(Hypertension. 2001;37:677.)
© 2001 American Heart Association, Inc.
Scientific Contributions |
Angiotensin II Increases Vesicular Trafficking in Brain Neurons
From the Department of Physiology, College of Medicine, and the University of Florida McKnight Brain Institute, Gainesville, Fla.
Correspondence to Mohan K. Raizada, PhD, Department of Physiology, University of Florida College of Medicine, PO Box 100274, Gainesville, FL 32610. E-mail mraizada{at}phys.med.ufl.edu
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Abstract |
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Our previous studies have established that angiotensin (Ang) II stimulates the release, uptake, and synthesis of norepinephrine (NE) in brain neurons involving distinct signal transduction pathways. However, little is known if this NE neuromodulatory effect is a result of Ang II activation of vesicular trafficking in the catecholaminergic neurons. Thus, the aim of this study was to determine if Ang II influences movement of vesicles in live neurons. Dopamine–ß-hydroxylase (DßH) fused to green fluorescence protein (GFP) has been used to trace vesicular movement in live neurons by confocal microscopy. Transfection by a plasmid containing GFP-DßH resulted in the expression of green fluorescence, representing
100 kDa GFP-DßH
fusion protein. The fluorescence was predominantly localized in
the perinuclear region of the neuronal soma, with a few neurites also
depicting the fluorescence. Ang II caused a dramatic
redistribution of fluorescence. The fluorescence was
translocated to the neurites in a time-dependent manner. As a result,
the number of neurites depicting fluorescence was significantly
increased. The translocation was blocked by losartan, an Ang II
type 1 receptor subtype–specific antagonist and not by
PD123319, an Ang II type 2 receptor subtype antagonist.
High-magnification confocal microscopic examination revealed that Ang
II treatment resulted in a distal movement of certain
fluorescent clusters in the neurites at an average rate of
0.84±0.2 µm/s. These observations suggest increased vesicular
trafficking is a key signaling event in Ang II stimulation of NE
neuromodulation.
Key Words: dopamine • norepinephrine • neuroregulators • hypothalamus • brain • receptors, angiotensin
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Introduction |
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The brain renin-angiotensin system (RAS) exerts profound physiological effects leading to the central control of blood pressure. They include activation of sympathetic pathways, dampening of baroreceptor reflexes, stimulation of vasopressin release, and regulation of dipsogenic responses.1 2 3 4 Despite our understanding of the physiological circuitry, the cellular and molecular mechanisms of the central control of blood pressure by the RAS remains poorly understood. Our research group had developed an in vitro neuronal cell culture system from the hypothalamus–brain stem areas of rat brain as a model in an attempt to investigate these mechanisms.2 4 These studies have established that sympathetic activation by angiotensin (Ang) II is associated with the stimulation of turnover, synthesis, and release of norepinephrine (NE) at a cellular level.5 6 7 Thus, these cultures have provided an excellent in vitro model system to investigate the signal transduction mechanism of Ang II regulation of NE neuromodulation. Our previous studies have led us to conclude that Ang II, through its activation of Ang II type 1 receptor (AT1 receptor), causes both evoked (acute) and enhanced (chronic) stimulation of NE neuromodulation.2 3 4 5 7 Evoked responses of Ang II involve inhibition of K+ channels and stimulation of Ca2+ channels through activation of protein kinase C
and
Ca2+-calmodulin–dependent
protein kinase
II.2 3 4
In contrast, enhanced stimulation of NE neuromodulation involves
activation of the Ras–Raf–MAP
kinase signaling
pathway.5 7 An important question that arises from these studies concerns the mechanism by which AT1 receptor stimulation leads to the release of NE. We have hypothesized that Ang II stimulates transport of vesicles to increase release of NE and thus stimulate neuromodulation in the catecholaminergic neuron. The present study was undertaken to provide evidence in support or against this hypothesis. Dopamine–ß-hydroxylase (DßH), a marker for vesicles in the neurons, was fused with the green fluorescent protein (GFP) to visualize the effects of Ang II on vesicular movements in live neurons.
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Methods |
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Materials
One-day-old Wistar-Kyoto rats (WKY) were obtained from our breeding colony, which originated from Harlan Sprague-Dawley. DMEM, plasma-derived horse serum, and 1x crystalline trypsin (10 000 BAEE/mg) were obtained from Central Biomedia. Ang II was purchased from Sigma Co. Agarose-conjugated protein A/G was from Santa Cruz Biotechnology, Inc. Rabbit polyclonal antibody to GFP was from Clontech. The DßH activity assay kit was from ALPCO. Lostartan was a gift from Dupont/Merck, and PD123319 was from RBI. All other reagents were purchased from Fisher Scientific.
Preparation of Neuronal Cultures
Neuronal cells in primary culture were prepared
essentially as described
previously.8 9
Briefly, hypothalamus–brain stem areas of 1-day-old WKY brain were
dissected, and brain cells were dissociated by trypsin. Cells were
plated onto
poly-L-lysine–precoated
tissue culture dishes (3x106 cells/35-mm
diameter; 2x107 cells/100
mm-diameter-dishes) in DMEM containing 10% plasma-derived horse serum,
essentially as described
elsewhere.8 9 In
addition, 1x105 cells were plated in
chambered cover glass for confocal analysis. Cultures were
allowed to establish for 7 to 10 days before the experiment. These
cultures contain about 85% to 90% neuronal cells and 10% to 15%
astrocytic glial cells and have been used extensively by us as an in
vitro model system to investigate Ang II regulation of NE
neuromodulation.2 4
Plasmids
The cDNA encoding the full-length coding region of
rat DßH with GFP were cloned in mammalian expression vector pCI-Neo
(Promega) under the control of the cytomegalovirus promoter. The entire
humanized GFP sequence, including a unique 5'
XbaI site followed by a Kozak
sequence and translation start site, was directionally cloned into
NheI- and
EcoRI-cleaved modified pCI-Neo
vector called pCI-Neo-GFP, as described
previously.10 This vector
allows expression of polymerase chain reaction products with
appropriate 5' EcoRI and 3'
SalI site as fusion protein
containing an N-terminal GFP.
The full length–coding region of rat DßH with intrinsic signal
sequence was obtained by polymerase chain reaction from rat adrenal
cDNA library, and 3' SalI site
was introduced in frame. The DßH cDNA was inserted into TA clone
vector and then subcloned into the pCI-Neo-GFP and produced the
pCI-GFP-DßH plasmid. The construct was verified by restriction
analysis and DNA sequencing.
Transfection of Neurons With pCI-GFP-DßH
Plasmid
Hypothalamus–brain stem neurons from WKY brain were
transfected by pCI-GFP-DßH plasmid, essentially as described
previously.11 Briefly, the
DNA/calcium phosphate precipitate was prepared by mixing 1 volume of
DNA in 25 mmol/L CaCl2 with an equal volume
of 2x HBS (274 mmol/L NaCl, 10 mmol/L KCl, 1.4 mmol/L
Na2HPO4, 15 mmol/L
D-glucose, 42 mmol/L
HEPES, pH 7.07). The precipitate was allowed to form for 30 minutes at
room temperature before addition to the cultures. The conditioned
culture medium was removed and saved. The cells were incubated with 2
mL fresh DMEM for 1 hour. Six micrograms of total plasmid DNA was used
for each 35-mm-diameter plate or chambered covered glass. One hundred
twenty microliters of DNA/calcium phosphate precipitate was added
dropwise to each dish and mixed gently. Dishes were then returned to a
37°C incubator set at 10% CO2/90% air
(vol/vol) for 8 hours. The incubation was stopped by "shocking" the
cells for 1 to 2 minutes with 1x HBS/10 mmol/L
MgCl2 in 4 mmol/L HEPES, pH 7.5/5%
glycerol. The saved conditioned medium was added back to each plate,
and the cells were returned to the incubator for an addition 2 days.
The transfection efficiency for neurons was 2%.
TE671 cells that were grown in 100-mm dishes were transfected with the GFP-DßH plasmid as follows: Cultures were transfected at 80% confluence with 250 mL of DNA/calcium phosphate precipitate containing 20 µg plasmid DNA per dish; cells were washed 2 times with DMEM 30 minutes after transfection; regular medium was added to the dish; and the cells were returned to incubator at 37°C for 2 days. Transfections of both neurons and TE671 cells with GFP plasmid were used as control.
DßH Immunoreactivity Measurement
Cells, grown in 100mm dishes, were transfected by
pCI-GFP-DßH plasmid (experimental) or pCI-GFP plasmid (control), as
described above. The cells were collected and incubated with lysis
buffer (50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 10%
glycerol, 1% NP-40, 0.5% sodium deoxycholate, 2 mmol/L EDTA,
1 mmol/L PMSF, 10 mg/mL aprotinin). After 20 minutes, lysates were
centrifuged for 10 minutes at
14 000g at 4°C, and
postnuclear supernatants containing 400 µg protein were
immunoprecipitated with anti-GFP polyclonal antibody for 1 hour. Immune
complexes were precipitated with protein A/G PLUS agarose and washed 3
times with lysis buffer, and protein was dissolved in the sample
buffer.5 7 Lysates
were subjected to SDS/PAGE, essentially as described
previously.7 Proteins were
transferred to nitrocellulose membrane, and nonspecific binding was
blocked by 5% nonfat dry milk in TBST (20 mmol/L Tris-HCl, pH
8.0, 150 mmol/L NaCl, and 0.05% Tween 20) for 1 hour. This was
followed by incubation with anti-DßH antibody at 1:1000 dilution for
1 hour at room temperature. Specific GFP-DßH fusion protein was
detected by chemiluminescence assay reagent after incubation with
anti-rabbit IgG-conjugated with horseradish peroxidase secondary
antibody and was visualized by
autoradiography.7
DßH Activity Assay
After 48 hours of transfection with control or
GFP-DßH plasmid, cells were collected and homogenized in
50 mmol/L acetate buffer, pH 6.0, containing 1% Triton-100,
quick-frozen, and thawed. The resulting homogenate was
centrifuged at 20 000g
for 10 minutes, and supernatants were used for the DßH
assay.12
Confocal Microscopic Analysis of
GFP-DßH Fluorescent Images of Neuronal Cells
Neuronal cultures were grown on chambered cover glass
(Nalge Nunc International). After transfection, GFP-DßH expression
was detected and photographed with a Bio-Rad 1024ES confocal scanning
laser microscope with a Fluotar 100x1.3 objective. The optical
sections were scanned with the 488-nm laser line at 10% intensity.
Sequential images were taken at indicated time periods. Two or three
1.0-mm z-sections were obtained
at each time point to ensure that GFP-DßH puncta were not eclipsed by
limited focal depth. To track the position of vesicles, data were
collected at a resolution of 1024x1024 pixels, and the time required
between each image was 10 minutes unless specified otherwise. Because
it was not possible to visualize individual vesicles in live neurons,
vesicular trafficking was tracked by following fluorescent
clusters that presumably represent clusters of vesicles. Thus,
the speed of fluorescent cluster movement represents
vesicular trafficking, determined by measuring the distance moved
between two sequential images. Images were captured and explored as TIF
files and analyzed with the Scion Image
Analyzer.
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Results |
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Expression of GFP-DßH Fusion Protein
TE671 cells were initially used to characterize the expression of GFP-DßH fusion protein and to determine if the resulting fusion protein expresses DßH activity. The principle rationale for selecting this cell line was our observation that these cells are relatively easy to transfect and that there is little or no endogenous DßH. Within 48 hours after transfection, 70% to 80% of these cells began to express bright green fluorescence. GFP-DßH plasmid–transfected cells showed a 100-kDa protein band that was recognized by anti-DßH antibody (Figure 1A). This band was absent in the GFP-transfected control cells. The 100-kDa band appears to be GFP-DßH fusion protein as expected, because GFP runs at
30 kDa and DßH at 70 kDa on
SDS-PAGE.
Figure 1B shows DßH activity in GFP-transfected and
GFP-DßH–transfected cells. The GFP-DßH–transfected cells, which
express the fusion protein, showed a DßH activity 10-fold greater
than those expressed in GFP-transfected cells (0.28±0.012 versus
0.03±0.013 NE/min). It appears that there is a disproportionate
increase in the DßH activity compared with the DßH protein levels.
This could be caused by the high enzyme activity/unit DßH protein. In
addition, differential sensitivity in the enzyme and Western assays may
explain this discrepancy. These data establish that pCI-GFP-DßH
vector expresses a fusion protein (GFP-DßH) that contains DßH
activity.
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Effects of Ang II on Neuronal DßH
Distribution
Having established that the GFP-DßH vector expresses
active GFP-DßH fusion protein, our first aim was to determine the
effects of Ang II on cellular localization of green
fluorescence that represents GFP-DßH in the neurons.
Neuronal cultures transfected with pCI-GFP-DßH were incubated with
100 nmol/L Ang II, and individual live neurons were examined by
confocal microscopy.
Figure 2 shows a confocal scan through a single live neuron.
Bright green fluorescence was primarily localized in the
perinuclear region of the neuronal cell soma. In addition, a few
neurites (generally 1 to 2) also exhibited green fluorescence
at zero time. Treatment with Ang II caused a dramatic redistribution of
fluorescence. A time-dependent increase of green
fluorescence was observed in the neurites. As a result, both
the intensity of fluorescence and the number of neurites
depicting fluorescence were increased with Ang II in a
time-dependent fashion. Quantification of fluorescence in the
neurites revealed a 2-fold increase by 100 nmol/L Ang II in 30 minutes
(Figure 2B). The fluorescence appears to exhibit
vesicular localization: (1) DßH itself is a vesicular protein, (2)
distribution of fluorescence was punctate, and (3) DßH
fluorescence was colocalized with synaptophysin (data not
shown). This observation was our first indication that Ang II may cause
vesicular trafficking from the cell body to the neurites.
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4 neurons. Results were normalized with value at 0 minutes as 1.Trafficking of vesicles containing GFP-DßH was examined next by examining neurites with resolution of 1024x1024 pixels at a normal scan speed by a laser scan unit, as described elsewhere.13 Figure 3 is a representative image after examination of 20 live neurons. An area of the neurite, outlined in Figure 3A, was examined. The choice of this area was primarily based on the presence of relatively low number of clusters of fluorescence, which made the examination of movement easier. Highly fluorescent-labeled neurites were excluded from examination because it was not possible to isolate individual clusters of fluorescence. Examination of the neurite for 10 minutes before addition of Ang II was used as movement of fluorescent vesicles independent of Ang II and was recorded at zero time (Figure 3B). A cluster of nonmobile fluorescence (represented by a vertical dotted line) was used as a guide to track the movement of 5 clusters. Treatment with Ang II caused some clusters (Nos. 1, 2, and 3) to move relatively faster than some other clusters (No. 4). On average, vesicular movement was 0.84±0.2 µm/s in the presence of Ang II. This is an average rate derived from the images taken from 20 neurites in 4 separate experiments. Vesicular clusters that did not show movement in the absence of Ang II for 10 minutes before treatment were considered in the calculation of this rate.
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Finally, we examined Ang II receptor subtype specificity in vesicular trafficking (depicted in Figure 4). Cotreatment of neurons with 100 nmol/L Ang II and 1 µmol/L losartan caused almost complete attenuation of neurite distribution of fluorescence. PD1233319, an AT2-receptor subtype–specific antagonist, did not block this translocation.
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Discussion |
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The significance of this study is that it demonstrates the trafficking of DßH-containing vesicles in live neurons. In addition, it establishes that stimulation of vesicular trafficking is part of the mechanism by which AT1 receptors regulate NE neuromodulation.
Our previous studies have established that both evoked and enhanced neuromodulatory responses of Ang II are associated with NE release.2 3 4 5 6 7 The signal transduction mechanisms that initiate these two responses are distinct: Evoked responses involve K+-Ca2+ channels and CAM kinase (CAMK) II, whereas enhanced responses involve Ras–Raf–MAP kinase–mediated transcriptional and posttranscriptional regulation.2 3 4 5 6 7 Despite distinct signaling systems, it appears that both responses converge in the final stage, resulting in the stimulation of transport of catecholamine containing vesicles. It is tempting to speculate that vesicles are bound to cytoskeletal elements and that dephosphorylated synapsin I keeps them in a state of reserve. Stimulation by Ang II activates a signaling cascade that phosphorylates synapsin I, thus releasing the vesicles from reserve to active state. This action allows exocytosis to occur, thus releasing NE. Further experiments will be needed to support or refute this hypothesis. What signaling kinases are involved in this activation? Are they similar to those previously identified (Ca2+–CAMK II, Ras–Raf–MAP kinase) or distinct? It appears that CAMK II may be ideally poised in this mechanism because Ang II stimulates calmodulin and CAMK II in these neurons,14 and this pathway is known to stimulate phosphorylation of synapsin I.15 16 Finally, inhibition of calmodulin by 100 µmol/L W-7 attenuates Ang II–induced translocation of GFP-DßH vesicles to neurites. In conclusion, the study provides strong evidence that Ang II stimulates vesicular trafficking in brain neurons.
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Acknowledgments |
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This research was supported by National Institutes of Health grant HL- 33610. Dr Xianyu Wang was a visiting fellow from Fuzhou, Medical University, Division of Cardiology and Hypertension, Fuzhou, Peoples Republic of China.
Received October 26, 2000; first decision November 30, 2000; accepted December 11, 2000.
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