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Rodriguez, Elvira Fernandez and Jose M. Valdivielso Eva Parisi, Yolanda Almadén, Mercé Ibarz, Sara Panizo, Anna Cardús, Maria

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Rodriguez, Elvira Fernandez and Jose M. Valdivielso Eva Parisi, Yolanda Almadén, Mercé Ibarz, Sara Panizo, Anna Cardús, Maria Eva Parisi, Yolanda Almadén, Mercé Ibarz, Sara Panizo, Anna Cardús, Mariano Rodriguez, Elvira Fernandez and Jose M. Valdivielso Am J Physiol Renal Physiol 296:1291-1296, 2009. First published Apr 8, 2009; doi:10.1152/ajprenal.90557.2008 You might find this additional information useful... This article cites 25 articles, 12 of which you can access free at: http://ajprenal.physiology.org/cgi/content/full/296/6/F1291#BIBL Updated information and services including high-resolution figures, can be found at: http://ajprenal.physiology.org/cgi/content/full/296/6/F1291 Additional material and information about AJP - Renal Physiology can be found at: http://www.the-aps.org/publications/ajprenal This information is current as of June 2, 2009 . Downloaded from ajprenal.physiology.org on June 2, 2009 AJP - Renal Physiology publishes original manuscripts on a broad range of subjects relating to the kidney, urinary tract, and their respective cells and vasculature, as well as to the control of body fluid volume and composition. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2005 by the American Physiological Society. ISSN: 0363-6127, ESSN: 1522-1466. Visit our website at http://www.the-aps.org/. Am J Physiol Renal Physiol 296: F1291–F1296, 2009. First published April 8, 2009; doi:10.1152/ajprenal.90557.2008. N-methyl-D-aspartate receptors are expressed in rat parathyroid gland and regulate PTH secretion Eva Parisi,1 Yolanda Almade´n,3 Merce´ Ibarz,4 Sara Panizo,1 Anna Cardu´s,1 Mariano Rodriguez,3 Elvira Fernandez,2* and Jose M. Valdivielso1* 1Research Laboratory, 2Nephrology Department, and 4Biochemistry Department Hospital Universitari Arnau de Vilanova, IRBLLEIDA, Lleida, Spain; and 3Unidad de Investigacio´n and Nephrology Department, Hospital Universitario Reina Sofia, Co´rdoba, Spain Submitted 17 September 2008; accepted in final form 5 April 2009 Parisi E, Almade´n Y, Ibarz M, Panizo S, Cardu´s A, Rodriguez biologically active mediators which suppressed PTH secretion M, Fernandez E, Valdivielso JM. N-methyl-D-aspartate receptors (23). This pathway can be modulated by an increase in intra- are expressed in rat parathyroid gland and regulate PTH secretion. cellular calcium that produces an activation of cPLA2 followed Am J Physiol Renal Physiol 296: F1291–F1296, 2009. First published by a release of AA (1), confirming the important role of free April 8, 2009; doi:10.1152/ajprenal.90557.2008.—N-methyl-D-aspar- intracellular calcium as a mediator of the inhibition of PTH tate receptors (NMDAR) are tetrameric amino acid receptors which secretion. act as membrane calcium channels. The presence of the receptor has Downloaded from been detected in the principal organs responsible for calcium ho- When serum calcium levels decrease, PTH is released and meostasis (kidney and bone), pointing to a possible role in mineral acts on its target organs, mainly kidney and bone, to bring back metabolism. In the present work, the presence of the receptor was calcium levels to normal. Hypocalcemia and the following determined in normal parathyroid glands (PTG) by real-time PCR, PTH release stimulate calcitriol production which contributes immunoprecipitation, and immunohistrochemistry. Healthy animals to the restoration of normocalcemia. Calcitriol also regulates showed a decrease in blood parathyroid hormone (PTH) levels 15 min PTH secretion through the vitamin D receptor (VDR) in the after the treatment with NMDA. This effect was also observed in PTG. Patients with chronic renal disease with persistent reduc- ajprenal.physiology.org animals with high levels of PTH-induced EDTA injection, but not in tions in serum calcitriol and calcium show an increase in PTH uremic animals with secondary hyperparathyroidism (2HPT). Normal synthesis and release. Continued stimulus of the PTG produces rat PTG incubated in media with low calcium concentration (0.8 mM hyperplasia and hypertrophy causing secondary hyperparathy- CaCl2) showed a decrease in PTH release when NMDA was added to the media. This effect of NMDA was abolished when glands were roidism (2HPT). In these hypertrophic PTG, the expression of coincubated with MK801 (a pharmacological blocker of the NMDA CaSR and VDR is downregulated (5), accounting for the lack channel) or PD98059 (an inhibitor of the ERK-MAPK pathway). of effect of Ca and vitamin D on severe 2HPT. Glands obtained from animals with 2HPT showed no effect of NMDA N-methyl-D-aspartate receptors (NMDAR) are tetrameric in the in vitro release of PTH, together with a decrease in the amino acid receptors which act as membrane calcium channels. expression of NMDAR1. In conclusion, NMDA receptor is present in The receptor has two subunits: R1 with the catalytic function on June 2, 2009 PTG and is involved in the regulation of the PTH release. The and R2 with regulatory properties. There are four types of the mechanism by which NMDAR exerts its function is through the subunit R2 (A, B, C, D) and their presence is different depend- activation of the MAPK cascade. In uremic 2HPT animals the recep- ing on the distribution, properties, and regulation of the recep- tor expression is downregulated and the treatment with NMDA does not affect PTH secretion. tor. The receptor is gated by the binding of L-glutamate and its cofactor L-glycine, allowing calcium to enter the cell. In secondary hyperparathyroidism; ERK-MAPK addition, there is a binding site for polyamines which can act as allosteric activators (16). The receptor has been well-described in the nervous system THE PARATHYROID GLANDS (PTG) play an essential role in min- where the entry of calcium produces nitric oxide (NO) synthase eral ion homeostasis because of their capacity to recognize and activation and NO production. However, little is known about respond to small changes in the extracellular ionized calcium NMDAR in other tissues. Recently, the presence of this recep- concentration. Calcium changes and parathyroid hormone tor has been shown in the kidney (6) where it plays a role in the (PTH) secretion are detected and regulated, respectively, by the maintenance of basal arterial tone and in the bone (3, 18) where cell surface calcium-sensing receptor (CaSR). The activation it contributes to the local regulation of bone cell function. The of CaSR by extracellular calcium results in a G -mediated q/11 presence of the receptor in the principal organs responsible for activation of phosphatidylinositol-phospholipase C (PI-PLC), calcium homeostasis led us to think that the NMDAR could be leading to intracellular calcium mobilization, protein kinase C involved in its regulation and also be present in the PTG. (PKC) activation, and a resulting PKC-mediated stimulation of In the present work, we show for the first time the presence the mitogen-activating protein kinase (MAPK) cascade. Acti- of the NMDAR in the PTG and its effect in the regulation of vated MAPK then phosphorylates and activates cPLA , which 2 PTH secretion. releases free arachidonic acid (AA) that is metabolized to MATERIALS AND METHODS * E. Fernandez and J. M. Valdivielso share senior authorship. Address for reprint requests and other correspondence: J. M. Valdivielso, Animals and samples. Experimental methods used on laboratory Laboratorio de Investigacio´n HUAV-UDL, Hospital Universitari Arnau de Vil- animals comply with the Law 5/1995, of June 21, by “Generalitat de anova, Rovira Roure 80, 25198 Lleida, Spain (e-mail: valdivielso@medicina. Catalunya” of protection of animals used for experimentation and udl.es). other scientific finalities and the Royal Decree 1201/2005, of October http://www.ajprenal.org 0363-6127/09 $8.00 Copyright © 2009 the American Physiological Society F1291 F1292 N-METHYL-D-ASPARTATE RECEPTORS IN RAT PARATHYROID GLAND 10, about the protection of animals used for experimentation and other horseradish peroxidase-conjugated secondary antibody (1:12,500; scientific finalities. Moreover, the present work was approved by the Amersham Biosciences) was added for an extra hour. Binding was Ethic Animal Experimentation Committee of the University of Lleida. detected with the ECL Advance Western Blotting Detection Kit The study was performed with 10 Sprague-Dawley rats (200–250 (Amersham Biosciences) and visualized at the VersaDoc Imaging g) per group maintained under standard conditions. Food and water system model 4000 (Bio-Rad Laboratories, GmbH, Mu¨nchen, Ger- were available ad libitum. Room temperature was at 21°C with a many). 12:12-h dark-light cycle. Immunohistochemistry. PTG were fixed with 4% paraformalde- The 5/6 nephrectomized animals were obtained following the hyde, included in paraffin, and sliced in 4-␮m sections. After being method performed by Perez-Ruiz et al. (19): animals were anesthe- blocked, sections were incubated with primary antibody for tized with isoflurane and suffered 2/3 nephrectomy in the left kidney NMDAR1 diluted in PBS 1:50 (overnight at 4°C), washed in PBS, by ligation of both poles. After 1 wk, contralateral nephrectomy was and incubated with biotinylated anti-mouse IgG (Vectastain ABC kit, performed. Vector Labs) diluted 1:250, for1hatroom temperature, and then in Animals with high levels of PTH were obtained by EDTA-induced Vectastain ABC reagent diluted 1:250 for 1 h. Tissue-bound peroxi- hypocalcemia performed following a modified protocol described by dase was visualized using 0.05% of 3,3Ј-diaminobenzidine and Thomas et al. (24): rats received one intramuscular injection of EDTA 0.003% hydrogen peroxide in 0.1 M Tris⅐HCl buffer (pH 7.6) for (300 mg/kg) which resulted in6hofsustained hypocalcemia. 5–10 min, under visual control. To obtain parathyroid tissue, on the day of the experiment, animals In vivo experiments. For the in vivo experiments, blood samples (controls and 5/6 nephrectomized) were killed with pentobarbital were obtained before and 15 and 60 min after intraperitoneal injection sodium (50 mg/kg) and PTG were dissected free of the thyroid glands with NMDA (10 mg/kg).
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