help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
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 PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gallinat, S.
Right arrow Articles by Sumners, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gallinat, S.
Right arrow Articles by Sumners, C.
Endocrinology Vol. 142, No. 3 1009-1016
Copyright © 2001 by The Endocrine Society

ARTICLES

Gene Expression Profiling of Rat Brain Neurons Reveals Angiotensin II-Induced Regulation of Calmodulin and Synapsin I: Possible Role in Neuromodulation1

Stefan Gallinat2, Silke Busche2, Hong Yang, Mohan K. Raizada and Colin Sumners

Department of Physiology, College of Medicine, and McKnight Brain Institute, University of Florida, Gainesville, Florida 32610

Address all correspondence and requests for reprints to: Colin Sumners, Ph.D., Department of Physiology, University of Florida, P.O. Box 100274, 1600 SW Archer Road, Gainesville, Florida 32610. E-mail: csumners{at}phys.med.ufl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiotensin (Ang II) activates neuronal AT1 receptors located in the hypothalamus and the brainstem and stimulates noradrenergic neurons that are involved in the control of blood pressure and fluid intake. In this study we used complementary DNA microarrays for high throughput gene expression profiling to reveal unique genes that are linked to the neuromodulatory actions of Ang II in neuronal cultures from newborn rat hypothalamus and brainstem. Of several genes that were regulated, we focused on calmodulin and synapsin I. Ang II (100 nM; 1–24 h) elicited respective increases and decreases in the levels of calmodulin and synapsin I messenger RNAs, effects mediated by AT1 receptors. This was associated with similar changes in calmodulin and synapsin protein expression. The actions of Ang II on calmodulin expression involve an intracellular pathway that includes activation of phospholipase C, increased intracellular calcium, and stimulation of protein kinase C. Taken together with studies that link calmodulin and synapsin I to axonal transport and exocytotic processes, the data suggest that Ang II regulates these two proteins via a Ca2+-dependent pathway, and that this may contribute to longer term or slower neuromodulatory actions of this peptide.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE OCTAPEPTIDE angiotensin II (Ang II) elicits important receptor-mediated effects via the central nervous system (1, 2, 3, 4). Ang II type 1 (AT1) receptors are located on noradrenergic neurons in the brainstem and hypothalamus (5, 6), and Ang II acts at these sites to elicit both short-term (rapid) and longer-term (slow) modulation of norepinephrine (NE) neurotransmission. These actions of Ang II contribute to physiological changes such as increased blood pressure, altered baroreflex modulation, and increased secretion of vasopressin (7, 8, 9). Neurons in primary culture from newborn rat brainstem and hypothalamus have begun to provide an understanding of the mechanisms through which Ang II influences NE transmission in the central nervous system. Incubation of these cultures with Ang II elicits an AT1 receptor-mediated increase in NE release, synthesis, and reuptake/metabolism (10, 11, 12). Further studies have begun to reveal the intracellular mechanisms that mediate these rapid (release) and long-term (synthesis/release/metabolism) neuromodulatory actions of Ang II. The rapid actions of Ang II involve stimulation of phospholipase C (PLC), increases in intracellular Ca2+ ([Ca2+]int) and activation of protein kinase C (PKC) and calcium/calmodulin-dependent kinase II (CaMKII) (13, 14). PKC and CaMKII subsequently elicit depolarization and transmitter release via selective modulation of K+ and Ca2+ currents (13, 14, 15). The longer-term actions of Ang II involve PKC- and extracellular signal-regulated kinase (Erk) mitogen-activated protein kinase induced synthesis of the AP-1 transcription factor, which subsequently directs the de novo synthesis of tyrosine hydroxylase (TH) and dopamine {beta}-hydroxylase (D{beta}H) (11, 16). Recent studies have indicated that Ang II induces the axonal translocation of D{beta}H-containing synaptic vesicles via a myristolated alanine-rich C kinase/PKC{beta}-dependent mechanism (17, 18). Although these studies are helping to define the effects of Ang II on the specific processes involved in NE synthesis, it is also important to understand whether Ang II can elicit regulation of the mechanisms that control axonal transport and release (exocytosis), two fundamental processes in neuromodulation. Thus, we employed complementary DNA (cDNA) microarrays for high throughput gene expression profiling to identify novel intracellular signaling molecules that are involved in the neuromodulatory process and are influenced by Ang II via the AT1 receptor. Here we assessed the effects of Ang II on gene expression in neuronal cultures. Results from these experiments reveal that Ang II (1 or 24 h) elicits respective increases and decreases in calmodulin and synapsin I messenger RNAs (mRNAs), both of which are involved in vesicular trafficking and exocytosis (19, 20, 21, 22). These effects of Ang II are also apparent at the protein level and suggest an important role for calmodulin and synapsin I in the fundamental processes that control the longer-term neuromodulatory actions of Ang II in the hypothalamus and brainstem.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
Neuronal cocultures were prepared from hypothalamus and brainstem of newborn Sprague Dawley rats exactly as described previously (23). Cells dissociated from the hypothalamus and brainstem were pooled, resuspended in DMEM containing 10% plasma-derived horse serum (PDHS), plated on 35-mm Nunc plastic culture dishes (Nunc, Copenhagen, Denmark), and incubated for 3 days. Neuronal selection was achieved using 1 µM cytosine arabinoside for 2 days, and cells were used after 14–17 days in culture. At this time, these cocultures consist of approximately 90% neurons and approximately 10% astrocytes, as determined by immunofluorescent staining. Neuronal cultures prepared in this way contain both AT1 and AT2 receptors that are localized exclusively to neurons (23, 24). Note that even though purified astrocytes contain high levels of AT1 receptors, the glia present within the neuronal cultures used here do not contain Ang II receptors (24) (Sumners, C., unpublished observations).

Preparation of synaptosomes
For the preparation of synaptosomes, quadruplicate control or drug-treated neuronal cultures were washed once with ice-cold PBS and homogenized in 0.32 M sucrose using a Dounce homogenizer (Kontes Co., Vineland, NJ). After centrifugation (1,000 x g at 4 C for 10 min), the supernatant was again centrifuged at 20,000 x g at 4 C for 20 min. The resulting pellet was used as the synaptosomal fraction.

cDNA microarrays
RNA isolation. For the cDNA microarrays, total RNA was extracted from quadruplicate control or drug-treated neuronal cultures with the use of Atlas Pure Total RNA isolation kits (CLONTECH Laboratories, Inc., Palo Alto, CA) with several modifications. As RNA purity is the critical factor for cDNA microarray experiments, three phenol/chloroform extractions followed by ethanol/sodium acetate precipitations were performed for each sample. At the end of the extraction, RNA pellets were resuspended in ribonuclease-free water, and the concentration was determined. Before carrying out deoxyribonuclease treatment followed by RNA reprecipitation, samples were again subjected to phenol/chloroform extraction for further purification. Finally, the quality of deoxyribonuclease-treated RNA samples was confirmed by OD measurements and denaturing agarose gel electrophoresis. Total RNA samples used for cDNA microarrays elicited an A260/A280 ratio between 1.9 and 2.1 and did not show any signs of degradation.

Probe synthesis and hybridization. For cDNA synthesis, 5 µg total RNA extracted from five independent experiments were pooled to generate representative probes. A master mix for each labeling reaction was prepared containing 5 x reaction buffer, 10 x deoxy-NTP mix, [{alpha}-32P]deoxy-ATP (3000 Ci/mmol), dithiothreitol (100 mM), and Moloney murine leukemia virus reverse transcriptase. Total RNA and the gene-specific primer mix were preheated at 70 C for 2 min and at 50 C for 2 min before the master mix was added. After RT for 25 min at 50 C, the reaction was stopped by adding 10 x termination mix. The purification of labeled cDNA probes was performed by column chromatography using Chroma Spin 200 columns (Qiagen Inc., Valencia, CA), and the fractions that contained the purified labeled probes were confirmed. To further verify RNA purity, blank membranes were probed with labeled cDNAs confirming the absence of genomic DNA impurities that would result in high background signals. The microarrays used in these studies were Atlas rat cDNA expression arrays (catalogue no. 7738–1, CLONTECH Laboratories, Inc.). cDNA or oligonucleotide microarrays represent a further development of the dot blot technique and provide the possibility for automated high throughput screening of gene expression. In the case of the CLONTECH Laboratories, Inc., microarrays used here, cDNA fragments of 300 bp, sequenced to verify their identity, were spotted onto nylon membranes and fixed via UV cross-linking. The cDNAs used did not show any cross-reactivity with other genes, and they were not able to create intramolecular hybridizations. Also, the arrays were designed such that the cDNAs exclusively bound to single target cDNAs in a 1:1 ratio. Each microarray contained 588 cDNAs, double spotted to confirm the consistency of the spotting process and to reduce the risk of detecting false positive changes. For the hybridization of microarrays, labeled probes were denatured with a solution containing 1 mM NaOH and 10 mM EDTA for 20 min at 68 C. Samples were neutralized and incubated for 10 min at 68 C. After hybridization overnight at 68 C, membranes were washed four times at 68 C with wash solution 1 (2 x SSC/1% SDS) and twice for 30 min with wash solution 2 (0.1 x SSC/0.1% SDS). After a final 5-min wash at room temperature with 2 x SSC, membranes were wrapped in plastic wrap, and arrays were exposed to a Bio-Rad Laboratories, Inc., phosphorimaging screen (Hercules, CA). The quantitation of changes in gene expression was performed using Atlas Image 1.0 software that is specifically designed to exclusively calculate gene expression patterns resulting from experiments carried out with CLONTECH Laboratories, Inc., microarrays. The respective genes are automatically identified after alignment of an underlying grid and the phosphorimager image of the hybridized array. In our experiments, gene expression was normalized either to {beta}-actin or via the global normalization method, which avoids the problems that arise through possible regulation of the housekeeping gene itself. Global normalization is based on the assumption that the vast majority of genes are not regulated by a certain stimulus. Therefore, the expression of a particular gene was calculated with respect to the sum of all gene intensities on the microarray, assuming that the sum of all intensities is not affected by the regulation of the gene of interest. Changes in gene expression of greater than 30% after normalization were considered significant (25).

Analysis of TH and D{beta}H mRNA levels
TH and D{beta}H gene expression was determined using RT-PCR as detailed previously (11, 16). In brief, extraction of total RNA from triplicate control or drug-treated neuronal cultures was performed using TRIzol reagent. Five micrograms of the extracted RNA were subjected to the RT reaction, which was followed by PCR using 2 µl RT solution for TH and D{beta}H, and 1 µl RT solution for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as a housekeeping gene. All PCR conditions were determined by cyclic lineage analysis as described previously (16). The TH and D{beta}H PCR products are normalized against GAPDH.

Analysis of calmodulin, synapsin, TH, and D{beta}H protein levels
This was achieved by Western blot analyses, after isolation of proteins from quadruplicate control or drug-treated neuronal cultures or from synaptosomes prepared from the cultures. Cultures were washed once with ice-cold PBS and lysed with 0.5 ml of a boiling lysis buffer consisting of 4% SDS, 0.25 M Tris HCl (pH 6.8), 10% glycerol, and 2% {beta}-mercaptoethanol. Cells were scraped and incubated at 100 C for 3 min, and the cell solution was ultrasonically homogenized for 15 sec to disrupt the DNA. After centrifugation, supernatants were transferred into new tubes, and samples were stored at -20 C until further processing. Synaptosomes prepared from control or drug-treated cultures were resuspended in 200 µl boiling lysis buffer and processed as described above. In all cases, protein concentrations were determined according to the method of Bradford (26) to ensure equal loading of samples.

For determination of calmodulin (17 kDa) expression, total cellular proteins (25 µg) were separated by SDS-PAGE using 18% Tris-HCl gels and were transferred onto nylon membranes for 2 h at 100 V. Membranes were washed in 1 x PBS-T (PBS containing 0.5% Tween-20) for 10 min and then blocked in 10% milk in PBS-T containing 1% BSA for 3 h. The membranes were incubated overnight in rabbit anticalmodulin antibody (concentration, 1 mg/ml; dilution, 1:1,000). After a 15-min wash in PBS-T, four 5-min washes in PBS-T were performed, and membranes were then incubated for 1 h in antirabbit peroxidase conjugate antibody (dilution, 1:16,000). After one 15-min and four 5-min washes in PBS-T membranes, visualization of bands was performed using Renaissance Western Blot Chemiluminescence Kits (NEN Life Science Products, Boston, MA) according to the manufacturer’s instructions.

To analyze the synapsin Ia and Ib protein (77 and 80 kDa) content, 20 µg synaptosomal protein were separated by SDS-PAGE using 10% Tris-HCl gels and were transferred onto nylon membranes for 1.5 h at 100 V. Membranes were incubated overnight in rabbit antisynapsin Ia and Ib antibody (concentration, 0.1 mg/ml; dilution, 1:2000), which recognizes both of these synapsin I isoforms (subsequently referred to as synapsin I). Blots were then treated as described above for calmodulin.

Analysis of TH (58 kDa) and D{beta}H (70 kDa) proteins in synaptosomes from Ang II- or drug-treated cultures was achieved as detailed previously (16), using monoclonal anti-TH antibodies (1:1000) and rabbit anti-D{beta}H antibodies (1:1000), respectively.

Quantification of calmodulin, synapsin I, TH, and D{beta}H blots was carried out by densitometry using a GS710 Calibrated Imaging densitometer (Bio-Rad Laboratories, Inc.).

Peptide and drug applications
Ang II and drugs were dissolved in the appropriate solvent, followed by dilution in DMEM. The final dilution was made into the DMEM/PDHS surrounding the neuronal cultures. The concentration of Ang II used for these experiments far exceeds (by ~10,000-fold) the basal level of endogenous Ang II (5–16 pg/ml) within the medium (Sumners, C., unpublished data).

Experimental groups and data analysis
For individual microarray experiments, each experimental treatment was performed in quadruplicate dishes of neuronal cultures. As stated above, for probe synthesis and microarray hybridization, we pooled the total RNA that had been extracted from five individual experiments. For the individual Western blot experiments, each experimental treatment was performed in quadruplicate dishes of neuronal cultures. Comparisons were made with the use of a one-way ANOVA, followed by Newman-Keuls test to assess statistical significance.

Materials
One-day-old Sprague Dawley rats were obtained from our breeding colony, which originated from Charles River Laboratories, Inc. (Wilmington, MA). DMEM and TRIzol reagent were obtained from Life Technologies, Inc. (Gaithersburg, MD). Atlas Rat cDNA Expression and Atlas Pure Total RNA Isolation Kits were obtained from CLONTECH Laboratories, Inc.. Losartan was provided by Dr. William Henckler (Merck & Co., Rahway, NJ). PD 123,319 and W-7 were purchased from Research Biochemicals International (Natick, MA). PDHS, Ang II, U-73122, KN-93, monoclonal anti-TH antibodies, and antirabbit peroxidase conjugate were obtained from Sigma (St. Louis, MO). 1,2-Bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester; BAPTA-AM) and chelerythrine were obtained from Merck & Co. Rabbit polyclonal anti-synapsin I and rabbit polyclonal anticalmodulin antibodies were purchased from Zymed Laboratories, Inc. (San Francisco, CA). Rabbit polyclonal anti-D{beta}H antibodies were purchased from Chemicon (Temecula, CA). Renaissance Western Blot Chemiluminescence Kits were obtained from NEN Life Science Products (Boston, MA). Oligonucleotide primers for TH (27), D{beta}H (28), and GAPDH (29) were synthesized by Gemini Biotech (Alachua, FL). The sequences of these primers are as follows: TH: sense, 5'-CTGGAGGCTGTGGTATTTGA-3'; antisense, 5'-GCCCTTCAGCGTGACATATA-3'; D{beta}H: sense, 5'-GAGGATGACACTGTCCATC-3'; antisense, 5'-GTAGTGTAGACGGATGCC-3'; and GAPDH: sense, 5'-CCCTTCATTGACCTCAACTACATGG-3'; antisense, 5'-GAGGGGCCATCCACAGTCTTCTG-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ang II modulation of gene expression in neuronal cultures
To identify genes involved in long-term effects of Ang II via the AT1 receptor, neuronal cultures from the hypothalamus/brainstem of 1-day-old rats were treated with 100 nM Ang II for 1 or 24 h in the absence or presence of the selective AT2 receptor antagonist, PD123,319 (10 µM). Hybridization of cDNA microarrays with probes derived from control or experimental cultures revealed that only a limited number of the 588 genes present on each array were regulated by Ang II. These are listed in Table 1Go as genes that are highly regulated (>=70%), regulated (>=50%), and moderately regulated (>=30%) compared with the control after Ang II treatments for 1 or 24 h. Among the genes found to be highly regulated by Ang II were calmodulin (Fig. 1Go) and synapsin I, both of which are known to play a fundamental role in the trafficking and exocytotic events that are a part of neuromodulatory processes (19, 20, 21, 22). Thus, for further studies we concentrated on the regulation of calmodulin and synapsin I. Our data indicate that levels of calmodulin mRNA were increased by Ang II after 1- and 24-h incubations (Fig. 2Go, A and B). This stimulatory effect of Ang II was not altered by the presence of the selective AT2 receptor antagonist PD123,319 (10 µM) in the incubation solution (Fig. 2Go, A and B), indicating that these Ang II-induced actions are mediated by AT1 receptors. It should be noted that the changes in calmodulin mRNA produced by Ang II were similar regardless of the method of normalization (Fig. 2Go, A and B).


View this table:
[in this window]
[in a new window]
  		Table 1. Gene regulation by Ang II in neuronal cultures, as determined by cDNA microarrays

 


View larger version (84K):
[in this window]
[in a new window]
  		Figure 1. Gene expression analysis using cDNA microarrays. Left, Hybridization of arrays with 32P-labeled cDNAs from control neuronal cultures resulted in an expression pattern reflecting basal gene expression. Genes were double spotted on arrays to confirm the accuracy of the cDNA spotting process and thus to minimize the risk of detecting false positive genes. Right, Computerized comparison of arrays from control and Ang II-treated neuronal cultures revealed the regulation of several genes. Genes that are up-regulated by Ang II compared with controls (e.g. calmodulin and Erk1) are shown in white. Most of the genes either were not regulated by Ang II, or their expression levels were too low to be calculated (dark gray).

 


View larger version (23K):
[in this window]
[in a new window]
  		Figure 2. Modulation of calmodulin gene expression in neuronal cultures by Ang II. Neuronal cultures were treated with 100 nM Ang II (Ang) for 1 or 24 h in the presence or absence of the AT2 receptor blocker PD123319 (10 µM). Total RNA isolated from five independent experiments was pooled to generate representative probes. Radioactively labeled cDNA was hybridized to cDNA arrays overnight. Quantification of gene expression was carried out as detailed in Materials and Methods. Data are from a single hybridization in each treatment situation. A, Data from 1-h incubations. B, Data from 24-h incubations. Data are calculated with respect to the control value (100%) and either globally normalized or standardized with respect to {beta}-actin.

 
Ang II modulation of calmodulin and synapsin protein levels in neuronal cultures
Western blot analyses demonstrated that the changes in Ang II-induced mRNA were also reflected at the protein level. As shown in Fig. 3Go, Ang II (100 nM) significantly increased calmodulin protein expression in neuronal cultures after incubations of 3, 6, and 24 h, with a maximal stimulation of 60% at 6 h. These effects were mediated by AT1 receptors, as they were entirely abolished by the selective AT1 receptor antagonist losartan (10 µM; pretreated for 3 min; Fig. 3Go).



View larger version (35K):
[in this window]
[in a new window]
  		Figure 3. Ang II elicits a time-dependent increase in calmodulin protein expression in neuronal cultures. Cultures were treated for the indicated times with Ang II (Ang; 100 nM) in the absence or presence of the selective AT1 receptor antagonist losartan (Los; 10 µM). A, Representative autoradiogram showing bands that correspond to calmodulin. B, Quantification of Ang II-induced effects on calmodulin expression. Bar graphs are the mean ± SEM from eight independent experiments. *, P < 0.05 compared with control values (100%). Treatment of neuronal cultures with losartan (10 µM) alone did not alter calmodulin expression (data not shown).

 
cDNA microarrays revealed that levels of synapsin I mRNA were decreased by Ang II (100 nM) to undetectable levels. After 1 h, the synapsin I signal intensity was reduced by 3858 U (normalized to actin) or 3793 U (globally normalized), whereas the synapsin signal intensity was diminished by 3991 U (normalized to actin) or 4343 U (globally normalized) after 24 h. The effect of Ang II on synapsin I mRNA was not altered by the AT2 receptor antagonist PD123,319 (10 µM). Incubation of neuronal cultures with 100 nM Ang II (1–24 h) also produced a time-dependent decrease in synapsin protein levels, as demonstrated by Western blot analysis of synaptosomal fractions (Fig. 4Go). This action of Ang II was maximal at 3 h, with a 33% decrease in synapsin I.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 4. Ang II decreases synapsin I protein expression in synaptosomes. Synaptosomes prepared from neuronal cultures were isolated after treatment of cells with Ang II (Ang; 100 nM) for the indicated times. A, Representative autoradiogram showing bands corresponding to synapsin I a and b. B, Quantification of Ang II-induced effects on synapsin I protein expression. Bar graphs are the mean ± SEM from seven independent experiments. *, P < 0.05 compared with control values (100%).

 
Ang II-induced increase in calmodulin involves a PLC/Ca2+/PKC mechanism
As described previously, the rapid and slow neuromodulatory actions of Ang II in neurons involve signaling pathways that include stimulation of PLC, with subsequent increases in [Ca2+]int and activation of PKC and CaMKII (13, 14, 15, 16, 17). Thus, we investigated the possible involvement of these signaling molecules in the stimulatory effect of Ang II on calmodulin expression in neuronal cultures. The data presented in Fig. 5Go clearly indicate that the general PLC inhibitor U-73122 (10 and 25 µM; pretreated for 30 min) completely abolished the increase in calmodulin protein elicited by Ang II (100 nM; 3 or 6 h).



View larger version (31K):
[in this window]
[in a new window]
  		Figure 5. Ang II-induced increase in calmodulin protein expression is mediated by PLC. Neuronal cultures were treated with 100 nM Ang II (Ang) for 3 or 6 h in the absence or presence of the PLC inhibitor, U-73122 (10 or 25 µM). A, Representative autoradiogram displaying calmodulin protein expression in each treatment situation. B, Quantification of effects on calmodulin protein levels. Bar graphs are the mean ± SEM from seven independent experiments. *, P < 0.05 compared with control values (100%). Treatment of neuronal cultures with U-73122 (10 and 25 µM) alone did not alter calmodulin expression (data not shown).

 
PLC activation and subsequent generation of IP3 result in an increase in [Ca2+]int. To determine whether Ca2+ ions have an impact on Ang II-induced calmodulin protein expression, neuronal cultures were pretreated for 30 min with BAPTA-AM, which is a cell-permeable Ca2+ ion-chelating agent. Although Ang II treatment (100 nM) increased calmodulin levels after 3 and 6 h, calmodulin expression in cells costimulated with Ang II (100 nM) and BAPTA-AM (10 µM) did not significantly differ from controls at either time point (Fig. 6Go), underlining a role for Ca2+ ions in calmodulin modulation.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 6. Ang II-induced increase in calmodulin protein expression is mediated by a calcium-dependent mechanism. Neuronal cultures were treated with 100 nM Ang II (Ang) for 3 or 6 h in the absence or presence of the cell-permeable Ca2+ chelator BAPTA-AM (10 µM). A, Representative autoradiogram showing calmodulin protein expression in each treatment situation. B, Quantification of BAPTA-AM effects on calmodulin protein levels. Bar graphs are the mean ± SEM from four independent experiments. *, P < 0.05 compared with control values (100%). Treatment of neuronal cultures with BAPTA-AM (10 µM) alone did not alter calmodulin expression (data not shown).

 
A prominent consequence of increased [Ca2+]int is the activation of serine/threonine kinases such as CaMKII and PKC, so we investigated whether these enzymes had a role in the Ang II-induced increase in calmodulin expression. Treatment of neuronal cultures with the selective CaMKII inhibitor KN-93 (10 µM; pretreated for 15 min) did not significantly alter the increase in calmodulin protein expression elicited by Ang II (100 nM, 3- or 6 h; Fig. 7Go). Next we investigated a possible role for PKC in Ang II-induced calmodulin modulation. Neuronal cultures were treated with Ang II (100 nM) for 3 and 6 h in the absence or presence of the PKC inhibitor chelerythrine (10 µM; pretreated for 15 min). Chelerythrine abolished the Ang II stimulation of calmodulin protein expression (Fig. 8Go), supporting the idea that the Ang II stimulation of calmodulin involves a PKC-dependent mechanism.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 7. Ang II effects on calmodulin expression are CaMKII independent. Neuronal cultures were treated with 100 nM Ang II (Ang) for 3 or 6 h in the absence or presence of the CaMKII inhibitor KN-93 (KN-93; 10 µM). A, Representative autoradiogram showing calmodulin levels in each treatment situation. B, Quantification of KN-93 effects on calmodulin protein levels. Bar graphs are the mean ± SEM from five independent experiments. *, P < 0.05 compared with control values (100%). Treatment of neuronal cultures with KN-93 (10 µM) alone did not alter calmodulin expression (data not shown).

 


View larger version (24K):
[in this window]
[in a new window]
  		Figure 8. Ang II-induced increase in calmodulin protein expression is mediated by a PKC-dependent mechanism. Neuronal cultures were treated with 100 nM Ang II (Ang) for 3 or 6 h in the absence or presence of the PKC inhibitor chelerythrine (Chel; 10 µM). A, Representative autoradiogram showing calmodulin levels in each treatment situation. B, Quantification of Chel effects on calmodulin protein levels. Bar graphs are the mean ± SEM from five independent experiments. *, P < 0.05 compared with control values (100%). Treatment of neuronal cultures with Chel (10 µM) alone did not alter calmodulin expression (data not shown).

 
Ang II-induced increase in TH and D{beta}H expression is not mediated by calmodulin
Our previous studies have shown that the Ang II-induced increase in the expression of TH and D{beta}H in neurons involves a pathway that includes activation of PKC and Erk mitogen-activated protein kinases, formation of Fos, and synthesis of the activator protein-1 transcription factor (11, 16). In this series of experiments we determined whether the Ang II-induced increase in calmodulin has a role in the stimulatory actions of this peptide on TH and D{beta}H synthesis. Incubation of neuronal cultures with 100 nM Ang II for 4 h produced a significant increase in the expression of TH and D{beta}H mRNAs (Fig. 9AGo). This action of Ang II was not altered by the selective calmodulin inhibitor W-7 (10 µM; Fig. 9AGo). Similarly, Ang II (100 nM; 6 h) elicited an increase in the expression of TH and D{beta}H protein that was not altered by the presence of 10 µM W-7 (Fig. 9BGo).



View larger version (29K):
[in this window]
[in a new window]
  		Figure 9. Ang II-induced increase in TH and D{beta}H expression is calmodulin independent. Neuronal cultures were treated with Ang II (Ang; 100 nM) in the absence or presence of the calmodulin antagonist W-7 (10 µM) for the indicated times, followed by analysis of TH and D{beta}H mRNA and protein levels as detailed in Materials and Methods. A, Top, Representative autoradiogram showing the effects of control solution, Ang II, W-7, and Ang II plus W-7 on TH, D{beta}H, and GAPDH mRNAs levels. Bottom, Bar graphs are means from three independent experiments. B, Top, Representative autoradiogram showing the effects of control solution, Ang II, W-7, and Ang II plus W-7 on TH and D{beta}H protein levels. Bottom, Bar graphs are the mean ± SEM from three independent experiments. *, P < 0.05 compared with respective control values.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is established from our previous studies that Ang II, acting via AT1 receptors, exerts regulatory effects on several genes that are involved in controlling the synthesis of NE in rat brain neurons (11, 16, 17). In addition, the intracellular signaling pathways involved in these actions of Ang II are quite well established. The overall aim of the present study was to determine whether Ang II elicits a long-term influence over the general processes that control axonal transport and release (exocytosis) in neurons. This would enable us to gain a better understanding of the molecular mechanisms that underlie Ang II-induced neuromodulatory processes in the central nervous system. The most significant finding of this study is that Ang II regulates the expression of both calmodulin and synapsin I, which are known to interact with one another to modulate the association of synaptic vesicles and the cytoskeleton, eventually resulting in exocytosis of neurotransmitters (19, 20, 21, 22). The demonstration that both calmodulin and synapsin I genes are regulated by Ang II is a unique finding that for the first time supports the idea that this peptide can influence the processes of vesicular trafficking and exocytosis.

The results obtained here indicate that Ang II increases calmodulin expression in neurons at both the mRNA and protein levels, as revealed by the cDNA microarray and Western blot analyses. In addition, our studies have begun to elucidate the intracellular signaling pathways that are involved in the Ang II-induced increase in calmodulin expression. Similar to its effects on K+ and Ca2+ currents and NE synthesis (13, 14, 15, 16), the Ang II-induced increase in calmodulin expression involves a PLC/Ca2+/PKC-dependent pathway. The mechanism through which Ca2+/PKC modulate calmodulin gene expression in this particular situation is unknown. However, analysis of the rat calmodulin gene reveals the presence of a serum response element (30). It is well known that serum response elements within gene promoter regions can be regulated by the binding of serum response factors, which are themselves regulated by Ca2+-dependent PKC (31, 32). Thus, it is possible that such a mechanism is responsible for the modulation of calmodulin gene expression by Ang II, and a putative pathway is presented in Fig. 10Go.



View larger version (30K):
[in this window]
[in a new window]
  		Figure 10. Putative signaling pathways for Ang II-induced changes in neuronal calmodulin- and synapsin I expression. CAM, Calmodulin; DAG, diacylglycerol; IP3, inositol trisphosphate; SRE, serum response element.

 
The results presented here also demonstrate that the Ang II-induced increase in TH and D{beta}H expression is not dependent upon calmodulin. Thus, it appears that Ang IIinduced increase in expression of calmodulin is not involved in the actual process of NE synthesis. However, in preliminary experiments we determined that the Ang II-induced axonal translocation of D{beta}H-containing vesicles is inhibited by the calmodulin antagonist W-7 (Yang, H., et al., unpublished observation). Thus, the Ang II-induced increase in calmodulin may allow for greater levels of axonal transport of catecholamines and, as discussed below, be involved in the general processes that control exocytosis.

The data also indicate that Ang II decreases the expression of synapsin I, a phosphoprotein that is localized exclusively in neurons (33), at both the mRNA and protein levels. The time course of Ang II action is similar to that observed for the changes in calmodulin. In the present study the mechanism by which Ang II regulates synapsin I expression has not been investigated in detail. However, it was previously demonstrated that activation of neuronal AT1 receptors causes stimulation of CaMKII (14), and that this kinase activates the cAMP-response element binding protein via phosphorylation (34). As the cAMP response element is present in the synapsin I promoter (35), it is possible that Ang II-induced synapsin I down-regulation uses this pathway.

As stated above, interactions between synapsin I and calmodulin are crucial in regulating exocytosis. Synapsin, which is located in the synaptic vesicle membranes, is responsible for anchoring the vesicles to the cytoskeleton (36, 37, 38). In this way synapsin aids in the transport of vesicles and prevents their association with the plasma membrane and exocytosis (19, 22, 39). It is also apparent that binding of calmodulin to synapsin I, or phosphorylation of synapsin I via a calmodulin/CaMKII mechanism, causes synaptic vesicles to dissociate from the cytoskeleton, allowing the exocytotic mechanism to proceed (20, 21, 22, 40, 41, 42). Thus, factors that alter the expression of calmodulin and synapsin should ultimately result in altered exocytosis. In effect, according to the literature, the observed reciprocal changes in expression of calmodulin and synapsin I in response to Ang II would result in increased neurotransmitter release, thus contributing to the long-term neuromodulatory actions of this peptide at a fundamental level.

In summary, we have identified calmodulin and synapsin I as two targets that are regulated by Ang II acting via its AT1 receptor in neurons. These data imply that Ang II can modulate via calmodulin and synapsin I the general processes that control vesicular exocytosis, independent of its specific enhancement of NE synthesis. Further, such regulation by Ang II may be required for its longer term neuromodulatory actions, but this idea will only be substantiated by further studies.


   Acknowledgments
 
The authors thank Jennifer Moore for preparation of neuronal cultures.


   Footnotes
 
1 This work was supported by NIH Grants NS-19441 and HL-49130 (to C.S.) and HL-33610 (to M.K.R.), Fellowship Bu 1238/1–1 from the German Research Foundation (to S.B.), and Fellowship 9920557V from the American Heart Association-Florida Affiliate (to S.G.). Back

2 S.G. and S.B. contributed equally to these studies. Back

Received August 24, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Culman J, Hohle S, Qadri F, Edling O, Blume A, Lebrun C, Unger T 1995 Angiotensin as neuromodulator/neurotransmitter in central control of body fluid and electrolyte homeostasis. Clin Exp Hypertens 17:281–293
  2. Muratani H, Teruya H, Sesoko S, Takishita S, Fukiyama K 1996 Brain angiotensin and circulatory control. Clin Exp Pharmacol Physiol 23:458–464[Medline]
  3. McKinley MJ, McAllen RM, Pennington GL, Smardencas A, Weisinger RS, Oldfield BJ 1996 Physiological actions of angiotensin II mediated by AT1 and AT2 receptors in the brain. Clin Exp Pharmacol Physiol Suppl 3:S99–S104
  4. Averill DB, Diz DI 2000 Angiotensin pepetides and barorelflex control of sympathetic outflow: pathways and mechanisms of the medulla oblongata. Brain Res Bull 51:199–128
  5. Lenkei Z, Palkovits M, Corvol P, Llorens-Cortes C 1997 Expression of angiotensin type-1 (AT1) and type-2 (AT2) receptor mRNAs in the adult rat brain: a functional neuroanatomical review. Front Neuroendocrinol 18:383–439[CrossRef][Medline]
  6. Yang SN, Lippoldt A, Jansson A, Phillips MI, Ganten D, Fuxe K 1997 Localization of angiotensin II AT1 receptor-like immunoreactivity in catecholaminergic neurons of the rat medulla oblongata. Neuroscience 81:503–515[CrossRef][Medline]
  7. Koepke JP, Bovy PR, McMahon EG, Olins G, Reitz DB, Salles K, Schuh JR, Trapani AJ, Blaine ED 1990 Central and peripheral actions of a nonpeptide angiotensin II receptor antagonist. Hypertension 15:841–847[Abstract/Free Full Text]
  8. Cunningham JT, Johnson AK 1991 The effects of central norepinephrine infusions on drinking behavior induced by angiotensin after 6-hydroxydopamine injections into the anteroventral region of the third ventricle (AV3V). Brain Res 558:112–116[CrossRef][Medline]
  9. Qadri F, Culman J, Veltmar A, Maas K, Rascher W, Unger T 1993 Angiotensin II- induced vasopressin release is mediated through alpha-1 adrenoceptors and angiotensin II AT1 receptors in the supraoptic nucleus. J Pharmacol Exp Ther 267:567–574[Abstract/Free Full Text]
  10. Lu D, Yu K, Paddy MR, Rowland NE, Raizada MK 1996 Regulation of norepinephrine transport system by angiotensin II in neuronal cultures of normotensive and spontaneously hypertensive rat brains. Endocrinology 137:763–772[Abstract]
  11. Yu K, Lu D, Rowland NE, Raizada MK 1996 Angiotensin II regulation of tyrosine hydroxylase gene expression in the neuronal cultures of normotensive and spontaneously hypertensive rats. Endocrinology 137:3566–3576[Abstract]
  12. Richards EM, Raizada MK, Gelband CH, Sumners C 1999 Angiotensin II type 1 receptor-modulated signaling pathways in neurons. Mol Neurobiol 19:25–41[Medline]
  13. Sumners C, Zhu M, Gelband CH, Posner P 1996 Angiotensin II type 1 receptor modulation of neuronal K+ and Ca2+ currents: intracellular mechanisms. Am J Physiol 271:C154–C163
  14. Zhu M, Gelband CH, Posner P, Sumners C 1999 Angiotensin II decreases neuronal delayed rectifier potassium current: role of calcium/calmodulin-dependent protein kinases II. J Neurophysiol 82:1560–1568[Abstract/Free Full Text]
  15. Wang D, Gelband CH, Sumners C, Posner P 1997 Mechanisms underlying the chronotropic effect of angiotensin II on cultured neurons from rat hypothalamus and brain stem. J Neurophysiol 78:1013–1020[Abstract/Free Full Text]
  16. Yang H, Lu D, Yu K, Raizada MK 1996 Regulation of neuromodulatory actions of angiotensin II in the brain neurons by the Ras-dependent mitogen-activated protein kinase pathway. J Neurosci 16:4047–4058[Abstract/Free Full Text]
  17. Lu D, Yang H, Lenox RH, Raizada MK 1998 Regulation of angiotensin II-induced neuromodulation by MARCKS in brain neurons. J Cell Biol 142:217–227[Abstract/Free Full Text]
  18. Wang XY, Yang H, Raizada MK 2000 Angiotensin II stimulates vesicular transport of dopamine {beta} Hydroxylase (D{beta}H) in hypothalamic-brainstem neurons. FASEB J 14:499.12 (Abstract)
  19. Goldenring JR, Lasher RS, Vallano ML, Ueda T, Naito S, Sternberger NH, Sternberger LA, De Lorenzo RJ 1986 Association of synapsin I with neuronal cytoskeleton. Identification in cytoskeletal preparations in vitro and immunocytochemical localization in brain of synapsin I. J Biol Chem 261:8495–8504[Abstract/Free Full Text]
  20. Llinas R, Gruner JA, Sugimori M, McGuiness TL, Greengard P 1991 Regulation by synapsin I and (Ca2+)-calmodulin-dependent protein kinase II of the transmitter release in squid giant synapse. J Physiol 436:257–282[Abstract/Free Full Text]
  21. Nicol S, Rahman D, Baines AJ 1997 Ca2+-dependent interaction with calmodulin is conserved in the synapsin family: identification of a high-affinity site. Biochemistry 36:11487–11495[CrossRef][Medline]
  22. Hilfiker S, Peirbone VA, Czernik AJ, Kao HT, Augustine GJ, Greengard P 1999 Synapsins as regulators of neurotransmitter release. Phil Trans R Soc B Biol Sci 354:269–279[CrossRef][Medline]
  23. Sumners C, Tang W, Zelezna B, Raizada MK 1991 Angiotensin II receptor subtypes are coupled with distinct signal transduction mechanisms in neurons and astrocytes from rat brain. Proc Natl Acad Sci USA 88:7567–7571[Abstract/Free Full Text]
  24. Gelband CH, Zhu M, Lu D, Reagan LP, Fluharty SJ, Posner P, Raizada MK, Sumners C 1997 Functional interactions between neuronal AT1 and AT2 receptors. Endocrinology 138:2195–2198[Abstract/Free Full Text]
  25. Gaasterland T, Bekiranov S 2000 Making the most of microarray data. Nat Genet 24:204–206[CrossRef][Medline]
  26. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  27. Grima B, Lamouroux A, Balnot F, Biguet NF, Mallet J 1985 Complete coding sequence of rat tyrosine hydroxylase mRNA. Proc Natl Acad Sci USA 82:617–621[Abstract/Free Full Text]
  28. McMahon A, Geertman R, Sabban EL 1990 Rat dopamine beta-hydroxylase: molecular cloning and characterization of the cDNA and regulation of the mRNA by reserpine. J Neurosci Res 25:395–404[CrossRef][Medline]
  29. Hussain SN, Giaid A, El Dawiri Q, Sakkal D, Hattori R, Guo Y 1997 Expression of nitric oxide synthases and GTP cyclohydrolase I in the ventilatory and limb muscles during endotoxemia. Am J Respir Cell Mol Biol 17:173–180[Abstract/Free Full Text]
  30. Nojima H, Sokabe H 1987 Structure of a gene for rat calmodulin. J Mol Biol 193:439–445[CrossRef][Medline]
  31. Graham R, Gilman M 1990 Distinct protein targets for signals acting at the c-fos serum response element. Science 251:189–192
  32. Soh JW, Lee EH, Prywes R, Weinstein IB 1999 Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element. Mol Cell Biol 19:1313–1324[Abstract/Free Full Text]
  33. Melloni RH, Degennaro LJ 1994 Temporal onset of synapsin I gene expression coincides with neuronal differentiation during the development of the nervous system. J Comp Neurol 342:449–462[CrossRef][Medline]
  34. Sheng M, Thompson MA, Greenberg ME 1991 CREB: a Ca(2+)-regulated transcription factor phosphorylated by calmodulin-dependent kinases. Science 252:1427–1430[Abstract/Free Full Text]
  35. Jungling S, Cibelli G, Czardybon M, Gerdes HH, Thiel G 1994 Differential regulation of chromogranin B and synapsin I gene promoter activity by cAMP and cAMP-dependent protein kinase. Eur J Biochem 226:925–935[Medline]
  36. Bahler M, Greengard P 1987 Synapsin I bundles F-actin in a phosphorylation-dependent manner. Nature 326:704–707[CrossRef][Medline]
  37. Petrucci TC, Morrow JS 1987 Synapsin I: an actin-bundling protein under phosphorylation control. J Cell Biol 105:1355–1363[Abstract/Free Full Text]
  38. Sikorski AF, Terlecki G, Zagon IS, Goodman SR 1991 Synapsin I-mediated interaction of brain spectrin with synaptic vesicles. J Cell Biol 114:313–318[Abstract/Free Full Text]
  39. Rosahl TW, Geppert M, Spillane D, Herz J, Hammer RE, Malenka RC, Sudhof TC 1993 Short-term synaptic plasticity is altered in mice lacking synapsin I. Cell 75:661–670[CrossRef][Medline]
  40. Okabe T, Sobue K 1987 Identification of a new 84/82 kDa calmodulin-binding protein, which also interacts with actin filaments, tubulin and spectrin, as synapsin I. FEBS Lett 213:184–188[CrossRef][Medline]
  41. Hayes NVL, Bennett AF, Baines AJ 1991 Selective Ca(2+)-dependent interaction of calmodulin with the head domain of synapsin I. Biochem J 275:93–97
  42. Petrucci TC, Morrow JS 1991 Actin and tubulin binding domains of synapsin Ia and Ib. Biochemistry 30:413–422[CrossRef][Medline]



This article has been cited by other articles:


Home page
 IOVSHome page
T. Kurihara, Y. Ozawa, K. Shinoda, N. Nagai, M. Inoue, Y. Oike, K. Tsubota, S. Ishida, and H. Okano
Neuroprotective Effects of Angiotensin II Type 1 Receptor (AT1R) Blocker, Telmisartan, via Modulating AT1R and AT2R Signaling in Retinal Inflammation
Invest. Ophthalmol. Vis. Sci., December 1, 2006; 47(12): 5545 - 5552.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
L. P. LaGrange, G. M. Toney, and V. S. Bishop
Chronic angiotensin II infusion attenuates the renal sympathoinhibitory response to acute volume expansion
Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2003; 284(4): R1098 - R1107.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. Busche, S. Gallinat, M. A. Fleegal, M. K. Raizada, and C. Sumners
Novel Role of Macrophage Migration Inhibitory Factor in Angiotensin II Regulation of Neuromodulation in Rat Brain
Endocrinology, November 1, 2001; 142(11): 4623 - 4630.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
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 PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gallinat, S.
Right arrow Articles by Sumners, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gallinat, S.
Right arrow Articles by Sumners, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals