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 , 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 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 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. and the following determined in normal parathyroid (PTG) by real-time PCR, PTH release stimulate production which contributes immunoprecipitation, and immunohistrochemistry. Healthy animals to the restoration of normocalcemia. Calcitriol also regulates showed a decrease in blood parathyroid (PTH) levels 15 min PTH secretion through the 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 (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 THE PARATHYROID GLANDS (PTG) play an essential role in min- where the entry of calcium produces nitric oxide (NO) synthase eral ion 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 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 glands with NMDA (10 mg/kg). After learning that the peak of inhibition was with a dissecting microscope. at 15 min in the animals with 2HPT and EDTA-induced hypocalce-

PTG incubation conditions. PTG were placed in individual wells, mia, we only assessed basal and 15 min. Downloaded from containing 1 ml of incubation medium, resting inside a nylon basket; Analytical determinations. PTH concentration in the PTG incuba- the glands were maintained at 37°C, with constant rocking and tion medium and in animal serum was measured using a Rat Intact shaking motions (1). The incubation medium contained 125 mM PTH Elisa Kit (Immunotopics, San Clemente, CA). NaCl, 5.9 mM KCl, 0.5 mM MgCl2, 1 mM sodium pyruvate, 4 mM Blood levels of total calcium were analyzed by the Biochemical glutamine, 10 ␮M glycine, 12 mM glucose, 25 mM HEPES, 0.1 IU/ml Unit of the Hospital Universitari Arnau de Vilanova de Lleida in the human , 0.1% bovine serum albumin, 100 IU/ml penicillin/ Roche/Hitachi modular analytic system. Statistical analysis. Differences between single groups were as- streptomycin, and 1 mM phosphate was obtained by adding NaH2PO4 ajprenal.physiology.org and Na2HPO4 in 1:2 proportion and finally the medium was buffered sessed by paired Student’s t-test. Multiple groups were analyzed by at pH 7.4. First, the glands were stabilized in 1.25 mM calcium, the ANOVA followed by Dunnett’s post hoc test. A P Ͻ 0.05 was media were collected, and PTH levels were measured and used as a considered statistically significant. basal levels of PTH. To promote PTH release, the following 3-h incubation media contained low Ca concentration (0.8 mM). In RESULTS addition to low calcium (used as control), PTG were incubated with A low calcium ϩ NMDA (500 ␮M), low calcium ϩ NMDA ϩ MK801 In Fig. 1 , we show the expression of all the NMDAR (100 nM; receptor antagonist), and low calcium ϩ NMDA ϩ subunits, R1, R2A, R2B, R2C, and R2D, in the PTG, compared PD98059 (10 ␮M; inhibitor for the MAPK-ERK kinase or MEK). The with levels present in kidney (taken as reference) assessed by media were changed every hour and collected for the subsequent RT-PCR. The main subunits present in PTG are NMDAR1 and on June 2, 2009 analysis. At the end of the experiment, some glands were used to NMDAR2A. determine pre-pro-PTH expression by real-time PCR analysis. Real-time PCR. Total RNA was extracted from tissue using the TRIzol method. Reverse transcription was performed with First Strand cDNA Synthesis Kit for RT-PCR (AMV; Roche) followed by a Taqman real-time PCR amplification with -specific primers for NMDAR or pre-pro-PTH, purchased specifically at Applied Biosys- tems (Branchburg, NJ) and human GAPDH as a reference. Forty cycles at 95°C for 15 s and 60°C for 1 min were performed with an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Triplicate readings were taken and the average was calculated. The results are in relationship to a control sample randomly selected that we consider as value ϭ 1. Immunoprecipitation. Protein extracts from parathyroid tissue were obtained as described previously. We performed immunoprecipitation because the low level of the NMDAR protein in the PTG precludes us from detecting a band in a normal Western blot. We immunoprecipi- tated 300 ␮g of parathyroid tissue and 60 ␮g of brain tissue (used as a positive control). After preclearing with G protein sepharose balls, protein extract was incubated overnight with 1 ␮g of specific antibody (Affinity Bioreagents, Golden, CO) and G protein sepharose balls (Amersham Biosciences, Uppsala, Sweden) at 4°C on rocking plat- form. After incubation, the mixture was placed 5 min at 95°C to Fig. 1. A: real-time PCR for N-methyl-D-aspartate receptor (NMDAR) sub- separate the protein from the sepharose balls. We loaded the whole units in the parathyroid glands (PTG; open bars) compared with renal tissue (filled bars). We detected the presence of all the subunits (especially R1 and immunoprecipitate in an 8% polyacrylamide-SDS gel. After running R2A). Data are means Ϯ SE. B: immunoprecipitation and Western blot for and transfer to PDVF membranes (Immobilon-P, Millipore, Bedford, NMDAR1 in the PTG and brain tissue. Three hundred micrograms of total MA), blots were incubated in 3% nonfat milk in TBST for 1 h. Then, protein extract from normal PTG or 60 ␮g of brain tissue were immunopre- primary antibody for NMDAR1 (1:100) was added and incubation cipitated with NMDAR1-specific antibody. Total immunoprecipitates were run was performed overnight at 4°C. After being washed with TBST, in a Western blot and probed with NMDAR1 antibody.

AJP-Renal Physiol • VOL 296 • JUNE 2009 • www.ajprenal.org N-METHYL-D-ASPARTATE RECEPTORS IN RAT PARATHYROID GLAND F1293 Moreover, we also detected the presence of the NMDAR1 protein by immunoprecipitation techniques (Fig. 1B) using brain tissue as a positive control. Immunohistochemistry analysis using specific antibody showed that most of the cells in the PTG are positive for NMDAR1 (see Fig. 5A). To investigate the effect of NMDAR activation on PTH release, we first used an ex vivo model. In Fig. 2A we can see that rat PTG incubated with low Ca (0.8 mM CaCl2) showed an increase in PTH release that was inhibited when NMDA was added to the incubation media. Moreover, in Fig. 2C we show that the effect of NMDA is dose dependent. This inhibitory

Fig. 3. PTH levels in the incubation media of glands obtained from normal animals and stimulated with low Ca (black solid lines), low calcium ϩ NMDA (500 ␮M; gray solid lines), low calcium ϩ NMDA (500 ␮M) ϩ PD98059 (10 ␮M; black discontinuous lines), and low calcium ϩ NMDA (500 ␮M) ϩ MK801 (100 nM; gray discontinuous lines). Values are percentage over the Ϯ Ͻ

basal (1.25 mM Ca) SE. *P 0.05 vs. glands incubated in low Ca media. Downloaded from

effect was abolished by coincubation with a NMDAR antago- nist (MK801), suggesting that the effect observed is mediated by an increase in Ca influx through the activated channel (Fig. 3). Furthermore, the inhibition mediated by NMDA was also abol-

ished by a pharmacological inhibitor of the MEK (PD98059), ajprenal.physiology.org suggesting that the MAPK pathway is involved in the NMDA- mediated inhibition of PTH secretion (Fig. 3). The incubation with only MK801 or PD98059 did not modify PTH secretion [0.8 mM CaCl2 ϭ 277.98% over the basal; MK801 ϭ 243.11%; PD98059 ϭ 221.81%; P ϭ not significant (n.s.)]. Real-time PCR of the glands at the end of the incubation period showed that the effect of NMDAR activation on PTH secretion was not mediated by a decrease in pre-pro-PTH gene expression (Fig. 2B). Table 1 shows the effect of a single intraperitoneal adminis- on June 2, 2009 tration of NMDA (10 mg/kg) on blood PTH, Ca, and phosphorus levels in different animal models. In normal animals, PTH levels showed a significant decrease 15 min after the administration of the drug. Sixty minutes after the administration of NMDA, PTH levels returned back to normal. Those changes on PTH levels were achieved with no significant changes in blood total calcium. We repeated the same approach with animals with 2HPT. Glands obtained from animals with 2HPT did not respond to incubation with NMDA (Fig. 4A). Furthermore, 2HPT animals injected with NMDA did not show decreases in blood PTH levels after 15 min (Table 1) contrary to what was observed in normal animals. When we analyzed NMDAR expression in the

Table 1. Biochemical parameters of normal, EDTA, and 2HPT rats before (basal) and 15 or 60 min after administration of 10 mg/kg NMDA

Fig. 2. A: PTG from normal animals were extracted and incubated in media Normal (n ϭ 10) 2HPT (n ϭ 7) EDTA (n ϭ 10) with low calcium (0.8 mM calcium; black lines) to stimulate parathyroid Ј Ј Ј Ј hormone (PTH) release. Another group of PTG was incubated with low Basal 15 60 Basal 15 Basal 15 ␮ calcium plus NMDA (500 M; gray lines). Values are presented as percentage PTH, pg/ml 180.5 115.75* 212.78 329.64* 323.58* 486.93* 314.06† Ϯ Ͻ over the basal (1.25 mM Ca) SE. *P 0.05 vs. glands incubated in control Ca, mg/dl 10.42 10.24 10.85 n.d. n.d. 9.63* 9.6* media. B: pre-pro-PTH mRNA levels in glands obtained from normal animals P, mg/dl 5.81 5.24 6.51 n.d. n.d. 7.96 7.56 and incubated in low Ca media (control) or low Ca media with 500 ␮M NMDA (NMDA). Data are means Ϯ SE. C: dose response to increasing EDTA, EDTA-induced hypocalcemia; 2HPT, secondary hyperparathyroid- concentrations of NMDA. Data are represented as percentage of reduction with ism; NMDA, N-methyl-D-aspartate; PTH, parathyroid hormone; n.d., not respect to low Ca media alone (0.8 mM Ca). Data are means Ϯ SE. *P Ͻ 0.05 determined; P, phosphorus. *P Ͻ 0.05 vs. normal basal. †P Ͻ 0.05 vs. EDTA vs. glands incubated in control media. basal.

AJP-Renal Physiol • VOL 296 • JUNE 2009 • www.ajprenal.org F1294 N-METHYL-D-ASPARTATE RECEPTORS IN RAT PARATHYROID GLAND be implicated in mineral metabolism regulation. In this context, our results demonstrate for the first time that PTG express NMDAR. Functional NMDAR require the presence of both NMDA R1 and NMDA R2 subunits. In our experiments, we identify mRNA for both NMDA R1 and all the subunits of NMDA R2 (mainly NMDA R2A) in parathyroid tissue, sug- gesting the presence of fully functional NMDAR and a new role for neuroexcitatory amino acids in PTG function. In the present work, functionality of the NMDAR in PTG has been proved both ex vivo and in vivo. In PTG incubated in low calcium, the addition of NMDA blunted the increase in PTH secretion. Furthermore, this effect could be blocked by the addition of MK801, a specific antagonist of NMDAR suggesting that the intracellular Ca increase induced by NMDAR activation inhibits PTH secretion. Our results agree with previously published reports, which described that acti- vation of Ca channels in the PTG can inhibit PTH release (7, 8, 17). In addition, raising intracellular calcium with thapsi-

gargin also blocks PTH release (1, 22). The mechanism of Downloaded from blocking PTH secretion by increases in intracellular Ca has been extensively studied. Kifor et al. (13) showed that in- creases in intracellular Ca elicited a rapid phosphorylation of Fig. 4. A: PTH levels in the incubation media of glands obtained from animals ERK1/2. Thus, activated ERK1/2 can phosphorylate its cyto- with secondary hyperparathyroidism (2HPT) and stimulated with low Ca (0.8 solic protein substrates [e.g., phospholipase A2 (PLA2)] (10, mM). Black lines: glands incubated in low Ca media. Gray lines: glands 14), resulting in increased production of AA which has been incubated in low Ca media plus NMDA (500 ␮M). Data are means Ϯ SE. directly implicated in the regulation of PTH release (1, 2). ajprenal.physiology.org B: percentage of decrease in mRNA levels of the different NMDAR subunits from PTG of animals with 2HPT with respect to PTG of normal animals. Data Thus, the inhibition of ERK1/2 activity has been proven to are means Ϯ SE. inhibit the activation of ERK1/2 induced by increases in intracellular Ca (13) and therefore, the decrease in PTH secre- glands obtained from 2HPT rats, we found that mRNA amount of most of the NMDAR subunits was significantly decreased with respect to the glands of normal animals (Fig. 4B). Only levels of NMDAR2D were increased, but basal expression of that particular subunit was very low, suggesting that changes in on June 2, 2009 its expression are not likely to play a significant role in receptor functionality. Immunohistochemistry for NMDAR1 also showed a decrease in staining in glands obtained from animals with 2HPT (Fig. 5B). In a third approach, we administered NMDA to rats with high levels of PTH but without 2HPT. Thus, animals with EDTA-induced hypocalcemia showed a decrease in blood PTH levels 15 min after the administration of NMDA (Table 1). This decrease in PTH levels was obtained even after the persistence of EDTA-induced hypocalcemia.

DISCUSSION NMDAR is a potent calcium channel of the central nervous system gated by the binding of glutamate and glycine (16). Glutamate is one of the most common amino acids found in nature and is the main component of many proteins and . The role of NMDAR has been widely studied in the central nervous system, and its presence has been detected in other tissues like , , , heart, aorta, and ileum (11, 15, 20, 21, 25). Recent studies described the presence of NMDAR in the kidney (6) where it plays a role in the maintenance of basal arterial tone and in the bone where it stimulates (3, 18). Thus, the presence and role of this receptor outside the nervous system are a new field to study. Fig. 5. Immunostaining of parathyroid tissue for NMDAR1 showing the The presence of NMDAR in organs that control calcium presence of the receptor in parathyroid cells of normal animals (A) or animals homeostasis (bone and kidney) indicates that the receptor could with 2HPT (B).

AJP-Renal Physiol • VOL 296 • JUNE 2009 • www.ajprenal.org N-METHYL-D-ASPARTATE RECEPTORS IN RAT PARATHYROID GLAND F1295 tion (4). Indeed, our results show that the decrease in PTH ACKNOWLEDGMENTS release induced by NMDA can be blocked by addition of The authors thank A. Martinez, P. Valcheva, M. Bozic, and M. Freixenet PD98059, an ERK1/2 phosphorylation inhibitor, pointing to a (IRB Lleida) for help and cooperation in the laboratory. role of MAPK in the effect of NMDAR activation in PTH GRANTS secretion, probably mediated by an increase in intracellular Ca after activation of the channel. Furthermore, the decrease of This work was supported by Fundacio´ Dr. Pifarre´ grants, FIS PI07/0427 and REDINREN (16/06). J. M. Valdivielso holds a contract from the Miguel Servet PTH release was not due to a decline in its synthesis because program. pre-pro-PTH expression was not inhibited, but to an inhibition of its release. REFERENCES The role of NMDAR on the regulation of PTH release was 1. Almaden Y, Canalejo A, Ballesteros E, Anon G, Canadillas S, Rodri- also demonstrated in our rat model in vivo, where animals were guez M. Regulation of arachidonic acid production by intracellular cal- treated with NMDA. Administration of NMDA to normal cium in parathyroid cells: effect of extracellular phosphate. JAmSoc Nephrol 13: 693–698, 2002. animals resulted in a decrease in blood PTH levels at 15 min, 2. Bourdeau A, Moutahir M, Souberbielle JC, Bonnet P, Herviaux P, Sachs which returned to basal levels at 60 min. Furthermore, all the C, Lieberherr M. Effects of lipoxygenase products of arachidonate metabo- changes in PTH levels were observed without changes in blood lism on parathyroid hormone secretion. 135: 1109–1112, 1994. calcium levels, suggesting again a direct effect of NMDA on 3. Chenu C, Serre CM, Raynal C, Burt-Pichat B, Delmas PD. Glutamate receptors are expressed by bone cells and are involved in bone resorption. the PTG. Bone 22: 295–299, 1998. Renal patients often suffer extrarenal complications that 4. Corbetta S, Lania A, Filopanti M, Vicentini L, Ballare E, Spada A. aggravate their overall health condition. One of them is 2HPT, Mitogen-activated protein kinase cascade in human normal and tumoral Downloaded from which is characterized by high turnover bone disease and the parathyroid cells. J Clin Endocrinol Metab 87: 2201–2205, 2002. 5. Cunningham J. Achieving therapeutic targets in the treatment of second- continuous secretion of excess PTH and driven by a decrease ary hyperparathyroidism. Nephrol Dial Transplant 19: 9–14, 2004. in Ca and calcitriol resulting from decreases in kidney paren- 6. Deng AH, Valdivielso JM, Munger KA, Blantz RC, Thomson SC. chyma (5). In later stages of 2HPT, the levels of CaSR and Vasodilatory N-methyl-D-aspartate receptors are constitutively expressed VDR in the PTG decrease, rendering the gland unresponsive to in rat kidney. J Am Soc Nephrol 13: 1381–1384, 2002. Ca and calcitriol modulation (9, 12). In our laboratory, we use 7. Fitzpatrick LA, Brandi ML, Aurbach GD. Control of PTH secretion is mediated through calcium channels and is blocked by pertussis toxin treatment ajprenal.physiology.org the 5/6 nephrectomized rats as a model to study 2HPT. These of parathyroid cells. Biochem Biophys Res Commun 138: 960–965, 1986. animals have only 1/6 of renal tissue and develop 2HPT in 8. Fitzpatrick LA, Yasumoto T, Aurbach GD. Inhibition of parathyroid several months. Contrary to the results obtained in the control hormone release by maitotoxin, a calcium channel activator. Endocrinol- group, when we treated uremic rats with NMDA, as well as ogy 124: 97–103, 1989. 9. Fukuda N, Tanaka H, Tominaga Y, Fukagawa M, Kurokawa K, Seino when we incubated the PTG of these animals with it, we did Y. Decreased 1,25-dihydroxyvitamin D3 receptor density is associated not see effects on PTH release. This difference could be with a more severe form of parathyroid hyperplasia in chronic uremic explained by the fact that the PTG obtained from animals with patients. J Clin Invest 92: 1436–1443, 1993. 2HPT showed a marked decrease in the expression of both of 10. Hazan I, Dana R, Granot Y, Levy R. Cytosolic phospholipase A2 and its

mode of activation in human neutrophils by opsonized zymosan. Corre- on June 2, 2009 the NMDAR subunits that are mainly expressed in the gland. lation between 42/44 kDa mitogen-activated protein kinase, cytosolic These results suggest that NMDAR show a similar behavior to phospholipase A2 and NADPH oxidase. Biochem J 326: 867–876, 1997. VDR and CaSR, which are also found to be downregulated in 11. Inagaki N, Kuromi H, Gonoi T, Okamoto Y, Ishida H, Seino Y, 2HPT. Thus, the gland could be unresponsive to NMDA Kaneko T, Iwanaga T, Seino S. Expression and role of ionotropic glutamate receptors in pancreatic islet cells. FASEB J 9: 686–691, 1995. treatment, like it is to Ca or calcitriol. 12. Kifor O, Diaz R, Butters R, Brown EM. The Ca2ϩ-sensing receptor In a final approach, we tried to demonstrate whether the (CaR) activates phospholipases C, A2, and D in bovine parathyroid and activation of NMDAR could reduce PTH secretion in a model CaR-transfected, human embryonic kidney (HEK293) cells. J Bone Miner of increased PTH release other than 2HPT. We chose a model Res 12: 715–725, 1997. 13. Kifor O, MacLeod RJ, Diaz R, Bai M, Yamaguchi T, Yao T, Kifor I, in which we can render the animal hypocalcemic by injection Brown EM. Regulation of MAP kinase by calcium-sensing receptor in of EDTA. Thus, the EDTA-induced hypocalcemia will stimu- bovine parathyroid and CaR-transfected HEK293 cells. Am J Physiol late an increase in PTH release by a decrease of the signaling Renal Physiol 280: F291–F302, 2001. through the CaSR. In this case, and contrary to the 2HPT, the 14. Leslie CC. Properties and regulation of cytosolic phospholipase A2. J Biol Chem 272: 16709–16712, 1997. gland does not show hypertrophy and conserves the same level 15. Leung JC, Travis BR, Verlander JW, Sandhu SK, Yang SG, Zea AH, of receptors. Treatment of these animals with NMDA also Weiner ID, Silverstein DM. Expression and developmental regulation of produced a decrease in PTH levels, even after the persistence the NMDA receptor subunits in the kidney and cardiovascular system. of hypercalcemia, proving that NMDAR activation can mod- Am J Physiol Regul Integr Comp Physiol 283: R964–R971, 2002. 16. Mori H, Mishina M. Structure and function of the NMDA receptor ulate PTH secretion independently of Ca levels and thus, of channel. Neuropharmacology 34: 1219–1237, 1995. CaSR activation. 17. Nemeth EF, Kosz LM. Adenine nucleotides mobilize cellular Ca2ϩ and In conclusion, our results demonstrate that the NMDAR inhibit parathyroid hormone secretion. Am J Physiol Endocrinol Metab are present in PTG and take part in the regulation of the PTG 257: E505–E513, 1989. function, inhibiting PTH release. The mechanism by which 18. Patton AJ, Genever PG, Birch MA, Suva LJ, Skerry TM. Expression of an N-methyl-D-aspartate-type receptor by human and rat and NMDAR exert their function is through allowing the en- suggests a novel glutamate signaling pathway in bone. Bone trance of calcium in the parathyroid cell with the following 22: 645–649, 1998. activation of the PLC and MAPK cascade. In uremic con- 19. Perez-Ruiz L, Ros-Lopez S, Cardus A, Fernandez E, Valdivielso JM. A ditions, the receptor expression is downregulated and con- forgotten method to induce experimental chronic renal failure in the rat by ligation of the renal parenchyma. Nephron Exp Nephrol 103: E126–E130, 2006. sistently the treatment with NMDA does not affect PTH 20. Said SI, Berisha HI, Pakbaz H. Excitotoxicity in the lung: N-methyl-D- secretion. aspartate-induced, nitric oxide-dependent, pulmonary edema is attenuated

AJP-Renal Physiol • VOL 296 • JUNE 2009 • www.ajprenal.org F1296 N-METHYL-D-ASPARTATE RECEPTORS IN RAT PARATHYROID GLAND

by vasoactive intestinal and by inhibitors of poly(ADP-ribose) 23. Silver J, Kilav R, Naveh-Many T. Mechanisms of secondary hyper- polymerase. Proc Natl Acad Sci USA 93: 4688–4692, 1996. parathyroidism. Am J Physiol Renal Physiol 283: F367–F376, 21. Shannon HE, Sawyer BD. Glutamate receptors of the N-methyl-D- 2002. aspartate subtype in the myenteric plexus of the guinea pig ileum. 24. Thomas S, Movsowitz C, Epstein S, Jowell P, Ismail F. The response of J Pharmacol Exp Ther 251: 518–523, 1989. circulating parameters of metabolism to ethanol- and EDTA- 22. Shoback D, Chen TH, Pratt S, Lattyak B. Thapsigargin stimulates induced hypocalcemia in the rat. Bone Miner 8: 1–6, 1990. intracellular calcium mobilization and inhibits parathyroid hormone re- 25. Yoneda Y, Ogita K. Localization of [3H]glutamate binding sites in rat lease. J Bone Miner Res 10: 743–750, 1995. adrenal medulla. Brain Res 383: 387–391, 1986. Downloaded from ajprenal.physiology.org on June 2, 2009

AJP-Renal Physiol • VOL 296 • JUNE 2009 • www.ajprenal.org