodotoxin (TTX, 1 µM). In the NFAT panel (red) of the stimulated slice, the contours of pericyte NFAT-positive (green dashed line) and NFAT-negative (white dashed line) nuclei are shown. B: summary data. *P < 0.001 vs. control, #P < 0.001 vs. EFS. n = 8.

View larger version (58K): [in this window] [in a new window]   Fig. 3. Metabotropic glutamate receptor (mGluR) activation induces NFATc3 nuclear accumulation in astrocytes and pericytes. A: representative immunofluorescence confocal microscopy images of control and (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD)-stimulated cortical brain slices. NFATc3 immunostaining is shown in red, glial fibrillary acidic protein (GFAP, astrocyte marker) in blue, and nuclei in green. White pixels represent colocalization of NFATc3 and nuclei. Scale bar represents 50 µm. Cortical brain slices were superfused with aCSF, and 50 µM t-ACPD was applied for 30 min. At the end of the experiments, slices were fixed with 4% formaldehyde in PBS, and the immunofluorescence was performed. *P < 0.01 vs. control; n = 4. B: mGluR activation induces NFATc3 nuclear accumulation in parenchymal microvessel pericytes. Cortical brain slices were superfused with aCSF, and t-ACPD was applied for 30 min. A subgroup of slices was preincubated for 10 min with mGluR I antagonists [50 µM 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) and 300 µM (RS)-1-aminoindan-1,5-dicarboxylic acid (Aida)] or calcineurin blockers [1 µM Cyclosporin A (CsA) and 1 µM FK-506 (FK)]. Slices were fixed with 4% formaldehyde in PBS, and the immunofluorescence was performed. *P < 0.001 vs. control, #P < 0.05 vs. t-ACPD; n = 10. C: cortical brain slices were superfused with aCSF, EFS was applied for 3 min (50 Hz, 0.3 ms, 150 V), and slices were continuously superfused for an additional 15 min to allow sufficient time for NFAT to translocate into the nucleus. A subgroup of slices was preincubated for 10 min with mGluR I antagonists (50 µM MPEP and 300 µM Aida). At the end of the experiments, slices were fixed with 4% formaldehyde in PBS, and the immunofluorescence was performed. *P < 0.01 vs. control, #P < 0.05 vs. EFS; n = 8. No significant differences were observed between control and ESF+MPEP+Aida.

Cellular targets of glutamate-induced signaling during mGluR stimulation. Because Gillard et al. (13) reported expression of mGluR on cerebral pericytes, we first determined whether, in our preparation, pericytes expressed these receptors. Consistent with the report of Gillard et al. (13), we found that pericytes expressed both the mGluR1 and the mGluR5 (Fig. 4A). However, it is possible that mGluR could also be expressed in astrocytic processes that are in direct contact with the pericyte membrane and are thus an ideal upstream target from the pericytes. To test this hypothesis, astrocytic function was disrupted by the gliotoxin LAA (2 mM). LAA is a structural homolog of glutamate (21) and has been efficiently used as a gliotoxin in both in vivo and in vitro studies (29, 47, 48, 55). Following a 40-min incubation period, slices were exposed to the mGluR receptor agonist t-ACPD in the presence and absence of LAA. In response to mGluR activation, the percentage of NFATc3-positive nuclei increased from 15.7 ± 5.3% (n = 10) to 68.6 ± 7.5% (n = 8). In contrast, t-ACPD failed to increase NFATc3 nuclei accumulation in the group preincubated with LAA (28.35 ± 9; n = 8). In a control group, no significant differences were observed between control and slices incubated with the LAA alone (% NFATc3-positive nuclei: 23.3 ± 12%; n = 4) (Fig. 4B). As a control experiment and to verify the specificity of the gliotoxin, we monitored [Ca²⁺]_i changes in response to t-ACPD (astrocytic stimulation) and elevated K⁺ (neuronal and vascular stimulation) in slices preincubated in LAA (>30 min). As shown in Fig. 4C, astrocytes failed to respond to t-ACPD in slices preincubated with the gliotoxin. On the other hand, both neurons and vascular cells responded with an increase in [Ca²⁺]_i following exposure to elevated K⁺ (supplemental figure for this article is available online at the American Journal of Physiology-Cell Physiology website).

View larger version (77K): [in this window] [in a new window]   Fig. 4. mGluR-induced NFATc3 nuclear accumulation in parenchymal pericytes requires functional astrocytes. A: representative confocal microscopy images showing expression of mGluR1 and mGluR5 in parenchymal microvessel pericytes. mGluR 1 and mGluR5 are shown in green, PDGFR is shown in red, and nuclei is shown in blue. White pixels represent colocalization between mGluR and PDGFR. Scale bar represents 50 µm. B: cortical brain slices were superfused with aCSF, and 50 µM t-ACPD was applied for 30 min. A subgroup of slices was preincubated for 40 min with L--aminoadipate (LAA, 2 mM). Slices were fixed with 4% formaldehyde in PBS, and the immunofluorescence was performed. *P < 0.001 control and t-ACPD+LAA; #P < 0.01 vs. t-ACPD; n = 8. C: astrocytic response to 100 µM t-ACPD before and after incubation in LAA (2 mM). a: regions of interest (ROIs) on astrocytes. b: traces corresponding to ROIs in a showing intracellular calcium changes in response to t-ACPD. c: pseudocolor images showing intracellular calcium changes in astrocytes in response to t-ACPD before (left) and after (right) LAA incubation.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Nitric oxide synthase and cyclooxygenase derivatives mediate t-ACPD-induced NFATc3 nuclear accumulation in parenchymal microvessel pericytes. Cortical brain slices were superfused with aCSF, and 50 µM t-ACPD was applied for 30 min. A subgroup of slices was preincubated for 10 min with N-nitro-L-arginine (L-NNA, 200 µM) or with indomethacin (Indo, 10 µM). Slices were fixed with 4% formaldehyde in PBS, and the immunofluorescence was performed. *P < 0.001 vs. control, #P < 0.01 and **P < 0.05 vs. t-ACPD; n = 10.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This is the first study to report expression of the transcription factor NFATc3 in pericytes and astrocytes from cortical brain slices. We have found that EFS-induced increase in neuronal activity results in NFATc3 nuclear accumulation in pericytes. This response was dependent on the activation of astrocytic mGluR. Moreover, glutamate-induced NFATc3 nuclear accumulation in pericytes was coupled to NOS and cyclooxygenase activity. In accordance with the reported effect of glutamate on NFATc3 transcriptional activity in cultured astrocytes (28), our results showed that, in native astrocytes, t-ACPD, a mGluR agonist, significantly increased NFATc3 nuclear accumulation.

Glutamate release on neuronal depolarization mediates NFATc3 nuclear accumulation in pericytes from cortical parenchymal microvessels. An increase in neuronal activity results in the release of glutamate from neurons and astrocytes and subsequent activation of glutamate receptors in target cells. Neurons and astrocytes express ionotropic and metabotropic glutamate receptors (4). Recently, mGluR expression was also shown in pericytes and endothelial cells within the microvasculature of the cortex (13), which suggests a possible direct role for glutamate in the cerebral circulation. Consistent with this report, our results also suggest the expression of mGluR1 and mGluR5 (group I) in pericytes of parenchymal microvessels. Interestingly, however, the expression of group I mGluRs was observed only on the outer surface membrane, likely in contact with astrocytic processes (endfeet). Thus, it is also possible that the expression of these receptors was not specific to pericytes but rather to astrocytic membrane as well. We found that enhanced neuronal activity induced by EFS significantly increased NFATc3 nuclear accumulation in pericytes. This latter event depended on the activation of mGluRs, although other pathways are possible since EFS likely induces the release of multiple neuronal signals. However, a similar response was induced by the selective mGluR agonist, t-ACPD, in pericytes and cortical astrocytes. As expected, t-ACPD-induced NFATc3 pericyte nuclear accumulation was blocked by antagonists of the group I mGluRs. In addition, glutamate-induced NFATc3 nuclear accumulation is dependent on the activation of the Ca²⁺/calmodulin-dependent phosphatase calcineurin, which is consistent with the requirement of NFAT dephosphorylation as part of its activation mechanism (reviewed in Refs. 5, 24, 26, and 46).

The observed expression of mGluR in pericytes raises the possibility that these are the direct target cells for the released glutamate during neuronal activation. It is also possible that the immunostaining did not provide accurate localization of the receptors and that the actual targets were upstream of the pericytes, such as the astrocytes. To address this important question, astrocyte function was abolished by exposing brain slices to a gliotoxin, LAA. At low concentrations, LAA is toxic only to glial cells, possibly via oxidative cell damage (47). Following LAA incubation, the mGluR agonist failed to induce NFATc3 nuclear accumulation in pericytes, which suggests that the target cell was indeed the astrocyte. These results suggest that signaling from the neurons to the astrocytes is an essential step leading to NFATc3 nuclear accumulation in pericytes.

Possible signaling pathways mediating NFATc3 nuclear accumulation in pericytes on mGluR activation. Different sources of NO have been demonstrated in the nervous system. Neurons, astrocytes, and endothelial cells have been shown to express constitutive NOS (neuronal and endothelial NOS isoforms) (6, 34, 36, 40, 49, 50). Activation of the group I mGluRs in astrocytes increases intracellular Ca²⁺, which, in turn, results in the release of Ca²⁺-dependent vasoactive substances such as prostaglandins and NO (56). The close proximity of pericytes to astrocytic endfeet provides a potential intercellular pathway by which astrocytes, in addition to other cellular sources such as endothelial cells, may communicate with pericytes through the release of NO and/or prostaglandins (30, 34, 36, 56). Previously, we showed that NFATc3 nuclear accumulation in VSMCs depends on the activation of PKG through endothelial NO (17). In these cells, PKG regulates NFATc3 nuclear export through the modulation of c-Jun kinase 2 activity (14, 17). As in VSMCs, we found that glutamate-induced NFATc3 nuclear accumulation in pericytes is also dependent on NO. The cellular sources of NO and the targets of NO (astrocytes vs. the pericytes) are still to be determined. For example, it is well known that, following neuronal N-methyl-D-aspartate receptor stimulation, NO is formed and released (8, 12) and that NO can then diffuse and signal neighboring cells. It has also been shown that signals that increase Ca²⁺ in astrocytes can induce the production of NO in these cells, and NO then acts on the astrocytes to stimulate Ca²⁺-influx pathways that contribute to the refilling of astrocytic Ca²⁺ stores (31). Furthermore, the rise in astrocytic Ca²⁺ has been linked to the release of prostaglandins (57), which further supports the intercellular communication between astrocytes and pericytes and our observations that blockade of NO signaling with L-NNA and inhibition of prostaglandin synthesis with Indo both inhibit NFATc3 nuclear accumulation in pericytes. Future studies on the precise cellular sources and targets of these proposed pathways will be needed.

In summary, this is the first report demonstrating NFATc3 expression in both native cortical astrocytes and pericytes from parenchymal microvessels. Increased neuronal activity or application of the mGluR agonist resulted in NFATc3 nuclear accumulation in both astrocytes and pericytes from native tissue. In pericytes, this response was mediated by group I mGluRs, required functional astrocytes, NOS, and cyclooxygenase activity. Our findings support the concept of a neuronal-glial-vascular communication mediated by glutamate (10, 56). Given the important physiological role that NFAT, including NFATc3, plays in the establishment of the vasculature, the present study provides an important initial step in our understanding of the upstream intercellular processes leading to NFATc3 nuclear accumulation in pericytes.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Institutes of Health Grants HL-44455, HL-63722, and DKK-53832 to M. T. Nelson, by a Postdoctoral Fellowship from the American Heart Association to L. V. Gonzalez Bosc (0225597T) and J. A. Filosa (0425923T), and by a grant from the Totman Trust for Medical Research.

	ACKNOWLEDGMENTS

 
We thank David Hill-Eubanks for helpful discussions.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. A. Filosa, Dept. of Psychiatry, Univ. of Cincinnati, 2170 E. Galbraith Rd., Room 239-A, Cincinnati, OH 45237 (e-mail: jessica.filosa{at}uc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
1. Ballabh P, Braun A, Nedergaard M. The blood-brain barrier: an overview: structure, regulation, and clinical implications. Neurobiol Dis 16: 1–13, 2004.[CrossRef][Web of Science][Medline]

2. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med 6: 389–395, 2000.[CrossRef][Web of Science][Medline]

3. Chow CW, Rincon M, Cavanagh J, Dickens M, Davis RJ. Nuclear accumulation of NFAT4 opposed by the JNK signal transduction pathway. Science 278: 1638–1641, 1997.[Abstract/Free Full Text]

4. Condorelli DF, Conti F, Gallo V, Kirchhoff F, Seifert G, Steinhauser C, Verkhratsky A, Yuan X. Expression and functional analysis of glutamate receptors in glial cells. Adv Exp Med Biol 468: 49–67, 1999.[Web of Science][Medline]

5. Crabtree GR, Olson EN. NFAT signaling: choreographing the social lives of cells. Cell 109, Suppl: S67–S79, 2002.

6. Dawson TM, Snyder SH. Gases as biological messengers: nitric oxide and carbon monoxide in the brain. J Neurosci 14: 5147–5159, 1994.[Abstract]

7. Doherty MJ, Canfield AE. Gene expression during vascular pericyte differentiation. Crit Rev Eukaryot Gene Expr 9: 1–17, 1999.[Web of Science][Medline]

8. East SJ, Garthwaite J. NMDA receptor activation in rat hippocampus induces cyclic GMP formation through the L-arginine-nitric oxide pathway. Neurosci Lett 123: 17–19, 1991.[CrossRef][Web of Science][Medline]

9. Emmel EA, Verweij CL, Durand DB, Higgins KM, Lacy E, Crabtree GR. Cyclosporin A specifically inhibits function of nuclear proteins involved in T cell activation. Science 246: 1617–1620, 1989.[Abstract/Free Full Text]

10. Filosa JA, Bonev AD, Nelson MT. Calcium dynamics in cortical astrocytes and arterioles during neurovascular coupling. Circ Res 95: e73–e81, 2004.[Abstract/Free Full Text]

11. Gallo V, Ghiani CA. Glutamate receptors in glia: new cells, new inputs and new functions. Trends Pharmacol Sci 21: 252–258, 2000.[CrossRef][Medline]

12. Garthwaite J, Garthwaite G, Palmer RM, Moncada S. NMDA receptor activation induces nitric oxide synthesis from arginine in rat brain slices. Eur J Pharmacol 172: 413–416, 1989.[CrossRef][Web of Science][Medline]

13. Gillard SE, Tzaferis J, Tsui HCT, Kingston AE. Expression of metabotropic glutamate receptors in rat meningeal and brain microvasculature and choroid plexus. J Comp Neurol 461: 317–332, 2003.[CrossRef][Web of Science][Medline]

14. Gomez MF, Gonzalez Bosc LV, Stevenson AS, Wilkerson MK, Hill-Eubanks DC, Nelson MT. Constitutively elevated nuclear export activity opposes Ca²⁺-dependent NFATc3 nuclear accumulation in vascular smooth muscle: role of JNK2 and Crm-1. J Biol Chem 278: 46847–46853, 2003.[Abstract/Free Full Text]

15. Gomez MF, Stevenson AS, Bonev AD, Hill-Eubanks DC, Nelson MT. Opposing actions of inositol 1,4,5-trisphosphate and ryanodine receptors on nuclear factor of activated T-cells regulation in smooth muscle. J Biol Chem 277: 37756–37764, 2002.[Abstract/Free Full Text]

16. Gonzalez Bosc LV, Layne JJ, Nelson MT, Hill-Eubanks DC. Nuclear factor of activated T cells and serum response factor cooperatively regulate the activity of an -actin intronic enhancer. J Biol Chem 280: 26113–26120, 2005.[Abstract/Free Full Text]

17. Gonzalez Bosc LV, Wilkerson MK, Bradley KN, Eckman DM, Hill-Eubanks DC, Nelson MT. Intraluminal pressure is a stimulus for NFATc3 nuclear accumulation: role of calcium, endothelium-derived nitric oxide, and cGMP-dependent protein kinase. J Biol Chem 279: 10702–10709, 2004.[Abstract/Free Full Text]

18. Graef IA, Chen F, Chen L, Kuo A, Crabtree GR. Signals transduced by Ca²⁺/calcineurin and NFATc3/c4 pattern the developing vasculature. Cell 105: 863–875, 2001.[CrossRef][Web of Science][Medline]

19. Graef IA, Chen F, Crabtree GR. NFAT signaling in vertebrate development. Curr Opin Genet Dev 11: 505–512, 2001.[CrossRef][Web of Science][Medline]

20. Graef IA, Mermelstein PG, Stankunas K, Neilson JR, Deisseroth K, Tsien RW, Crabtree GR. L-type calcium channels and GSK-3 regulate the activity of NF-ATc4 in hippocampal neurons. Nature 401: 703–708, 1999.[CrossRef][Medline]

21. Guidetti P, Schwarcz R. Determination of -aminoadipic acid in brain, peripheral tissues, and body fluids using GC/MS with negative chemical ionization. Mol Brain Res 118: 132–139, 2003.[Medline]

22. Herman IM, D'Amore PA. Microvascular pericytes contain muscle and nonmuscle actins. J Cell Biol 101: 43–52, 1985.[Abstract/Free Full Text]

23. Hernandez GL, Volpert OV, Iniguez MA, Lorenzo E, Martinez-Martinez S, Grau R, Fresno M, Redondo JM. Selective inhibition of vascular endothelial growth factor-mediated angiogenesis by cyclosporin A: roles of the nuclear factor of activated T cells and cyclooxygenase 2. J Exp Med 193: 607–620, 2001.[Abstract/Free Full Text]

24. Hill-Eubanks DC, Gomez MF, Stevenson AS, Nelson MT. NFAT regulation in smooth muscle. Trends Cardiovasc Med 13: 56–62, 2003.[CrossRef][Web of Science][Medline]

25. Ho AM, Jain J, Rao A, Hogan PG. Expression of the transcription factor NFATp in a neuronal cell line and in the murine nervous system. J Biol Chem 269: 28181–28186, 1994.[Abstract/Free Full Text]

26. Hogan PG, Chen L, Nardone J, Rao A. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev 17: 2205–2232, 2003.[Free Full Text]

27. Horsley V, Pavlath GK. Prostaglandin F2 stimulates growth of skeletal muscle cells via an NFATC2-dependent pathway. J Cell Biol 161: 111–118, 2003.[Abstract/Free Full Text]

28. Jones EA, Sun D, Kobierski L, Symes AJ. NFAT4 is expressed in primary astrocytes and activated by glutamate. J Neurosci Res 72: 191–197, 2003.[CrossRef][Web of Science][Medline]

29. Kato S, Sugawara K, Matsukawa T, Negishi K. Gliotoxic effects of -aminoadipic acid isomers on the carp retina: a long term observation. Neuroscience 36: 145–153, 1990.[CrossRef][Web of Science][Medline]

30. Kobari M, Fukuuchi Y, Tomita M, Tanahashi N, Takeda H. Role of nitric oxide in regulation of cerebral microvascular tone and autoregulation of cerebral blood flow in cats. Brain Res 667: 255–262, 1994.[CrossRef][Web of Science][Medline]

31. Li N, Sul JY, Haydon PG. A calcium-induced calcium influx factor, nitric oxide, modulates the refilling of calcium stores in astrocytes. J Neurosci 23: 10302–10310, 2003.[Abstract/Free Full Text]

32. Lindahl P, Johansson BR, Leveen P, Betsholtz C. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science 277: 242–245, 1997.[Abstract/Free Full Text]

33. Lovick TA, Brown LA, Key BJ. Neuronal activity-related coupling in cortical arterioles: involvement of astrocyte-derived factors. Exp Physiol 90: 131–140, 2005.[Abstract/Free Full Text]

34. Lovick TA, Key BJ. Distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase staining in intraparenchymal blood vessels of the rat brain. Neurosci Lett 196: 113–115, 1995.[CrossRef][Web of Science][Medline]

35. Moroni F, Lombardi G, Thomsen C, Leonardi P, Attucci S, Peruginelli F, Torregrossa SA, Pellegrini-Giampietro DE, Luneia R, Pellicciari R. Pharmacological characterization of 1-aminoindan-1,5-dicarboxylic acid, a potent mGluR1 antagonist. J Pharmacol Exp Ther 281: 721–729, 1997.[Abstract/Free Full Text]

36. Murphy S, Simmons ML, Agullo L, Garcia A, Feinstein DL, Galea E, Reis DJ, Mincgolomb D, Schwartz JP. Synthesis of nitric oxide in Cns glial-cells. Trends Neurosci 16: 323–328, 1993.[CrossRef][Web of Science][Medline]

37. Nag S. Morphology and molecular properties of cellular components of normal cerebral vessels. In: The Blood-Brain Barrier: Biology and Research Protocols, edited by Nag S., Totowa, NJ: Humana, 2003, p. 3–36.

38. Nehls V, Drenckhahn D. The versatility of microvascular pericytes: from mesenchyme to smooth muscle? Histochemistry 99: 1–12, 1993.[CrossRef][Web of Science][Medline]

39. Ohkawa Y, Hayashi K, Sobue K. Calcineurin-mediated pathway involved in the differentiated phenotype of smooth muscle cells. Biochem Biophys Res Commun 301: 78–83, 2003.[CrossRef][Web of Science][Medline]

40. Oka M, Wada M, Yamamoto A, Itoh Y, Fujita T. Functional expression of constitutive nitric oxide synthases regulated by voltage-gated Na⁺ and Ca²⁺ channels in cultured human astrocytes. Glia 46: 53–62, 2004.[CrossRef][Web of Science][Medline]

41. Okamura H, Aramburu J, Garcia-Rodriguez C, Viola JP, Raghavan A, Tahiliani M, Zhang X, Qin J, Hogan PG, Rao A. Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity. Mol Cell 6: 539–550, 2000.[CrossRef][Web of Science][Medline]

42. Pardridge WM. Blood-brain barrier biology and methodology. J Neurovirol 5: 556–569, 1999.[Web of Science][Medline]

43. Parpura V, Basarsky TA, Liu F, Jeftinija K, Jeftinija S, Haydon PG. Glutamate-mediated astrocyte neuron signaling. Nature 369: 744–747, 1994.[CrossRef][Medline]

44. Peppiatt CM, Howarth C, Mobbs P, Attwell D. Bidirectional control of CNS capillary diameter by pericytes. Nature 443: 700–704, 2006.[CrossRef][Medline]

45. Ramsauer M, Krause D, Dermietzel R. Angiogenesis of the blood-brain barrier in vitro and the function of cerebral pericytes. FASEB J 16: 1274–1276, 2002.[Abstract/Free Full Text]

46. Rao A, Luo C, Hogan PG. Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol 15: 707–747, 1997.[CrossRef][Web of Science][Medline]

47. Re DB, Boucraut J, Samuel D, Birman S, Kerkerian-Le Goff L, Had-Aissouni L. Glutamate transport alteration triggers differentiation-state selective oxidative death of cultured astrocytes: a mechanism different from excitotoxicity depending on intracellular GSH contents. J Neurochem 85: 1159–1170, 2003.[CrossRef][Web of Science][Medline]

48. Roon RJ, Koerner JF. Persistent depression of synaptic responses occurs in quisqualate sensitized hippocampal slices after exposure to L-aspartate-β-hydroxamate. Brain Res 734: 223–228, 1996.[CrossRef][Web of Science][Medline]

49. Roufail E, Stringer M, Rees S. Nitric oxide synthase immunoreactivity and NADPH diaphorase staining are co-localised in neurons closely associated with the vasculature in rat and human retina. Brain Res 684: 36–46, 1995.[CrossRef][Web of Science][Medline]

50. Shareef S, Sawada A, Neufeld AH. Isoforms of nitric oxide synthase in the optic nerves of rat eyes with chronic moderately elevated intraocular pressure. Invest Ophthalmol Vis Sci 40: 2884–2891, 1999.[Abstract/Free Full Text]

51. Shepro D, Morel NM. Pericyte physiology. FASEB J 7: 1031–1038, 1993.[Abstract]

52. Sims DE. Diversity within pericytes. Clin Exp Pharmacol Physiol 27: 842–846, 2000.[CrossRef][Web of Science][Medline]

53. Stevenson AS, Gomez MF, Hill-Eubanks DC, Nelson MT. NFAT4 movement in native smooth muscle. A role for differential Ca²⁺ signaling. J Biol Chem 276: 15018–15024, 2001.[Abstract/Free Full Text]

54. Wada H, Hasegawa K, Morimoto T, Kakita T, Yanazume T, Abe M, Sasayama S. Calcineurin-GATA-6 pathway is involved in smooth muscle-specific transcription. J Cell Biol 156: 983–991, 2002.[Abstract/Free Full Text]

55. Xu HL, Koenig HM, Ye S, Feinstein DL, Pelligrino DA. Influence of the glia limitans on pial arteriolar relaxation in the rat. Am J Physiol Heart Circ Physiol 287: H331–H339, 2004.[Abstract/Free Full Text]

56. Zonta M, Angulo MC, Gobbo S, Rosengarten B, Hossmann KA, Pozzan T, Carmignoto G. Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation. Nat Neurosci 6: 43–50, 2003.[CrossRef][Web of Science][Medline]

57. Zonta M, Sebelin A, Gobbo S, Fellin T, Pozzan T, Carmignoto G. Glutamate-mediated cytosolic calcium oscillations regulate a pulsatile prostaglandin release from cultured rat astrocytes. J Physiol 553: 407–414, 2003.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. de Frutos, C. H. Nitta, E. Caldwell, J. Friedman, and L. V. Gonzalez Bosc Regulation of soluble guanylyl cyclase-{alpha}1 expression in chronic hypoxia-induced pulmonary hypertension: role of NFATc3 and HuR Am J Physiol Lung Cell Mol Physiol, September 1, 2009; 297(3): L475 - L486. [Abstract] [Full Text] [PDF]

				U. Panzer, O. M. Steinmetz, J.-E. Turner, C. Meyer-Schwesinger, C. von Ruffer, T. N. Meyer, G. Zahner, C. Gomez-Guerrero, R. M. Schmid, U. Helmchen, et al. Resolution of renal inflammation: a new role for NF-{kappa}B1 (p50) in inflammatory kidney diseases Am J Physiol Renal Physiol, August 1, 2009; 297(2): F429 - F439. [Abstract] [Full Text] [PDF]

				S. de Frutos, L. Duling, D. Alo, T. Berry, O. Jackson-Weaver, M. Walker, N. Kanagy, and L. Gonzalez Bosc NFATc3 is required for intermittent hypoxia-induced hypertension Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2382 - H2390. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Figure All Versions of this Article: 293/6/C1797    most recent 00554.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (4) Citing Articles via Google Scholar Google Scholar Articles by Filosa, J. A. Articles by Gonzalez Bosc, L. V. Search for Related Content PubMed PubMed Citation Articles by Filosa, J. A. Articles by Gonzalez Bosc, L. V.