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* Department of Physiology and Functional Genomics and McKnight Brain Institute, University of Florida, Gainesville, Florida, USA; and
Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
1Correspondence: Department of Physiology and Functional Genomics, College of Medicine, Box 100274, 1600 SW Archer Rd., University of Florida, Gainesville, FL 32610-0274, USA. E-mail: csumners{at}phys.med.ufl.edu
SPECIFIC AIMS
Angiotensin II (ANG II) acts via its ANG II type 1 receptor (AT1-R) to increase the expression of macrophage migration inhibitory factor (MIF) within hypothalamic neurons in culture and in normotensive rat hypothalamus in vivo. In turn, MIF acts intracellularly to counteract the AT1-R-mediated chronotropic effect of ANG II in hypothalamic neurons in vitro, an effect mediated by the thiol-protein oxidoreductase (TPOR) activity of the MIF molecule. The objective of this study was to determine the actions of MIF in regulating the cardiovascular, behavioral, and neurophysiological actions of ANG II mediated by the brain in vivo.
PRINCIPAL FINDINGS
1. Central nervous system injection of ANG II increases MIF expression in the paraventricular nucleus
Our first objective was to determine which area(s) within the hypothalamus express MIF after central nervous system (CNS) injections of ANG II. Intracerebroventricular (icv) injection of ANG II (10 pmol/2 µl) into Sprague-Dawley (SD) or Wistar Kyoto (WKY) rats elicited a significant (P<0.001) 
3-fold increase in MIF protein exclusively in the PVN, without affecting MIF levels in the anterior or lateral hypothalamic nuclei.
2. Role of AT1-R within the paraventricular nucleus in the pressor and dipsogenic actions of icv-injected ANG II
Evidence indicates that icv-injected ANG II increases blood pressure and drinking in part by stimulating forebrain sites that subsequently promote local actions of ANG II at AT1-R expressing paraventricular nucleus (PVN) neurons. Considering the above data on ANG II-induced MIF expression in the PVN, the objective here was to determine the role of the PVN in the pressor and dipsogenic actions of ANG II. Our data indicate that icv injection of ANG II (1–100 pmol/2 µl) into SD rats produces a dose-dependent increase in MAP and water intake, reaching a maximal effect at 10 pmol ANG II (P<0.001). Injection of the AT1-R antagonist losartan (2.1 nmol/µl) directly into the PVN significantly (P<0.001) reduced the pressor and drinking effects produced by subsequent icv injection of 10 pmol ANG II. Based on all of the above data, our subsequent studies with MIF were focused on the PVN.
3. Overexpression of MIF within the PVN blunts the pressor and dipsogenic actions of icv-injected ANG II
A gene transfer approach was used to over express MIF within the PVN of SD rats and determine its effects on the pressor and drinking responses induced by icv-injected ANG II. Microinjection of an adenoviral vector containing enhanced GFP (EGFP), driven by the neuron specific promoter synapsin (Ad5-Syn-EGFP; 1 µl of 1x107 ifu), into the PVN resulted in highly efficient and localized gene transfer within neurons. Transduction of MIF into PVN neurons was achieved via a similar Ad-Syn vector system (Ad5-Syn-MIF). Bilateral microinjection of Ad5-Syn-MIF (1 µl of 1x107 ifu) into the PVN produced an increase in MIF transgene expression and MIF protein levels within 3–4 days, reaching maximal levels by 7 days, and declining to basal levels by 14 days.
Seven days after viral transduction, basal mean arterial pressure (MAP) was not different between rats microinjected with either Ad5-Syn-MIF or Ad5-Syn-EGFP, or control (no virus) rats, as measured using radiotelemetry devices implanted into the abdominal aorta (controls, 94.9±1.3 mmHg; Ad5-Syn-EGFP, 91.4±1.8 mmHg; Ad5-Syn-MIF, 94.2±1.8 mmHg; n=10 rats per group). Injection of ANG II (10 pmol/2 µl icv) into Ad5-Syn-enhanced GFP-treated rats 7 days after viral transduction elicited a significant pressor action compared with icv injections of 2 µl 0.9% saline (Fig. 1
). Similar pressor responses were obtained in control rats (no viral injection; Fig. 1
). In contrast, the pressor action of icv-injected ANG II was diminished by >50% (P<0.001) in the Ad5-Syn-MIF-treated rats, failing to reach the same maximal response as observed in the Ad5-Syn-enhanced GFP-treated or control rats (Fig. 1)
. By 14 days postviral injections, when MIF levels in the PVN have returned to baseline, the ANG II-induced pressor action was not significantly different between the Ad5-Syn-MIF and Ad5-Syn-enhanced GFP-treated rats (Fig. 1)
.
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Overexpression of MIF within the PVN as above for 7 days also significantly reduced (P<0.01) the water intake induced by ANG II (10 pmol/2 µl icv) compared with that of control or Ad5-Syn-EGFP-treated rats (controls, 11.33±0.65 ml/30 min; Ad5-Syn-EGFP, 10.33±1.23 ml/30 min; Ad5-Syn-MIF, 3.7±0.39 ml/30 min; n=10–12 rats in each group).
4. MIF depresses electrophysiological responses to ANG II in PVN neurons via a TPOR mechanism
The previously observed inhibitory actions of MIF on ANG II-induced neuronal firing in hypothalamic neurons in culture were produced by increasing the intracellular levels of MIF or MIF-(50–65), a peptide that harbors the TPOR activity of MIF. Here, the aim was to determine the effects of MIF or MIF-(50–65) on the discharge responses of identified PVN sympathetic regulatory neurons to ANG II. Whole-cell patch-clamp recordings were made from PVN neurons contained within hypothalamic slices. These neurons were retrogradely labeled with rhodamine-labeled latex microspheres from the ipsilateral rostral ventrolateral medulla (RVLM). The peak increase in neuronal discharge produced by bath-applied ANG II (1 µM) in the presence of intracellular rMIF (0.8 nM) was significantly blunted (40%) compared with control neurons (Fig. 2
A, B). In addition, the maximum increase in neuronal discharge produced by locally applied ANG II (2 pmol) was significantly reduced after 15 min of intracellular dialysis with 0.8 nM MIF-(50–65) (Fig. 2C, D
). Lastly, intracellular perfusion of 0.8 nM MIF-(50–65) significantly reduced the stimulatory effect of locally applied ANG II (2 pmol) on inward current (Fig. 2E, F
). In contrast, the control inward current response to ANG II in a separate group of neurons was unaltered after intracellular exposure to C57S/C60S-MIF-(50–65), which does not exhibit TPOR activity (Fig. 2E, F
). Collectively, these data indicate that MIF depresses the electrophysiological responses to ANG II in PVN sympathetic regulatory neurons via a TPOR mechanism.
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CONCLUSIONS AND SIGNIFICANCE
The major findings from this study are 1) ANG II increases MIF levels in the rat PVN; 2) adenoviral-mediated overexpression of MIF within rat PVN blunts the pressor and drinking actions induced by centrally administered ANG II; and 3) MIF, acting intracellularly via a TPOR-mediated mechanism, can depress the electrophysiological effects of ANG II at PVN sympathetic regulatory neurons. As such, these studies support our previous in vitro studies and demonstrate for the first time that MIF within the PVN, acting via TPOR, can operate as a novel intracellular negative regulator of the cardiovascular and body fluid regulatory actions of ANG II. These studies are significant because it appears that neuronal AT1-R are not down-regulated or desensitized via the mechanisms that are established for G protein-coupled receptors. Thus, little is known about how neuronal AT1-R-mediated responses are regulated or switched off. By identifying MIF as a negative regulator of ANG II actions in the PVN, the current studies provide evidence for a potential regulatory pathway of ANG II actions in the brain. Furthermore these studies provide the first demonstration of an intracellular enzymatic action of MIF, via its TPOR activity, in neurons.
Several important issues arise from these studies. The first concerns the time course over which MIF might act in vivo. Based on the time course of MIF expression after icv injection of ANG II (1–3 h), it is unlikely that MIF will attenuate an initial increase in neuronal activity produced by ANG II. However, it is possible that MIF provides a longer term regulation of ANG II actions. Another question is whether the actions of MIF are limited to the PVN or whether ANG II actions at other AT1-R-containing nuclei such as the RVLM and subfornical organ are also tempered by MIF. A further issue is the mechanism of action of MIF in regulating the neuronal actions of ANG II. Our data indicate that MIF is working via its intrinsic TPOR activity to exert an inhibitory action over the effects ANG II in vitro and in vivo. Since the viral mediated overexpression of MIF in SD rat neurons in culture prevents the ANG II-induced accumulation of reactive oxygen species (ROS) and the centrally mediated pressor and dipsogenic actions of ANG II depend on the availability of intracellular ROS, the inhibitory action of MIF in the brain in vivo may involve a TPOR-mediated ROS scavenging action. However, other modes of action of MIF, such as direct effects at membrane K+ and Ca2+ channels that control neuronal activity, at signaling intermediates involved in the AT1-R signaling pathway (e.g., PKC, CaMKII, and NADPH oxidase) or at AT1-R cannot be discounted at this time. Our future investigations will address these points.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5836fje
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