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Endocrinology Vol. 142, No. 11 4623-4630
Copyright © 2001 by The Endocrine Society

ARTICLES

Novel Role of Macrophage Migration Inhibitory Factor in Angiotensin II Regulation of Neuromodulation in Rat Brain

Silke Busche1, Stefan Gallinat1, Melissa A. Fleegal, Mohan K. Raizada and Colin Sumners

Department of Physiology and Functional Genomics, 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, 1600 SW Archer Road, P.O. Box 100274, Gainesville, Florida 32610. E-mail: csumners{at}phys.med.ufl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previously we determined that angiotensin II (Ang II) activates neuronal AT1 receptors, located in the hypothalamus and the brainstem, to stimulate noradrenergic pathways. To link Ang II to the regulation of norepinephrine metabolism in neurons cultured from newborn rat hypothalamus and brainstem we have used cDNA arrays for high throughput gene expression profiling. Of several genes that were regulated, we focused on macrophage migration inhibitory factor (MIF), which has been associated with the modulation of norepinephrine metabolism. In the presence of the selective AT2 receptor antagonist PD123,319 (10 µM), incubation of cultures with Ang II (100 nM; 1–24 h) elicited an increase in MIF gene expression. Western immunoblots further revealed that Ang II (100 nM; 1–24 h) increased neuronal MIF protein expression. This effect was inhibited by the AT1 receptor antagonist losartan (10 µM), the PLC inhibitor U-73122 (10 or 25 µM), the PKC inhibitor chelerythrine (10 µM), and the Ca2+ chelator 1,2-bis-[2-aminophenoxy]-ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (10 µM). Taken together with our observation that MIF is expressed in the terminal fields of noradrenergic neurons (hypothalamus) and that Ang II increases the expression of MIF in this region in vivo, our data may suggest a novel role of Ang II in norepinephrine metabolism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE OCTAPEPTIDE HORMONE angiotensin II (Ang II) exerts a variety of actions via its Ang II type 1 (AT1) receptor in the central nervous system. These Ang II-induced effects lead to physiological alterations such as increased blood pressure, altered baroreflex function, and increased secretion of aldosterone and vasopressin (1, 2, 3, 4, 5, 6, 7, 8). Within the brain AT1 receptors are located on noradrenergic neurons in the hypothalamus and brainstem (8, 9) and have been shown to elicit both short-term (release) and long-term modulation of norepinephrine (NE) neurotransmission. Studies using neuronal cocultures from 1-d-old rat brainstem and hypothalamus revealed that long-term Ang II treatment induces an AT1 receptor-mediated increase in NE release, synthesis, and reuptake/metabolism (10, 11, 12).

The intracellular signaling mechanisms leading to Ang II-induced de novo synthesis of tyrosine hydroxylase and dopamine ß-hydroxylase involve activation of PKCß and ERK MAPKs (10, 13). Recent studies have further shown that Ang II induces the axonal transport of dopamine ß-hydroxylase-containing synaptic vesicles via a myristolated alanine-rich C kinase/PKCß-dependent mechanism (14, 15). Finally, targets such as calmodulin and synapsin, which are both involved in vesicular trafficking and exocytosis (16), are regulated by Ang II. Overall, these studies are beginning to provide a more detailed insight into the effects of Ang II on the molecular events underlying neurotransmission.

To further investigate the role of Ang II in NE metabolism we used cDNA microarrays for high throughput gene expression profiling to assess the longer-term effects of Ang II on gene expression in neuronal cultures. We identified macrophage migration inhibitory factor (MIF) as a gene that is up-regulated after Ang II stimulation for 1–24 h. The increased MIF gene expression in our study was paralleled by an enhanced MIF protein expression after Ang II incubation, which was mediated by the AT1 receptor through a PLC/Ca2+/PKC-dependent mechanism.

MIF has a molecular mass of 12.5 kDa (17) and was among the first of the lymphocyte-derived cytokines to be described. It was initially isolated from supernatants of antigen-activated lymphocytes as a factor that inhibited the random movement of macrophages and monocytes (18, 19, 20). MIF is widely expressed in the brain (21) and has been linked to the modulation of NE metabolism by virtue of its ability to catalyze the conversion of toxic catecholaminechromes to indoledihydroxyderivates (22, 23). Thus, the regulation of MIF by Ang II may be a further mechanism through which this peptide can influence NE neurotransmission.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Atlas Rat cDNA Expression Arrays and Atlas Pure Total RNA Isolation Kits were obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA). DMEM and TRIzol reagent were purchased from Life Technologies, Inc. (Gaithersburg, MD). Losartan was provided by Dr. William Henckler (Merck & Co., Inc. (Rahway, NJ). Plasma-derived horse serum, angiotensin II, PD 123,319, U-73122, KN-93, and antirabbit peroxidase conjugate antibody 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., Inc.. Rabbit polyclonal anti-MIF antibody was purchased from Torrey Pines Biolabs, Inc. (San Diego, CA). Renaissance Western blot chemiluminescence kits were obtained from NEN Life Science Products (Boston, MA).

Cell culture
Neuronal cocultures were prepared from hypothalamus and brainstem of newborn Sprague Dawley rats exactly as described previously (24). Cells were dissociated from the hypothalamus and brainstem, pooled, and resuspended in DMEM containing 10% plasma-derived horse serum. They were then plated on plastic culture dishes and used after 14–17 d in culture. During this period, the cocultures consisted of 90% neurons and 10% astrocytes and microglia, as determined by immunofluorescent staining. Neuronal cultures prepared in this way contain both AT1 and AT2 receptors that are localized exclusively to neurons (25). Thus, any observed effects of Ang II are neuronal, and not glial, in origin.

Animals
Adult male and female rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA) and used as a breeding colony to produce a supply of 1-d-old rats. Adult male rats (~200–220 g) purchased from Charles River Laboratories, Inc. (Wilmington, MA), were used for the surgical/injection procedures described below. Rats were housed two per cage in light-controlled facilities. Animals were fed ad libitum and had free access to water. All procedures were approved by the University of Florida institutional animal use and care committee.

Surgical procedures and intracranial injections
Adult male rats (200–250 g) were anesthetized with ketamine (30 mg/kg BW) and xylazine (6 mg/kg BW) im and placed in a David Kopf rat stereotaxic frame (Tujunga, CA). Using stereotaxic coordinates (bregma, -1.30 mm; lateral, 1.50 mm) obtained from Paxinos and Watson (26), a hole was drilled in the skull over the right lateral cerebroventricle. Intracerebroventricular (icv) injections of 0.9% saline (2 µl) or Ang II (10 ng/2 µl 0.9% saline) were made via a Hamilton (Reno, NV) microsyringe (needle gauge, 26; volume, 5 µl) that was lowered into position through the hole (from skull -4.50 mm). One minute postinjection the syringe was removed, the hole was plugged with bone wax, and the wound was cleaned and closed using stainless steel wound clips. Six hours after the icv injection rats were killed, and their brains were removed. The hypothalamus, brainstem, cerebellum, and cortex were dissected free and used for analysis of MIF protein levels as described below.

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, with several modifications. As RNA purity is the critical factor for cDNA microarray experiments, three phenol/chloroform extractions followed by ethanol/NaOAc 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 gave an A260/A280 ratio between 1.9–2.1 and did not show any signs of degradation.

Probe synthesis and hybridization. For cDNA synthesis, 5 µg total RNA that had been extracted from five independent experiments were pooled to generate representative probes. For each labeling a reaction master mix was prepared containing 5x reaction buffer, 10x 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 heated to 70 C for 2 min and to 50 C for 2 min before adding the master mix. RT was carried out for 25 min at 50 C, and the reaction was stopped by adding 10x termination mix [0.1 M EDTA (pH 8.0) and 1 mg/ml glycogen]. Labeled cDNA probes were purified by column chromatography using Chroma Spin 200 columns (QIAGEN, Valencia, CA), and the fractions that contained the purified labeled probes were collected. Finally, blank membranes were hybridized with labeled cDNAs, confirming the absence of genomic DNA impurities that would generate high background signals.

The microarrays used in these studies were Atlas rat cDNA expression arrays (catalogue no. 7738-1; CLONTECH Laboratories, Inc., Palo Alto, CA). 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 do not show any cross-reactivity with other genes, and they are not able to create intramolecular hybridizations. Also, the arrays are designed such that the cDNAs exclusively bind to single target cDNAs in a 1:1 ratio. Each microarray contains 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 (2x SSC/1% SDS) and twice for 30 min each time with wash solution 2 (0.1x SSC/0.1% SDS). After a final 5-min wash at room temperature with 2x SSC, membranes were wrapped in plastic wrap, and arrays were exposed to a phosphorimaging screen (Bio-Rad Laboratories, Inc., Richmond, 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 via a global normalization method using Atlas Image 1.0 software. Global normalization is based on the assumption that the vast majority of genes are not regulated by a certain stimulus and avoids the problems that arise through possible regulation of a housekeeping gene. 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 regulation of the gene of interest. Changes in gene expression of greater than 30% after normalization were considered significant (27).

Analysis of MIF protein levels
MIF protein levels in neuronal cultures or rat brain areas were assessed by Western blot analyses. Proteins were isolated from quadruplicate control or drug-treated neuronal cultures or from brain areas as follows. Cultures were washed once with ice-cold PBS and treated with 100 µl of a boiling lysis buffer consisting of 4% SDS, 0.25 M Tris HCl (pH 6.8), 10% glycerol, and 2% ß-mercaptoethanol. Cells were then scraped, incubated at 100 C for 3 min, and ultrasonically disrupted for 15 sec. Brain areas were treated with 5 ml of the same lysis buffer, homogenized for 15 sec using a Tekmar Tissuemizer (Cincinnati, OH), boiled for 3 min, and ultrasonically disrupted as described above. After homogenization, cell culture or brain samples were centrifuged at 14,000 rpm for 5 min, supernatants were transferred into new tubes, and protein concentrations were determined according to the method described by Bradford (28).

To determine MIF (12.5 kDa) expression, 25 µg (for neuronal cultures) or 20 µg (for brain areas) total cellular proteins were separated by SDS-PAGE using 18% Tris-HCl gels and were transferred onto nitrocellulose membranes for 2 h at 100 V. After a 10-min wash in 1x PBS-T (PBS containing 0.5% Tween 20), membranes were blocked in PBS-T containing 10% milk and 1% BSA for 3 h, followed by an overnight incubation in rabbit anti-MIF antibody (concentration, 1 mg/ml; dilution, 1:500). After a 15-min wash in PBS-T, four 5-min washes in PBS-T were carried out, and membranes were then incubated for 1 h in antirabbit peroxidase conjugate antibody (dilution, 1:16,000). Membranes were washed as described above, and bands were visualized using Renaissance Western blot chemiluminescence kits according to the manufacturer’s instructions (NEN Life Science Products, Inc.). MIF protein expression was quantified by densitometry using a calibrated imaging densitometer (model GS710, Bio-Rad Laboratories, Inc.).

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 a Newman-Keuls or Bonferroni test to assess statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
cDNA expression arrays
To identify genes that play a role in cell-cell communication triggered by Ang II via the AT1 receptor, neuronal cultures from the hypothalamus/brainstem of 1-d-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). After hybridizing the cDNA microarrays with probes derived from control or experimental cultures, only 16 of the 588 genes present on each array were found to be regulated by more than 30%. Genes that were highly regulated (>=70%) were MIF, calmodulin, synapsins I and II, and certain ribosomal proteins, whereas copper-zinc-superoxide dismutase-1 and polyubiquitin were moderately regulated (>=50%). A full list of the genes that were regulated by Ang II has been published previously (16). In further studies we focused on one of these genes, MIF, which shows high levels of basal expression in the brain and is proposed to be involved in regulating the breakdown of a NE metabolite. The data presented in Fig. 1Go indicate that Ang II (100 nM; 1 or 24 h) increases the expression of MIF mRNA in neuronal cultures. Coincubation of cultures with PD123,319 at a concentration that completely inhibits AT2 receptors (10 µM) partially blocked the Ang II-induced increase in MIF expression (Fig. 2Go). This suggests that the Ang II-induced increase in MIF gene expression may be mediated via both AT2 and AT1 receptors.



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  		Figure 1. Modulation of MIF gene expression in neuronal cultures by Ang II. Neuronal cultures were treated with 100 nM Ang II for 1 or 24 h in the presence or absence of the AT2 receptor blocker PD123,319 (PD; 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 was carried out by phosphorimaging, and gene expression levels in different experimental groups were determined using Atlas Image 1.0 software. Data are means from each treatment situation and are calculated with respect to the control value (100%) and normalized via the global method as detailed in Materials and Methods.

 


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  		Figure 2. Ang II elicits a time-dependent increase in MIF protein expression in neuronal cultures. Cultures were treated for the indicated times with Ang II (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 MIF. B, Quantification of Ang II-induced effects on MIF expression. Bar graphs are the mean ± SEM from five independent experiments for Ang II and three independent experiments for Ang II/Los. *, P < 0.05 compared with control values (100%).

 
Ang II increases MIF protein expression in neuronal cultures
To verify that the Ang II effects on MIF mRNA expression observed in the microarray studies are also reflected at the protein level, we performed Western blot analyses. Cells were treated for 1–24 h with control solution or Ang II, and the MIF protein expression was studied. Figure 2Go shows that Ang II (100 nM) exerted time-dependent effects and increased MIF protein expression significantly after incubations of 1, 3, 6, and 24 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. 2Go). In a further series of experiments cultures were incubated with Ang II (1–100 nM; 6 h) to test the concentration dependency of this effect. The results presented in Fig. 3Go indicate that 1 nM Ang II produced an increase in MIF expression, although the effect was not statistically significant. At a concentration of 10 nM, Ang II produced a significant increase in MIF levels that was not different from the effect obtained with 50 and 100 nM of this peptide.



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  		Figure 3. Ang II elicits a concentration-dependent increase in MIF protein expression in neuronal cultures. Cultures were treated for 6 h with Ang II at 1, 10, 50, or 100 nM. A, Representative autoradiogram showing bands that correspond to MIF in each treatment situation. B, Quantification of Ang II-induced effects on MIF expression. Bar graphs are the mean ± SEM from four independent experiments. *, P < 0.05 compared with control values (100%).

 
Ang II-induced increase in MIF involves a PLC/Ca2+/PKC mechanism
In neuronal cultures Ang II induces signaling pathways that include stimulation of PLC, with subsequent increases in intracellular Ca2+ ([Ca2+]int) and activation of PKC and Ca2+/calmodulin-dependent kinase II (CaMKII) (10, 13, 14, 15).

To study the effects of Ang II on MIF expression in more detail we investigated whether these signaling molecules have an impact on the enhanced MIF expression after Ang II stimulation. Figure 4Go demonstrates that the general PLC inhibitor U-73122 (10 and 25 µM; 30-min pretreatment) completely suppresses the increase in MIF protein elicited by Ang II (100 nM; 3 or 6 h).



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  		Figure 4. Ang II-induced increase in MIF protein expression is mediated by PLC. Neuronal cultures were treated with 100 nM Ang II for 3 or 6 h in the absence or presence of the PLC inhibitor, U-73122 (U; 10 or 25 µM). A, Representative autoradiogram displaying MIF protein expression in each treatment situation. B, Quantification of effects on MIF protein levels. Bar graphs are the mean ± SEM from three independent experiments. *, P < 0.05 compared with control values (100%).

 
PLC activation generates IP3 and subsequently leads to an increase in [Ca2+]int. We used a cell-permeable Ca2+ ion-chelating agent to determine whether Ca2+ ions influence the Ang II-induced increase in MIF protein expression. Thus, neuronal cocultures were pretreated for 30 min with BAPTA-AM. The data presented in Fig. 5Go indicate that in the presence of BAPTA-AM, the Ang II-stimulated increase in MIF protein levels is entirely abolished. In fact, at 6 h the expression of MIF fell below control levels under these treatment conditions. BAPTA-AM alone did not alter MIF protein expression. Overall, these data point to a role of Ca2+ ions in MIF modulation by Ang II.



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  		Figure 5. Ang II-induced increase in MIF protein expression is mediated by a calcium-dependent mechanism. Neuronal cultures were treated with 100 nM Ang II for 3 or 6 h in the absence or presence of the cell-permeable Ca2+ chelator BAPTA-AM (BAPTA; 10 µM). A, Representative autoradiogram showing MIF protein expression in each treatment situation. B, Quantification of BAPTA-AM effects on MIF protein levels. Bar graphs are the mean ± SEM from four independent experiments. *, P < 0.05 compared with control values (100%).

 
Increased [Ca2+]int levels in neurons can lead to the activation of CaMKII or PKC, which are known to exert a variety of effects in these cells. To investigate whether these kinases modulate Ang II-induced MIF expression, cultures were treated with Ang II (100 nM) in the absence or presence of the CaMKII inhibitor KN-93 (10 µM; pretreated for 15 min). Figure 6Go demonstrates that KN-93 did not have a significant impact on the increase in MIF protein expression elicited by Ang II.



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  		Figure 6. Ang II effects on MIF expression are CaMKII independent. Neuronal cultures were treated with 100 nM Ang II for 3 or 6 h in the absence or presence of the CaMKII inhibitor KN-93 (KN; 10 µM). A, Representative autoradiogram showing MIF levels in each treatment situation. B, Quantification of KN-93 effects on MIF protein levels. Bar graphs are the mean ± SEM from three independent experiments. *, P < 0.05 compared with control values (100%).

 
Generation of diacylglycerol and increased [Ca2+]int after PLC activation are known to activate PKC. As our results point to an involvement of PLC and [Ca2+]int in the AT1 receptor signaling, our next aim was to investigate a possible role of PKC in Ang II-induced MIF modulation. Neuronal cultures were stimulated 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 MIF protein expression (Fig. 7Go), suggesting the involvement of PKC in the Ang II-induced signaling events leading to increased MIF expression.



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  		Figure 7. Ang II-induced increase in MIF protein expression is mediated by a PKC-dependent mechanism. Neuronal cultures were treated with 100 nM Ang II for 3 or 6 h in the absence or presence of the PKC inhibitor chelerythrine (Chel; 10 µM). A, Representative autoradiogram showing MIF levels in each treatment situation. B, Quantification of Chel effects on MIF protein levels. Bar graphs are the mean ± SEM from four independent experiments. *, P < 0.05 compared with control values (100%).

 
Ang II increases expression of MIF in rat hypothalamus
To determine whether the Ang II-induced increases in MIF expression occur in the brain in vivo, we investigated the effects of intracranial injection of Ang II. Male rats received a single icv injection of either 0.9% saline (2 µl) or Ang II (10 ng/2 µl) as described in Materials and Methods. Six hours later brains were removed, and MIF expression was analyzed in the hypothalamus, brainstem, cortex, and cerebellum. From Fig. 8AGo it is clear that MIF is expressed in all of these areas in control rats, except the brainstem. Ang II injection elicited a significant increase in MIF expression in the hypothalamus in all rats, as shown in Fig. 8Go, B and C. By contrast, Ang II had no effect on MIF expression in the brainstem (Fig. 8BGo), and the effects of this peptide on cortical and cerebellar MIF expression were variable. For example, Ang II increased the expression of MIF in the cortex and cerebellum of two of the five rats tested (not shown).



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  		Figure 8. Ang II increases the expression of MIF protein in rat hypothalamus. Male Sprague Dawley rats were injected into the right lateral cerebroventricle (icv) with either 2 µl 0.9% saline (n = 5) or 2 µl 0.9% saline containing 10 ng Ang II (n = 5), and MIF expression was analyzed 6 h later in the hypothalamus (hypo), brainstem, cortex, and cerebellum (cereb) as detailed in the text. A, Representative Western blot showing the expression of MIF in all areas from a control rat. B, Western blots showing the expression of MIF in the hypothalamus of all saline- and Ang II-treated rats. Numbers in parentheses indicate the animal reference number. C, Densitometric analysis (mean ± SEM; n = 5 rats per group) of the hypothalamic data from B. The mean increase in hypothalamic MIF expression in response to Ang II was 4.68-fold.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies from our laboratory using neuronal cultures from newborn rat brainstem and hypothalamus have shown that Ang II, acting via the AT1 receptor, regulates genes involved in the synthesis, release, and reuptake/metabolism of NE (10, 11, 12, 13, 14, 15, 16). The aim of this study was to investigate whether Ang II modulates the long-term molecular events underlying NE turnover. As neuromodulation comprises a variety of complex processes, cDNA microarrays represent a very effective approach for high throughput gene expression profiling because they allow for the simultaneous and efficient screening of possible targets under given experimental conditions. Based on a high throughput gene expression profiling we identified several genes that were differentially regulated by Ang II via its AT1 receptor in neuronal cultures (16). Our results clearly show that Ang II increases MIF mRNA and protein expression as determined by cDNA microarrays and Western blot analyses, respectively. This suggests that regulation of MIF, which has been linked to NE metabolism (22, 23), may be a further mechanism through which Ang II can influence NE neurotransmission.

Based upon the data presented here, the intracellular signaling pathways that mediate the Ang II-induced increase in MIF expression include a PLC/Ca2+/PKC-dependent mechanism, similar to the effects of this peptide on calmodulin gene and protein expression (16), K+ and Ca2+ currents (29, 30, 31), and NE transporter synthesis (10) in neurons. The conclusion from this may be that some of the longer-term neuromodulatory actions of Ang II and also the short-term actions of this peptide share certain signaling molecules. However, further investigation is needed to determine whether the isozymes of PLC and PKC that are involved are the same. Furthermore, the exact mechanisms by which MIF gene expression is influenced by Ang II are not known, but include a number of possibilities. For example, one possibility is a direct action of PKC at the promoter region of the MIF gene to elicit an increase in transcription. In addition, we cannot exclude the idea that the increase in MIF protein levels by Ang II involves calmodulin, because the actions of Ang II on both proteins involve PKC and calcium. A further possibility is that the Ang II-induced increase in MIF protein expression is mediated through PKA, because the promoter region of the MIF gene is known to contain a cAMP response element (32). This latter idea is plausible, because we have determined that Ang II increases PKA activity in neuronal cultures via a PKC-dependent mechanism (Busche, S., et al., unpublished observations). The above possibilities are summarized in Fig. 9.

The multifunctional protein MIF is constitutively expressed within both glial and neuronal cells in the rat brain (21), but its role in the central nervous system is not yet understood. Although MIF’s name originated from its association with the immune system, it appears to be involved in cell proliferation (33, 34), differentiation (35), regeneration (36, 37), apoptosis, and various types of cancer (34, 38). Interestingly, Ang II has also been implicated in many of the above-mentioned fields (39, 40).

Of particular interest is the observation that MIF has been associated with NE turnover. Matsunaga et al. (22, 23) demonstrated that MIF can act as an enzyme and catalyze the conversion of toxic quinones to less toxic compounds. For example, NE can be oxidized to form toxic norepinephrinechrome, which can then be converted by MIF to 3,5,6-trihydroxyindole. Thus, our observation that Ang II enhances MIF expression may indicate that this peptide is able to promote the metabolism of norepinephrinechromes by increasing neuronal MIF levels.

This hypothesis is further supported by the finding that MIF expression is restricted to certain brain areas. We investigated the MIF protein expression in the adult rat brain and confirmed other studies (21, 41) by showing that MIF protein is present in the hypothalamus, cortex, and cerebellum, but not in the brainstem. MIF is thus expressed in the terminal fields of the catecholaminergic neurons (hypothalamus), but not in the neuronal cell bodies, supporting the idea that the Ang II-induced up-regulation of MIF protein expression may play a role in detoxification of catecholamine metabolites. Further support for this idea comes from the demonstration that Ang II increases the expression of MIF protein in rat hypothalamus (Fig. 8Go). However, further studies, such as determining the effects of Ang II on the formation of trihydroxyindoles in rat brain, are required to substantiate the above idea. In addition, it will be important to determine whether the actions of Ang II on MIF expression in rat hypothalamus occur via the PLC/Ca2+/PKC pathway.

Finally, Ang II-induced MIF regulation may represent a mechanism that contributes to the development/maintenance of hypertension. Previous reports have shown that Ang II-induced neuromodulation is enhanced in spontaneously hypertensive rats compared with normotensive Wistar-Kyoto rats, as evidenced by increased NE transporter synthesis (42, 43). Preliminary data (Busche, S., S. Gallinat, M. K. Raizada, and C. Sumners, unpublished observations) show that Ang II increases MIF protein levels in Wistar-Kyoto rats, but not in spontaneously hypertensive rats after 3 and 6 h. Together with a higher NE synthesis in spontaneously hypertensive rats, this potential decrease in NE breakdown would subsequently lead to higher noradrenergic responses and higher blood pressure.

In summary, we have identified MIF as a target that is regulated by Ang II acting via its AT1 receptor in neurons and have elucidated intracellular signaling pathways that are involved in this response. These data imply that Ang II can modulate NE metabolism via a novel mechanism that involves MIF-mediated NE breakdown. Further, this process may be involved in pathophysiological changes leading to hypertension, but this idea will only be substantiated by further studies.



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  		Figure 9. Flow diagram showing putative signaling pathways for Ang II- induced changes in neuronal MIF and calmodulin expression. CAM, Calmodulin; SRE, serum response element; CREB, Ca2+/cAMP response element. Solid arrows represent established pathways, and dashed arrows represent putative mechanisms.

 

   Acknowledgments
 
The authors thank Wenrong Zheng for preparation of neuronal cultures, and Thanh Mai Carroll for technical assistance.


   Footnotes
 
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.).

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

Abbreviations: Ang II, Angiotensin II; BAPTA-AM, 1,2-bis-[2-aminophenoxy]-ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester; [Ca2+]int, intracellular Ca2+; CaMKII, calmodulin-dependent kinase II; icv, intracerebroventricular; MIF, migration inhibitory factor; NE, norepinephrine; 1x PBS-T, PBS containing 0.5% Tween 20.

Received March 13, 2001.

Accepted for publication July 30, 2001.


   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 3(Suppl):S99–S104
  4. Averill DB, Diz DI 2000 Angiotensin peptides and baroreflex 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. 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]
  9. 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]
  10. Yu K, Lu D, Rowland NE, Raizada MK 1996 Regulation of norepinephrine transport system by angiotensin II in neuronal cultures of normotensive and spontaneously hypertensive rat brains. Endocrinology 137:3566–3576[Abstract]
  11. 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]
  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. Yang H, Lu D, Yu K, Raizada MK 1996 Regulation of neuromodulatory actions of angiotensin II in brain neurons by the Ras-dependent mitogen-activated protein kinase pathway. J Neurosci 16:4047–4058[Abstract/Free Full Text]
  14. Wang XY, Yang H, Raizada MK 2000 Angiotensin II stimulates vesicular transport of dopamine 36 hydroxylase (D 36 H) in hypothalamic-brainstem neurons. FASEB J 14:499.12 (Abstract)
  15. 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]
  16. Gallinat S, Busche S, Yang H, Raizada MK, Sumners C 2001 Gene expression profiling of rat brain neurons reveals angiotensin II-induced regulation of calmodulin and synapsin I: possible role in neuromodulation. Endocrinology 142:1009–1016[Abstract/Free Full Text]
  17. Bernhagen J, Calandra T, Bucala R 1998 Regulation of the immune response by macrophage migration inhibitory factor: biological and structural features. J Mol Med 76:151–161[CrossRef][Medline]
  18. George M, Vaughan JH 1962 In vitro cell migration as a model for delayed hypersensitivity. Proc Soc Exp Biol Med 111:514–521
  19. Bloom BR, Bennet B 1966 Mechanism of a reaction in vitro associated with delayed-type hypersensitivity. Science 153:80–82[Abstract/Free Full Text]
  20. David JR 1966 Delayed hypersensitivity in vitro: its mediation by cell free substances formed by lymphoid cell-antigen interaction. Proc Natl Acad Sci USA 56:72–77[Free Full Text]
  21. Bacher M, Meinhardt A, Lan HY, Dhabhar FS, Mu W, Metz CN, Chesney JA, Gemsa D, Donnelly T, Atkins RC, Bucala R 1998 MIF expression in the rat brain: implications for neuronal function. Mol Med 4:217–230[Medline]
  22. Matsunaga J, Sinha D, Pannell L, Santis C, Solano F, Wistow GJ, Hearing VJ 1999 Enzyme activity of macrophage migration inhibitory factor toward oxidized catecholamines. J Biol Chem 274:3268–3271[Abstract/Free Full Text]
  23. Matsunaga J, Sinha D, Solano F, Santis C, Wistow GJ, Hearing VJ 1999 Macrophage migration inhibitory factor (MIF): its role in catecholamine metabolism. Cell Mol Biol 45:1035–1040
  24. 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
  25. 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]
  26. Paxinos G, Watson C 1998 The rat brain in stereotaxic coordinates, 4th ed. New York: Academic Press
  27. Gaasterland T, Bekiranov S 2000 Making the most of microarray data. Nat Genet 24:204–206[CrossRef][Medline]
  28. 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]
  29. 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
  30. Zhu M, Gelband CH, Posner P, Sumners C 1999 Angiotensin II decreases neuronal delayed rectifier potassium current: role of calcium/calmodulin-dependent protein kinase II. J Neurophysiol 82:1560–1568[Abstract/Free Full Text]
  31. Wang D, Gelband CH, Sumners C, Posner P 1997 Mechanisms underlying the chronotropic effect of angiotensin II on neuronal cultures from rat hypothalamus and brain stem. J Neurophysiol 78:1013–1020[Abstract/Free Full Text]
  32. Waeber G, Thompson N, Chautard T, Steinmann M, Nicod P, Pralong FP, Calandra T, Gaillard RC 1998 Transcriptional activation of the macrophage migration-inhibitory factor gene by the corticotropin-releasing factor is mediated by the cyclic adenosine 3',5'-monophosphate responsive element-binding protein CREB in pituitary cells. Mol Endocrinol 12:698–705[Abstract/Free Full Text]
  33. Wistow GJ, Shaughnessy MP, Lee DC, Hodin J, Zelenka PS 1993 A macrophage migration inhibitory factor is expressed in the differentiating cells of the eye lens. Proc Natl Acad Sci USA 90:1272–1275[Abstract/Free Full Text]
  34. Mitchell RA, Metz CN, Peng T, Bucala R 1999 Sustained mitogen-activated protein kinase (MAPK) and cytoplasmic phospholipase A2 activation by macrophage migration inhibitory factor (MIF). J Biol Chem 25:18100–18106
  35. Shimizu T, Abe R, Nakamura H, Ohkawara, Suzuki M, Nishihira J 1999 High expression of macrophage migration inhibitory factor in human melanoma cells and its role in tumor cell growth and angiogenesis. Biochem Biophys Res Commun 264:751–758[CrossRef][Medline]
  36. Nishio Y, Minami A, Kato H, Kaneda K, Nishihira J 1999 Identification of macrophage migration inhibitory factor (MIF) in rat peripheral nerves: its possible role in nerve regeneration. Biochim Biophys Acta 1453:74–82[Medline]
  37. Abe R, Shimizu T, Ohkawara A, Nishihira J 2000 Enhancement of macrophage migration inhibitory factor (MIF) expression in injured epidermis and cultured fibroblasts. Biochim Biophys Acta 1500:1–9[Medline]
  38. Hudson JD, Shoaibi MA, Maestro R, Carnero A, Hannon GJ, Beach DH 1999 A proinflammatory cytokine inhibits p53 tumor suppressor activity. J Exp Med 190:1375–1382[Abstract/Free Full Text]
  39. Griendling KK, Lassegue B, Alexander RW 1996 Angiotensin receptors and their therapeutic implications. Annu Rev Pharmacol Toxicol 36:281–306[Medline]
  40. Gallinat S, Busche S, Raizada MK, Sumners C 2000 The angiotensin II type 2 receptor: an enigma with multiple variations. Am J Physiol 278:E357–E374
  41. Nishibori M, Nakaya N, Mori S, Saeki K 1997 Immunohistochemical localization of macrophage migration inhibitory factor (MIF) in tanycytes, subcommissural organ and choroid plexus in the rat brain. Brain Res 758:259–262[CrossRef][Medline]
  42. Yang H, Raizada MK 1999 Role of phosphatidylinositol 3-kinase in angiotensin II regulation of norepinephrine neuromodulation in brain neurons of the spontaneously hypertensive rat. J Neurosci 19:2413–2423[Abstract/Free Full Text]
  43. 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



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