Am J Physiol Cell Physiol Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 294: C306-C312, 2008. First published November 21, 2007; doi:10.1152/ajpcell.00214.2007
0363-6143/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/1/C306    most recent
00214.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Damon, D. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Damon, D. H.

NERVOUS SYSTEM CELL BIOLOGY

TH and NPY in sympathetic neurovascular cultures: role of LIF and NT-3

Deborah H. Damon

Department of Pharmacology, University of Vermont, Burlington, Vermont

Submitted 24 May 2007 ; accepted in final form 14 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The sympathetic nervous system is an important determinant of vascular function. The effects of the sympathetic nervous system are mediated via release of neurotransmitters and neuropeptides from postganglionic sympathetic neurons. The present study tests the hypothesis that vascular smooth muscle cells (VSM) maintain adrenergic neurotransmitter/neuropeptide expression in the postganglionic sympathetic neurons that innervate them. The effects of rat aortic and tail artery VSM (AVSM and TAVSM, respectively) on neuropeptide Y (NPY) and tyrosine hydroxylase (TH) were assessed in cultures of dissociated sympathetic neurons. AVSM decreased TH (39 ± 12% of control) but did not affect NPY. TAVSM decreased TH (76 ± 10% of control) but increased NPY (153 ± 20% of control). VSM expressed leukemia inhibitory factor (LIF) and neurotrophin-3 (NT-3), which are known to modulate NPY and TH expression. Sympathetic neurons innervating blood vessels expressed LIF and NT-3 receptors. Inhibition of LIF inhibited the effect of AVSM on TH. Inhibition of neurotrophin-3 (NT-3) decreased TH and NPY in neurons grown in the presence of TAVSM. These data suggest that vascular-derived LIF decreases TH and vascular-derived NT-3 increases or maintains NPY and TH expression in postganglionic sympathetic neurons. NPY and TH in vascular sympathetic nerves are likely to modulate NPY and/or norepinephrine release from these nerves and are thus likely to affect blood flow and blood pressure. The present studies suggest a novel mechanism whereby VSM would modulate sympathetic control of vascular function.

vascular smooth muscle; sympathetic nervous system; neurotrophin-3; leukemia inhibitory factor; tyrosine hydroxylase; neuropeptide Y

THE SYMPATHETIC NERVOUS SYSTEM is a major determinant of cardiovascular function (13, 30). Catecholamines and neuropeptide Y (NPY) act as neurotransmitters at postganglionic sympathetic nerve terminals (3, 9), mediating the actions of the sympathetic nervous system. In the vasculature, catecholamines and NPY promote vasoconstriction (3, 9, 15) and growth of vascular endothelial cells (40) and vascular smooth muscle cells (VSM) (39). These actions are important for normal cardiovascular function and are implicated in the etiology of cardiovascular disease (11, 16, 18). The mechanisms or mediators that regulate the expression of catecholamines and NPY in postganglionic sympathetic neurons innervating the cardiovascular system are not well understood.

It is known that catecholamine and NPY expression can be regulated (2, 4, 20, 21, 22, 26, 27, 35). It is also known that target tissues modulate neurotransmitter and neuropeptide expression (27). Postganglionic sympathetic neurons innervating blood vessels express tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine synthesis, and express more NPY than postganglionic sympathetic neurons innervating most other targets (5, 19, 24). Matsumoto et al. (21) reported that media conditioned by VSM increased the number of sympathetic neurons that express NPY. These observations suggest that interactions between vascular cells and postganglionic sympathetic neurons induce or maintain the expression of NPY and/or catecholamines.

In the present study, in vitro coculture models were used to test the hypothesis that VSM promote or maintain NPY and/or catecholamines in postganglionic sympathetic neurons. The effects of VSM isolated from arteries of adult rats on NPY and TH were studied. The hypotheses that vascular-derived leukemia inhibitory factor (LIF) and neurotrophin-3 (NT-3) determine sympathetic NPY and TH expression were also tested.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Sympathetic ganglia and arteries were obtained from neonatal (male and female) and adult (female) rats. The use of these animals was in accordance with the National Institutes of Health guidelines for the humane care and use of animals in research and was approved by the Institutional Animal Care and Use Committee of the University of Vermont.

Cell culture. Postganglionic sympathetic neurons were isolated from superior cervical ganglia of neonatal rats. Ganglia were collected and dissociated for 10 min at 37°C in a collagenase/hyaluronidase solution (10 mg/ml bovine serum albumin, 4 mg/ml collagenase, 1 mg/ml hyaluronidase) and then for 10 min in trypsin (3 mg/ml). Dissociated cells were resuspended in neuronal media [DMEM/F12 supplemented with 5% FBS, 10% NuSerum (Collaborative), 50 ng/ml nerve growth factor, and penicillin/streptomycin] and applied to collagen-coated tissue culture dishes. The cells were allowed to attach overnight in a humidified 5% CO2 environment maintained at 37°C. Nonneuronal cells were then growth arrested with mitomycin C (10 µg/ml for 1 h).

VSM were isolated from explants of aorta and tail arteries of adult postpartum female Sprague Dawley rats (29). These cells exhibited characteristic "hill and valley" growth patterns and immunohistochemical labeling with a monoclonal antibody for smooth muscle-specific {alpha}-actin. VSM were used from passage 2 to 3. Vascular cells were grown in low-glucose DMEM supplemented with 10% FBS, 100 units of penicillin, and 100 units of streptomycin. Cells were maintained at 37°C in a humidified 5% CO2 environment.

VSM were added to neuronal cultures after washout of mitomycin C to generate neuronal/VSM cocultures. These cocultures were grown for 7 days in neuronal media. At this time, the VSM were confluent.

Compartmentalized cocultures were also prepared (6). Neurons were plated in one compartment and grown in neuronal media until processes extended into the second compartment (7–10 days). VSM were then added to the second compartment, which contained neuronal processes but not cell bodies. The cocultures were then grown in neuronal media for an additional 7 days.

Western analysis. Cells were lysed with three cycles of rapid freeze thawing and sonicated in phosphate-buffered saline (PBS) containing protease inhibitors. Cell lysates (10 µl) were diluted with an equal volume of electrophoresis running buffer, boiled for 5 min, and electrophoresed on 10 or 12% acrylamide gels. Gels were transferred to nitrocellulose membrane. After transfer, the gels were stained (GELCODE Blue Stain Reagent, Pierce) to verify that equal amounts of protein were loaded for each sample. The membranes were blocked with PBS containing 3% nonfat dry milk. After a brief rinsing, the membranes were incubated with shaking for 60 min at room temperature in PBS containing 3% nonfat dry milk and primary antibody [0.26 µg/ml smooth muscle {alpha}-actin (Sigma), 1 µg/ml growth-associated protein 43 (GAP43), 4 µg/ml glycoprotein 130 (gp130) (R & D Systems), 0.1 µg/ml LIF receptor (LIFR; Santa Cruz Biotechnology), 0.3 µg/ml NT-3 (Alomone Labs), 1 µg/ml TrkC (Upstate), 0.9 µg/ml TH (Sigma)]. After a brief rinsing, the membranes were incubated with shaking in PBS containing 3% nonfat dry milk and 0.67 µg/ml appropriate horseradish peroxidase-conjugated secondary antibodies for 60 min at room temperature. The horseradish peroxidase was detected with enhanced chemiluminescence (Pierce) and documented on autoradiographic film. Signals were quantified densitometrically.

Radioimmunoassay. Cell-associated NPY (extracts prepared as for Western analysis) was measured using a commercially available radioimmunoassay (RIA) kit (Peninsula Laboratories). Assays were performed in duplicate.

Immunohistochemistry. Cells and tissues were fixed (4% formaldehyde in PBS) and permeabilized (0.2% Triton X-100 in PBS). Cells and tissues were then incubated for 30 min with 5% FBS (in PBS) to block nonspecific labeling and then incubated overnight at 4°C with primary antibody [8 µg/ml GAP43, 0.4 µg/ml gp130 (R & D Systems), 0.4 µg/ml LIFR (Santa Cruz Biotechnology)]. Unbound primary antibody was removed with 3 washes (PBS). Cells were then incubated with 4 µg/ml corresponding secondary antibodies. Cells were visualized on an upright fluorescence microscope (Olympus BX50). Cell images were recorded digitally with an Olympus camera (model U-ULH) and Magnafire software. Nerve fibers on the adventitial surfaces of tail arteries were visualized with a Zeiss LSM510 META Laser Scanning Microscope, and digital images were captured with Zeiss LSM META camera and software.

Western and RIA data presentation and analysis. All data for TH were obtained from Western analyses of TH and GAP43. All data for NPY were obtained from RIAs for NPY and Western analyses of GAP43. Western analyses of TH and GAP43 and RIAs of NPY were performed on the same cell extracts. Within each experiment, NPY and TH were normalized to the amount of GAP43 in each sample. GAP43 was used as an index of sympathetic neuronal protein in each sample. GAP43 expression as measured by Western analysis did not reproducibly change under the experimental conditions used in the present studies. In addition, in a few experiments, cell number was determined. In these experiments, cell number and GAP43 expression were closely correlated. Within each experiment, NPY/GAP43 and TH/GAP43 were then expressed as a percentage of control, control being NPY/GAP43 and TH/GAP43 measured in neurons grown in the absence of VSM. This allowed for comparisons between experiments. All data are presented as means ± SE. Statistical differences were assessed using one-sample Wilcoxon signed-rank or two-tailed Mann-Whitney tests. Differences were considered significant if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
VSM modulate NPY and TH in postganglionic sympathetic neurons. To determine the effects of VSM on sympathetic NPY and TH, NPY and TH were assessed in cultures of postganglionic sympathetic neurons grown for 7 days in the absence and presence of aortic (AVSM) and tail artery VSM (TAVSM). These two arteries were chosen because they are differentially innervated by the sympathetic nervous system. The aorta is sparsely innervated, whereas the tail artery is heavily innervated (8, 31). The data for AVSM are shown in Fig. 1A. A representative Western analysis of TH and GAP43 is shown (Fig. 1A, left), with corresponding quantitative data (% of –VSM control) indicated in parentheses at bottom (mean ± SE of 7 independent analyses). TH expression in neurons grown in the presence of AVSM was significantly less than that in neurons grown in the absence of VSM (P < 0.05; 1-sample, 2-tailed Wilcoxon signed-rank test). RIA data for cell-associated NPY are shown in Fig. 1A, right (mean ± SE of 7 independent analyses). AVSM did not affect NPY. Corresponding data for TAVSM are shown in Fig. 1B. Like AVSM, TAVSM decreased TH expression (Fig. 1B, left; P < 0.05; 1-sample, 2-tailed Wilcoxon signed-rank test). However, TH expression in the presence of TAVSM (76 ± 10% of control) was greater than that observed in the presence of AVSM (39 ± 12% of control) (P < 0.05; 2-tailed Mann-Whitney test). TAVSM increased NPY (P < 0.05; 2-tailed Wilcoxon signed-rank test).


Figure 1
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 1. Effects of vascular smooth muscle cells (VSM) on tyrosine hydroxylase (TH) and neuropeptide Y (NPY) expression in postganglionic sympathetic neurons. A: effects of aortic VSM (AVSM) on TH [representative Western blot (n = 7; shown at left) with quantitative data shown in parentheses at bottom] and cell-associated NPY measured by radioimmunoassay (RIA) (quantitative data shown at right; n = 7). B: effects of tail artery VSM (TAVSM) on TH [representative Western blot (n = 10; shown at left) with quantitative data shown in parentheses at bottom] and cell-associated NPY measured by RIA (quantitative data shown at right; n = 10). C: effects of AVSM (open bars; n = 3–5) and TAVSM (solid bars; n = 4) on TH (measured by Western blot) and NPY (measured by RIA) in postganglionic sympathetic neurons grown in compartmentalized chambers. VSM were added to the axon/nerve terminal compartment. *NPY or TH in the neurons grown in the presence of VSM (+VSM) was significantly different from that in neurons grown in the absence of VSM (–VSM) (P < 0.05; 1-sample Wilcoxon signed-rank test).

 
At sympathetic neurovascular junctions, VSM interact with axons or nerve terminals of postganglionic sympathetic axons. The VSM do not interact with neuronal cell bodies, which are located in ganglia a considerable distance from the blood vessels. In the culture model used to obtain the data shown in Fig. 1, A and B, the VSM potentially interacted with cell bodies as well as axons and nerve terminals. To determine whether VSM interactions with sympathetic axons or terminals modulated TH and/or NPY, experiments were performed in compartmentalized cultures (6), in which VSM were plated in the axonal compartment of the cultures. The results of these experiments are shown in Fig. 1C. Data for TH and NPY in neurons grown in the presence of VSM are expressed as a percentage of corresponding control expression in neurons grown in the absence of VSM. These data show that when VSM interacted with sympathetic axons and nerve terminals but not cells bodies, AVSM decreased TH (P < 0.05; 1-sample, 1-tailed Wilcoxon signed-rank test) and had no effect on NPY, and TAVSM did not affect TH or NPY.

LIF, VSM, and sympathetic NPY and TH. Data in Fig. 1 indicate that VSM decrease TH. LIF is a peptide produced by some sympathetic targets that decreases TH and NPY expression (1, 27). The role of LIF in VSM modulation of TH and NPY was evaluated. Figure 2A shows representative (n = 5–6) Western analyses of LIF expression in cultured VSM and arteries. Corresponding GAPDH Western blots are also shown to demonstrate that approximately equal amounts of protein were present in each lane of the LIF Western blot. Quantitative analyses indicate that LIF expression in TAVSM was 33 ± 15% of that in AVSM (n = 6; P < 0.05; 2-tailed, 1-sample t-test) and that LIF expression in tail artery was 46 ± 21% of that in aorta (n = 5; P < 0.05; 1-tailed, 1-sample t-test). These data indicate that LIF is present in AVSM and TAVSM and in aortas and tail arteries and suggest that more LIF is present in sparsely innervated aortas.


Figure 2
View larger version (32K):
[in this window]
[in a new window]

  		Fig. 2. Leukemia inhibitory factor (LIF) modulates NPY and TH expression in sympathetic neurovascular cultures. A: representative Western analyses of LIF (n = 5–6) and LIF receptor (LIFR) expression in VSM and postganglionic sympathetic neurons (n = 3–4). For LIF expression, data from cultured VSM and intact arteries are shown. For LIFRs, data from dissociated neurons (d), neonatal sympathetic ganglia (n), and adult sympathetic ganglia (a) are shown. Representative GAPDH and TH Western blots are also presented to demonstrate that approximately equal amounts of VSM (GAPDH) or sympathetic neuronal protein (TH) were present. A, aorta; TA, tail artery. B: representative (n = 2) immunohistochemical analyses of LIFR expression in cultures of postganglionic sympathetic neurons. C: representative (n = 2) immunohistochemical analyses of gp130 expression in cultures of postganglionic sympathetic neurons. D: representative (n = 2) immunohistochemical analyses of LIFR expression in sympathetic nerve fibers on the adventitial surface of rat tail arteries. Corresponding GAP43 immunohistochemistry of nerve fibers is shown. E: TH and NPY measured in sympathetic neurovascular cultures grown in the presence of control antibodies (Ab) or antibodies that neutralized the activity of LIF. Data measured in aortic (n = 4; open bars) and tail artery (n = 4; solid bars) neurovascular cultures are shown. *NPY or TH in the neurons grown in the presence of VSM was significantly different from that in neurons grown in the absence of VSM (P < 0.05; 1-sample Wilcoxon signed-rank test).

 
The effects of LIF are mediated via two receptors; LIF binds to LIFRs. LIF/LIFR complexes then bind to gp130s, which activate intracellular signaling (1, 25). Western analyses (representative of 3–4 independent analyses) shown in Fig. 2A indicate that the dissociated neurons used in the present study express both LIFRs. These analyses also indicate that these receptors are expressed in the ganglia of neonatal and adult rats. Corresponding TH Western analyses are also shown to demonstrate that approximately equal amounts of sympathetic neuronal protein were present in each lane of the gp130 and LIFR Western blots. Quantitative analyses [LIFR/TH (gp130/TH) normalized to LIFR/TH (gp130/TH) in dissociated neurons] indicate that there were no reproducible differences in expression of LIFR or gp130 (P > 0.05; 1-sample t-test or unpaired t-test assuming unequal variances). Figure 2, B and C, shows immunocytochemical analyses of LIFR and gp130 expression in cultures of postganglionic sympathetic neurons. These analyses indicate that both LIFRs are present on cell bodies and axons of the dissociated neurons used in the present study. Figure 2D shows immunohistochemical analyses of gp130 and LIFR expression in nerve fibers located on the adventitial surface of rat tail arteries. GAP43 staining of the fibers is shown in red; control IgG, gp130 antibody (Ab), and LIFR Ab staining of the arteries are shown in green. Figure 2, A–D, clearly indicates that LIF is expressed by VSM and that LIFRs are expressed by postganglionic sympathetic neurons both in vitro and in vivo. Figure 2E shows the effects of inhibition of LIF on VSM modulation of TH and NPY. Data obtained with AVSM and data obtained with TAVSM are shown. Control data were obtained in the presence of 10 µg/ml nonimmune rabbit IgG; LIF Ab data were obtained in the presence of 10 µg/ml LIF Ab (23, 27). As noted earlier in Fig. 1, in the absence of LIF Ab, TH expression in the presence of AVSM was less than that in the absence of AVSM (P < 0.05; 1-tailed, 1-sample Wilcoxon signed-rank test; n = 4). When LIF is inhibited (LIF Ab), TH in the presence of AVSM (98.8 ± 36% of control) is not different from control (P > 0.05; 1-tailed, 1-sample Wilcoxon signed-rank test; n = 4). In this set of experiments, TAVSM did not affect TH in the absence or presence of LIF Ab. Inhibition of LIF did not affect AVSM modulation of NPY. In the absence of LIF Ab, TAVSM increased NPY (P < 0.05; 1-tailed, 1-sample Wilcoxon signed-rank test; n = 4). In the presence of LIF Ab, TAVSM did not significantly affect NPY.

NT-3, VSM, and sympathetic NPY and TH. VSM have been reported to produce NT-3 (10), and NT-3 has been reported to modulate TH (2) and NPY (21). Does VSM-derived NT-3 modulate NPY and/or TH? Figure 3A shows a representative (n = 3) Western analysis of NT-3 in AVSM and TAVSM as well as in corresponding neonatal and adult arteries. Prominent immunoreactive NT-3 bands were detected at ~33 and 20 kDa, corresponding to the molecular masses of the proform of NT-3 (12, 17) and a partially processed form of NT-3 (12), respectively. In all three experiments, NT-3 in the adult aorta was considerably less than that in VSM, neonatal arteries, and adult tail artery. NT-3 mediates its effects via binding to TrkC receptors (2, 10). Western analyses (Fig. 3A) indicate that the neurons used in the present study express TrkC receptors. TrkC was not detected by immunohistochemical analyses with two different antibodies (Santa Cruz and Upstate). This is likely due to high background staining associated with the nonimmune IgG controls (data not shown). The presence of NT-3 in the VSM and the presence of NT-3 receptors on the neurons suggest that VSM-derived NT-3 could contribute to the observed effects of VSM on NPY and/or TH (Fig. 1). To test this hypothesis, the effects of inhibiting NT-3 on VSM modulation of NPY and TH were studied (Fig. 3B). NT-3 was inhibited with a soluble chimeric form of TrkC (5 µg/ml; R & D Systems, TrkC-Fc) that decreases NT-3 binding to its receptors on the neurons. Control experiments were performed with Fc (5 µg/ml; R & D Systems). AVSM similarly inhibited TH in the presence of TrkC-Fc and Fc, indicating that inhibition of NT-3 did not affect AVSM modulation of TH. TAVSM did not inhibit TH in the presence of Fc but markedly inhibited TH in the presence of TrkC-Fc. AVSM did not affect NPY, either in the absence or presence of TrkC-Fc. TAVSM increased NPY in the presence of Fc (P < 0.05; 1-tailed, 1-sample Wilcoxon signed-rank test; n = 4) but decreased NPY in the presence of TrkC-Fc (P < 0.05; 2-tailed, 1-sample Wilcoxon signed-rank test; n = 5).


Figure 3
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 3. Neurotrophin-3 (NT-3) modulates NPY and TH expression in sympathetic neurovascular cultures. A: representative (n = 3) Western analysis of NT-3 in VSM and arteries from neonatal (n) and adult (a) rats is shown at top. Corresponding smooth muscle {alpha}-actin Western blots are shown to demonstrate that approximately equivalent amounts of vascular smooth muscle protein were present in each sample. Representative (n = 3) Western analysis of TrkC expression in dissociated sympathetic neurons (d) and in superior cervical ganglia from neonatal (n) and adult (a) rats is shown at bottom. Corresponding analysis of TH is shown to demonstrate that approximately equal amounts of postganglionic sympathetic neuronal protein were present in each sample. B: TH and NPY measured in sympathetic neurovascular cultures grown in the presence of soluble control Fc or TrkC-Fc. The TrkC-Fc should neutralize the activity of NT-3. Data measured in aortic (n = 4; open bars) and tail artery (n = 5; solid bars) neurovascular cultures are shown. *NPY or TH in the neurons grown in the presence of VSM was significantly different from that in neurons grown in the absence of VSM (P < 0.05; 1-tailed, 1-sample Wilcoxon signed-rank test; n = 4–5). {blacklozenge}TH and NPY expression in the presence of TrkC-Fc was significantly different from that in the presence of Fc (P < 0.05; 2-tailed Mann-Whitney test; n = 4–5).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present studies provide evidence that VSM affect TH and NPY at sympathetic neurovascular junctions. Using in vitro sympathetic neurovascular culture models, we demonstrated that VSM derived from two different arteries differentially modulated the expression of TH and NPY. These studies thus suggest novel sympathetic neurovascular interactions that are likely to affect sympathetic control of blood pressure.

We found that aortic VSM decreased TH. This effect was observed in standard and compartmentalized cultures, suggesting that the effect is likely to occur at sympathetic neurovascular junctions in the rat aorta. These data suggest that VSM would decrease the production and release of norepinephrine from sympathetic neurons innervating the aorta. The aorta is sparsely innervated by the sympathetic nervous system (31). It is likely that the sympathetic nervous system and norepinephrine release from postganglionic sympathetic neurons are not important determinants of the function of this vessel.

LIF is a member of the IL-6 family of cytokines that modulates cardiovascular, endocrine, immune, and neural development and function (1, 14, 23, 25, 27, 28, 33, 35). It is well known that LIF decreases TH in postganglionic sympathetic neurons (27). The present studies demonstrate that LIF is produced by VSM, that LIFRs are expressed by postganglionic sympathetic neurons (Fig. 2, A–D), and that LIF from aortic VSM decreased TH in sympathetic neurovascular cultures (Fig. 2E). The present studies also demonstrate that LIFRs are present on the sympathetic neurons used in the present studies and on sympathetic nerve fibers innervating blood vessels (Fig. 2D). This suggests that VSM-derived LIF is a determinant of norepinephrine content of vascular sympathetic innervation and thus a determinant of sympathetic control of blood pressure and blood flow. This is the first report that LIF is produced by VSM. It is likely that VSM-derived LIF has additional important functions.

LIF is also known to decrease NPY in postganglionic sympathetic neurons (27). Aortic VSM did not decrease NPY (Fig. 1A). Furthermore, inhibition of LIF in aortic neurovascular cocultures did not alter NPY (Fig. 2E). This suggests that, although VSM-derived LIF determines TH in the neurovascular cultures used in the present study, it is not a primary determinant of NPY. It is not clear why this is the case. It is known that NPY and TH may be regulated independently (22). It is likely that VSM-derived factors other than LIF are affecting NPY, but not TH. The identity of these possible factors has yet to be determined.

We found that the effects of tail artery VSM on TH expression in sympathetic neurovascular cultures differed from that of aortic VSM. In standard cultures, tail artery VSM decreased TH slightly, but this effect was less than that of aortic VSM. In compartmentalized cultures, which are more representative of sympathetic neurovascular junctions, tail artery VSM did not affect TH. Differential expression of LIF could account for this differential modulation of TH. Tail artery VSM in culture and tail arteries expressed considerably less LIF than aortic VSM and aorta (Fig. 2A).

NT-3 has been detected in blood vessels (10) and has been reported to modulate TH and NPY expression in postganglionic sympathetic neurons (2, 4). The present studies suggest that VSM-derived NT-3 maintains TH and NPY expression in the postganglionic sympathetic neurons that innervate them. Inhibition of the activity of NT-3 decreased NPY and TH in neurons grown in the presence of tail artery VSM (Fig. 3B). Several lines of evidence support the present observation that vascular-derived NT-3 modulates the function of the postganglionic sympathetic neurons innervating blood vessels. NT-3 affects survival (32, 37), axon growth (2), and neuropeptide (26) and neurotransmitter (2) expression of these neurons. NT-3 is expressed in blood vessels (10), and studies by Zhou et al. (38) indicate that vascular-derived NT-3 is retrogradely transported by postganglionic sympathetic neurons innervating blood vessels. An important role for vascular-derived NT-3 is suggested by the studies of Zhang and Rush (36). These investigators reported that NT-3 is increased in blood vessels of spontaneously hypertensive rats. This increase in vascular-derived NT-3 could increase TH and NPY in vascular sympathetic nerves and thereby contribute to the development of hypertension in these animals.

The present data suggest that NT-3 increases or maintains TH. This is consistent with the observations of Belliveau et al. (2), who found that NT-3 increased TH mRNA expression in dissociated postganglionic sympathetic neurons. The present observations and those of Belliveau et al. differ from those reported by Brodski et al. (4), who found that NT-3 decreased TH mRNA. There are several differences in the studies that could account for the divergent results. The studies of Brodski et al. were performed on explants of embryonic chicken sympathetic ganglia. The present studies and those of Belliveau et al. were performed on dissociated ganglia from postnatal rats. Thus there are differences in species, age, and preparation. In support of an age-dependent difference in NT-3 effect, the studies of Brodski et al. indicate that NT-3 decreased TH in embryonic day 12 sympathetic explants but not in embryonic day 7.5 explants.

NPY and TH in vascular sympathetic nerves are likely to determine NPY and norepinephrine release and, as such, are likely to affect blood flow and blood pressure. The present studies indicate that VSM likely determine sympathetic NPY and TH and thus suggest a novel mechanism whereby VSM would modulate sympathetic control of vascular function. The data indicate that VSM produce both stimulators (NT-3) and inhibitors (LIF) of NPY and TH and suggest that the net effect will be determined by the balance between stimulation and inhibition. The data also indicate that vascular production of these factors is heterogeneous, and that the net effect of VSM on NPY and TH is heterogenous.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-68009 and HL-76774.


   ACKNOWLEDGMENTS
 
I would like to acknowledge the expert technical assistance of Joshua M. Halman, Steve Marko, Rachel Poole, and Jenn Wlodarski. I would also like to thank Marilyn Cipolla, Tony Morielli, and David Hill-Eubanks for invaluable discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. H. Damon, Dept. of Pharmacology, Univ. of Vermont, 89 Beaumont Ave., Given Bldg., Burlington, VT 05405 (e-mail: Deborah.Damon{at}uvm.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
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Auerhammer CJ, Melmed S. Leukemia-inhibitory factor–neuroimmune modulator of endocrine function. Endocr Rev 21: 313–345, 2000.[Abstract/Free Full Text]

2. Belliveau DJ, Krivko I, Kohn J, LaChance C, Pozniak C, Rusakov D, Kaplan D, Miller FD. NGF and neurotrophin-3 both activate trk A on sympathetic neurons but differentially regulate survival and neuritogenesis. J Cell Biol 136: 375–388, 1997.[Abstract/Free Full Text]

3. Bradley E, Law A, Bell D, Johnson CD. Effects of varying impulse number on cotransmitter contributions to sympathetic vasoconstriction in rat tail artery. Am J Physiol Heart Circ Physiol 284: H2007–H2014, 2003.[Abstract/Free Full Text]

4. Brodski C, Schnurch H, Dechant G. Neurotrophin-3 promotes the cholinergic differentiation of sympathetic neurons. Proc Natl Acad Sci USA 97: 9683–9688, 2000.[Abstract/Free Full Text]

5. Browning KN, Zheng Z, Kreulen DL, Travagli RA. Two populations of sympathetic neurons project selectively to mesenteric artery or vein. Am J Physiol Heart Circ Physiol 276: H1263–H1272, 1999.[Abstract/Free Full Text]

6. Campenot RB. Development of sympathetic neurons in compartmentalized cultures. II. Local control of neurite growth by nerve growth factor. Dev Biol 93: 1–12, 1982.[CrossRef][Web of Science][Medline]

7. Creedon DJ, Tuttle JB. Synergistic increase in nerve growth factor secretion by cultured vascular smooth muscle cells treated with injury-related growth factors. J Neurosci Res 47: 277–286, 1997.[CrossRef][Web of Science][Medline]

8. Damon DH. Sympathetic innervation promotes vascular smooth muscle differentiation. Am J Physiol Heart Circ Physiol 288: H2785–H2791, 2005.[Abstract/Free Full Text]

9. Donoso MV, Steiner M, Huidobro-Toro JP. BIBP 3226, suramin and prazosin identify neuropeptide Y, adenosine 5'-triphosphate and noradrenaline as sympathetic cotransmitters in the rat arterial mesenteric bed. J Pharmacol Exp Ther 282: 691–698, 1997.[Abstract/Free Full Text]

10. Donovan MJ, Miranda RC, Kraemer r McCaffrey TA, Tessarollo L, Mahadeo D, Sharif S, Kaplan DR, Tsoulfas P, Parada L, et al. Neurotrophin and neurotrophin receptors in vascular smooth muscle cells. Regulation of expression in response to injury. Am J Pathol 147: 309–324, 1995.[Abstract]

11. Erlinge D, Ekman R, Thulin T, Edvinsson L. Neuropeptide Y-like immunoreactivity and hypertension. J Hypertens 10: 1221–1225, 1992.[CrossRef][Web of Science][Medline]

12. Farhadi HF, Mowla SJ, Petrecca K, Morris SJ, Seidah NG, Murphy RA. Neurotrophin-3 sorts to the constitutive secretory pathway of hippocampal neurons and is diverted to the regulated secretory pathway by coexpression with brain-derived neurotrophic factor. J Neurosci 20: 4059–4068, 2000.[Abstract/Free Full Text]

13. Guyenet PG. The sympathetic control of blood pressure. Nat Rev Neurosci 7: 335–346, 2006.[Web of Science][Medline]

14. Hamanaka I, Saito Y, Nishikimi T, Magaribuchi T, Kamitani S, Kuwahara K, Ishikawa M, Miyamoto Y, Harada M, Ogawa E, Kajiyama N, Takahashi N, Izumi T, Shirakami G, Mori K, Inobe Y, Kishimoto I, Masuda I, Fukuda K, Nakao K. Effects of cardiotrophin-1 on hemodynamics and endocrine function of the heart. Am J Physiol Heart Circ Physiol 279: H388–H396, 2000.[Abstract/Free Full Text]

15. Han S, Yang CL, Chen X, Naes L, Cox BF, Westfall T. Direct evidence for the role of neuropeptide Y in sympathetic nerve stimulation-induced vasoconstriction. Am J Physiol Heart Circ Physiol 274: H290–H294, 1998.[Abstract/Free Full Text]

16. Karvonen MK, Valonen VP, Lakka TA, Salonen R, Koulu M, Pesonen U, Tuomainen TP, Kauhanen J, Nyyssonen K, Lakka HM, Uusitupa MI, Salonen JT. Leucine7 to proline7 polymorphism in the preproneuropeptide Y is associated with the progression of carotid atherosclerosis, blood pressure and serum lipids in Finnish men. Atherosclerosis 159: 145–151, 2001.[CrossRef][Medline]

17. Lee R, Kermani P, Teng KK, Hempstead BL. Regulation of cell survival by secreted proneurotrophins. Science 294: 1945–1948, 2001.[Abstract/Free Full Text]

18. Li L, Lee EW, Ji H, Zukowska Z. Neuropeptide Y-induced acceleration of postangioplasty occlusion of rat carotid artery. Arterioscler Thromb Vasc Biol 23: 1204–1210, 2003.[Abstract/Free Full Text]

19. Lundberg JM, Terenius L, Hokfelt T, Goldstein M. High levels of neuropeptide Y in peripheral noradrenergic neurons in various mammals including man. Neurosci Lett 42: 167–142, 1983.[CrossRef][Web of Science][Medline]

20. MacPhedran SE, Hall AK. Influence of non-neuronal cells on establishment of neuropeptide Y expression by sympathetic neurons in vitro. Dev Neurosci 23: 307–317, 2001.[CrossRef][Web of Science][Medline]

21. Matsumoto SG, Gruener RP, Kreulen DL. Neurotransmitter properties of guinea-pig sympathetic neurons grown in dissociated cell culture. II. Fetal and embryonic neurons: regulation of neuropeptide Y expression. Neuroscience 57: 1147–1157, 1993.[CrossRef][Web of Science][Medline]

22. May V, Brandenburg CA, Braas KM. Differential regulation of sympathetic neuron neuropeptide Y and catecholamine content and secretion. J Neurosci 15: 4580–4591, 1995.[Abstract]

23. Mi H, Haeberle H, Barres BA. Induction of astrocyte differentiation by endothelial cells. J Neurosci 21: 1538–1547, 2001.[Abstract/Free Full Text]

24. Morris JL, Zhu BS, Givvins IL, Blessing WW. Subpopulations of sympathetic neurons project to specific vascular targets in the pinna of the rabbit ear. J Comp Neurol 412: 147–160, 1999.[CrossRef][Web of Science][Medline]

25. Ng YP, He W, Ip NY. Leukemia inhibitory factor receptor signaling negatively modulates nerve growth factor-induced neurite outgrowth in PC12 cells and sympathetic neurons. J Biol Chem 278: 38731–38739, 2003.[Abstract/Free Full Text]

26. Ramirez-Ordonez R, Barreto-Estrada JL, Garcia-Arraras JE. Growth factors effects on the expression of morphological and biochemical properties of avian embryonic sympathetic cells. Emphasis on NGF. Dev Brain Res 114: 27–36, 1999.[CrossRef][Medline]

27. Rao MS, Sun Y, Escary JL, Perreau J, Tresser S, Patterson PH, Zigmond RE, Brulet P, Landis SC. Leukemia inhibitory factor mediates an injury response but not a target-directed developmental transmitter switch in sympathetic neurons. Neuron 11: 1175–1185, 1993.[CrossRef][Web of Science][Medline]

28. Rolfe BE, Stamatiou S, World CJ, Brown L, Thomas AC, Bingley JA, Worth NF, Campbell JH. Leukaemia inhibitory factor retards the progression of atherosclerosis. Cardiovasc Res 58: 222–230, 2003.[Abstract/Free Full Text]

29. Ross R. The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers. J Cell Biol 50: 172–186, 1971.[Abstract/Free Full Text]

30. Sauzeau V, Sevilla MA, Rivas-Elena JV, de Álava E, Montero MJ, López-Novoa JM, Bustelo XR. Vav3 protooncogene deficiency leads to sympathetic hyperactivity and cardiovascular dysfunction. Nat Med 12: 841–845, 2006.[CrossRef][Web of Science][Medline]

31. Stassen FR, Maas RG, Schiffers PM, Janssen GM, De Mey JG. A positive and reversible relationship between adrenergic nerves and alpha-1A adrenoceptors in rat arteries. J Pharmacol Ther 284: 399–405, 1998.[Abstract/Free Full Text]

32. Tafreshi AP, Zhou XF, Rush RA. Endogenous nerve growth factor and neurotrophin-3 act simultaneously to ensure the survival of postnatal sympathetic neurons in vivo. Neuroscience 83: 373–380, 1999.[Web of Science]

33. Takahashi E, Fukuda K, Miyoshi S, Murata M, Kato T, Ita M, Tanabe T, Ogawa S. Leukemia inhibitory factor activates cardiac L-type Ca2+ channels via phosphorylation of serine 1829 in the rabbit cav1.2 subunit. Circ Res 94: 1242–1248, 2004.[Abstract/Free Full Text]

34. Topouzis S, Majesky MW. Smooth muscle lineage diversity in the chick embryo. Dev Biol 15: 430–435, 1996.

35. Ure DR, Campenot RB, Acheson A. Cholinergic differentiation of rat sympathetic neurons in culture: effects of factors applied to distal neurites. Dev Biol 154: 388–395, 1992.[CrossRef][Web of Science][Medline]

36. Zhang SH, Rush RA. Neurotrophin 3 is increased in the spontaneously hypertensive rat. J Hypertens 19: 2251–2256, 2001.[CrossRef][Web of Science][Medline]

37. Zhou XF, Rush R. Sympathetic neurons in neonatal rats require endogenous neurotrophin-3 for survival. J Neurosci 15: 6521–6530, 1995.[Abstract/Free Full Text]

38. Zhou XF, Chie ET, Deng YS, Rush RA. Rat mature sympathetic neurones derive neurotrophin 3 from peripheral effector tissues. Eur J Neurosci 9: 2753–2764, 1997.[CrossRef][Web of Science][Medline]

39. Zukowska-Grojec Z, Pruszczyk P, Colton C, Yao J, Shen GH, Myers AK, Wahlestedt C. Mitogenic effect of neuropeptide Y in rat vascular smooth muscle cells. Peptides 14: 263–268, 1993.[CrossRef][Web of Science][Medline]

40. Zukowska-Grojec Z, Karwatowska-Prokopczuk E, Rose W, Rone J, Movafagh S, Ji H, Yeh Y, Chen WT, Kleinman HK, Grouzmann E, Grant DS. Neuropeptide Y: A novel angiogenic factor from the sympathetic nerves and endothelium. Circ Res 83: 187–195, 1998.[Abstract/Free Full Text]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/1/C306    most recent
00214.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Damon, D. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Damon, D. H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.