HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 16/6/610 01-0531fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by CHARRON, G. Articles by MOUGINOT, D. Search for Related Content PubMed PubMed Citation Articles by CHARRON, G. Articles by MOUGINOT, D.

Acute sodium deficit triggers plasticity of the brain angiotensin type 1 receptors¹

Centre de Recherche du CHUL, Unité de Neuroscience, Université Laval, Québec, G1V 4G2, Canada; and
^* Université de Montréal, Département de Pharmacologie, Québec, H3C 3J7, Canada

²Correspondence: Centre de recherche du CHUL, Unité de neuroscience, RC 9800, 2705 Blvd. Laurier, Québec, G1V 4G2, Canada. E-mail: didier.mouginot{at}crchul.ulaval.ca

SPECIFIC AIMS

The renin-angiotensin system is involved in salt appetite, a behavioral response motivated by a Na⁺ deficit in the extracellular fluid compartment. The present study aimed to demonstrate that furosemide-induced Na⁺ deficit alter the expression of type 1 angiotensin receptors (AT₁) in forebrain structures that contribute to thirst and salt appetite. Detection of hypothalamic neurons whose metabolism was activated by acute Na⁺ depletion was simultaneously undertaken in order to clarify neuronal mechanisms that contribute to detection or translation of the sensory message.

PRINCIPAL FINDINGS

1. Na⁺ depletion increased the expression of AT_1A receptor subtype in the lamina terminalis and hypothalamus
Using an in situ hybridization technique (radiolabeled riboprobe directed against the AT_1A receptor mRNA), we demonstrated that furosemide-induced Na⁺ depletion enhanced expression of the AT_1A receptor mRNA in two structures of the lamina terminalis, the subfornical organ (SFO; saline, n=15; furo, n=13) and the median preoptic nucleus (MnPO; saline, n=14; furo, n=14), as well as in the parvocellular division of the paraventricular nucleus of the hypothalamus (PVH; saline, n=16; furo, n=15; Fig. 1 , left panels). The enhanced mRNA expression was maximal 4 h after the start of Na⁺ depletion, indicating that the cellular changes were quickly initiated after the perturbation of hydromineral balance. The increased turnover of AT_1A mRNA was a long-lasting process because it was still observed under prolonged systemic stress (24 h of Na⁺ depletion mediated by furosemide treatment combined with low Na⁺ diet, 0.05% NaCl).

View larger version (107K): [in this window] [in a new window]   Figure 1. Furosemide-induced Na+ depletion increased expression of AT1 receptor in the SFO, MnPO, and PVN. Representative coronal brain sections illustrate the distribution of AT1A receptor mRNA (left panels) and AT1 binding sites (right panels). The increase in optical density reflecting the increase in the expression of AT1A receptor mRNA or in AT1 binding sites is represented by false colors (blue<green<yellow). Although a high basal level of AT1A receptor mRNA was present in the SFO, furosemide injections increased expression of AT1A receptor mRNA 4 h after the beginning of hydromineral perturbation. The constitutive level of AT1A receptor mRNA was weak in both the MnPO and the PVN. Furosemide treatment increased expression of the AT1A receptor mRNA in the ventral and dorsal regions of the MnPO as well as in the PVN. SFO, MnPO, and the PVN displayed constitutive levels of AT1 binding sites. The furosemide treatment, associated with a low Na+ diet significantly increased the number of AT1 binding sites in the same brain regions, as evaluated 12 h after the onset of the furosemide treatment. 3V: third ventricle; LV: lateral ventricles; AC: anterior commissure.

Autoradiographic binding assay carried out with I¹²⁵(Sar¹-Ile⁵-Ile⁸)-AngII performed 12 h after the onset of Na⁺ depletion showed that the enhancement of AT_1A receptor mRNA expression was associated with an increase in AT₁ binding sites in the SFO (saline, n=5; furo, n=6), MnPO (saline, n=6; furo, n=7), and PVN (saline, n=6; furo, n=6; Fig. 1 , right panels). This experiment demonstrated that Na⁺ deficit induced an up-regulation of AT_1A mRNA, followed by an enhanced transcription of AT₁ receptors in the same hypothalamic structures.

2. FRA-ir, AT_1A receptor mRNA and Na⁺ depletion.
Using an antibody directed against Fos-related antigen (FRA) proteins, we determined the number of FRA immunoreactive cells after saline and furosemide injections. Na⁺ depletion activated the metabolism in a neuronal subpopulation within the MnPO. Although AT_1A mRNA was constitutively expressed in a subpopulation of neurons in the ventral and dorsal parts of MnPO, the number of cells expressing AT_1A mRNA was not increased after furosemide treatment. Experiments carried out with a double-staining protocol (AT_1A mRNA+FRA-ir) indicated that in the MnPO, cells expressing AT_1A mRNA and those activated by acute Na⁺ depletion belong to two distinct neuronal populations (saline, n=5; furo, n=7).

Detailed analysis of the staining pattern revealed that few cells expressing AT_1A mRNA were observed in parvocellular division of the PVN (pPVN) under basal conditions (Fig. 2 A, B). Furosemide-induced Na⁺ depletion dramatically increased the number of cells that expressed AT_1A mRNA in the pPVN (Fig. 2A, B ) and the number of cells immunopositive to FRA (Fig. 2A, C ). Experiments carried out with a double-staining protocol (AT_1A mRNA+FRA ir) indicated that the number of double-stained cells in the pPVN was strongly increased after furosemide treatment (saline, n=5; furo, n=6; Fig. 2A, D ).

View larger version (54K): [in this window] [in a new window]   Figure 2. Furosemide-induced Na+ depletion increased the number of cells expressing AT1A receptor mRNA in the parvocellular division of the PVN. A) Representative photomicrographs of pPVN neurons expressing FRA proteins (orange arrow), AT1A receptor mRNA (gray arrow), or both (yellow arrow). B) Furosemide treatment increased the number of cells that expressed AT1A mRNA vs. control conditions. C) Furosemide-induced Na+ depletion simultaneously increased neuronal activity (metabolism) within the pPVN, as indicated by the significant increase in cells expressing FRA proteins. D) The distribution histogram of double-stained cells (FRA and AT1A mRNA) indicated that pPVN neurons activated by Na+ depletion (FRA positive cells) displayed a higher expression of the AT1A mRNA (dots). *P < 0.05 compared to saline, Mann-Whitney U test.

DISCUSSION

A major finding of our study was the up-regulation of AT_1A mRNA within the hypothalamus in response to acute Na⁺ deficit in the extracellular fluid compartment. The increased expression of AT_1A mRNA was maximal 4 h after the onset of Na⁺ depletion, indicating that cellular changes were quickly initiated after perturbation of the hydromineral balance. The up-regulation process was apparently a long-lasting mechanism because the turnover of AT_1A mRNA remained at a high level after a relatively long period of Na⁺ deficit (furosemide treatment combined with low Na⁺ diet for 20 h). This suggests that the expression of AT_1A mRNA is maintained at a high rate as long as there is no correction for the perturbation. Our study also indicates that the increase in expression of AT_1A mRNA in the hypothalamus was followed by functional post-transcription of enhanced mRNA production. Previous studies have showed that AT_1A mRNA expressed at the periphery could be modulated by dietary Na⁺ changes. Sodium deficiency was associated with enhanced plasma renin level and reciprocal changes in the expression of peripheral AT_1A receptor mRNA. These and our results suggest that peripheral and central AT_1A receptor gene could be differentially regulated (down- vs. up-regulation of AT_1A mRNA, respectively). At least two parameters might account for the apparent discrepancy between peripheral and central AT₁ mRNA expression. Either the nature of Na⁺ depletion (chronic vs. acute) differentially influenced expression of the AT_1A receptor mRNA, likely via dynamic changes in plasma renin, corticosteroids, and Na⁺ concentration, or the regulation of AT_1A mRNA induced by Na⁺ deficit was organ specific (brain vs. periphery). Expression of new AT₁ receptors throughout the neuronal populations of the MnPO and PVH reveals the existence of two different cellular mechanisms that are not exclusive: either Na⁺ deficit increased the turnover of constitutive brain AT₁ receptors within distinct neuronal subpopulations or Na⁺ deficit switched on AT₁ receptor gene expression in neurons that did not express AT₁ receptors under basal conditions. In the MnPO, the number of cells that expressed AT_1A receptor mRNA was stable before and after furosemide-induced Na⁺ depletion. This indicates that basal expression of the AT₁ receptor gene was enhanced in a neuronal population of the MnPO that did express AT_1A receptors under normal conditions (Fig. 3 , upper panel). In the pPVN, our results demonstrate that the number of cells that expressed AT_1A mRNA was significantly increased in Na⁺-depleted animals. However, we could not rule out the possibility that the constitutive expression of AT_1A mRNA within the pPVN was also enhanced in some neurons (Fig. 3 , lower panel).

View larger version (98K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating differential expression of AT1A receptor gene in preoptic area and paraventricular nucleus of the hypothalamus. Acute Na+ depletion mediated by furosemide injections triggered an up-regulation of constitutive AT1A receptor gene in the MnPO. Acute Na+ depletion stimulated production of AT1A receptors in parvocellular neurons (pPVN) that did not express these receptors under control conditions. Up-regulation of constitutive AT1A receptors may also take place in the pPVN.

In the MnPO and PVN, a putative role for the AT_1A receptor up-regulation in hydromineral homeostasis has to be located downstream the stimulation of circumventricular organs (CVOs) by blood-borne AngII. Indeed, cerebral AngII (synthesized within the CVOs) does act as a neuromodulator on MnPO and PVN neurons to increase their excitability. Activation of these cells and up-regulation of AT_1A receptors might be relevant for endocrine responses involved in Na⁺ intake such as synergistic action of mineralocorticoids or regulation of peptide release from magnocellular neuroendocrine cells. It was demonstrated recently that PVN cells expressing AT₁ receptors sent efferents mainly to the median eminence; thus, the excitatory action of AngII might enhance production and release of CRF in the circulation.

Experiments combining in situ hybridization technique with immunohistochemistry (ICC) were performed to localize cell populations that expressed AT_1A receptors and those stimulated by acute Na⁺ depletion. ICC data confirmed that furosemide treatment increased the metabolism of neurons in the pPVN and MnPO, indicating that neurons located in the hypothalamus were likely stimulated during acute Na⁺ depletion. Data obtained with the double-staining protocol (AT_1A mRNA and FRA-ir) gave additional information on the cellular mechanisms that underlie acute Na⁺ depletion. Our results clearly demonstrate the presence of two potential mechanisms, one for each integrative structure (MnPO and pPVN) contributing to fluid and electrolyte homeostasis. In the MnPO, we rarely observed double-stained cells after Na⁺ depletion whereas the number of these neurons was significantly enhanced in the pPVH. The weak numbers of double-labeled cells in the MnPO suggest the presence of at least two different populations of neurons recruited in response to acute Na⁺ depletion: a population of neurons displaying up-regulation of constitutive AT_1A receptors, but without expressing the FRA protein, and a metabolically activated group of neurons (FRA-ir neurons) that did not express AT_1A receptors. It is possible these two populations might underlie the existence of two distinct pathways that would be activated in response to acute Na⁺ depletion: an angiotensinergic pathway materialized by SFO projections and a metabolically activated group of neurons (FRA-ir neurons) that were likely activated by synaptic inputs, as MnPO is richly interconnected with other hypothalamic and brainstem structures involved in hydromineral balance. Results obtained in the pPVN with the double-staining protocol indicate that acute Na⁺ depletion increased the population of neurons expressing the AT_1A receptor gene. Because almost all the parvocellular neurons expressing AT_1A mRNA were also FRA immunopositive, one could postulate that FRA protein might arise from the intracellular cascade after the binding of AngII on the newly synthesized AT_1A receptors. This putative excitatory role of AngII on PVN neurons agrees with previous studies reporting that AngII stimulates excitability of parvocellular cells and modulates expression of CHR mRNA in these neurons via angiotensinergic SFO-PVN projection.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0531fje; to cite this article, use FASEB J. (February 25, 2002) 10.1096/fj.01-0531fje

This article has been cited by other articles:

				D. M. Dietz, K. S. Curtis, and R. J. Contreras Taste, Salience, and Increased NaCl Ingestion after Repeated Sodium Depletions Chem Senses, January 1, 2006; 31(1): 33 - 41. [Abstract] [Full Text] [PDF]

				S. J. Swenson, R. C. Speth, and J. P. Porter Effect of a perinatal high-salt diet on blood pressure control mechanisms in young Sprague-Dawley rats Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2004; 286(4): R764 - R770. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 16/6/610 01-0531fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by CHARRON, G. Articles by MOUGINOT, D. Search for Related Content PubMed PubMed Citation Articles by CHARRON, G. Articles by MOUGINOT, D.