Substrate-dependent regulation of MAO-A in rat mesangialcells: involvement of do - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:25 |只看该作者 |倒序浏览 |打印

作者：NathaliePizzinat, SophieMarchal-Victorion, AgnesMaurel, CatherineOrdener, GuyBompart,  AngeloParini作者单位：Institut National de la Santé et de la RechercheMédicale U38 Institut Louis Bugnard, Centre HospitalierUniversitaire Rangueil, 31403 Toulouse Cedex 0 France
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      【摘要】
   In the present study, we investigatedthe existence of a back-regulation of the catecholamine-degradingenzyme monoamine oxidase (MAO)-A by dopamine in rat renal cells. Inproximal tubule cells, MAO-A expression was not modified after dopaminereceptor stimulation. In contrast, in mesangial cells, enzyme assay and Western blots showed that MAO activity and protein increased by ~80痶er 48-h incubation with the D 2 -like receptor agonistbromocriptine and quinpirole but not with the D 1 -likereceptor agonist SKF-38393. This effect was prevented by theD 2 -receptor antagonist sulpiride and domperidone. Theincrease in MAO-A protein was preceded by an augmentation of MAO-A mRNAthat was prevented by the transcriptional inhibitor actinomycin D. Bromocriptine effect was mimicked by the PKA inhibitor H89 andinhibited by the PKA activator 8-bromo-cAMP. These results show for thefirst time the existence of a dopamine-dependent MAO-A regulationinvolving D 2 -like receptors, inhibition of the cAMP-PKApathway, and an ex novo enzyme synthesis. , F7 v+ |+ c7 X+ x8 F2 {  F
      【关键词】 dopamine receptors proximal tubule cells monoamine oxidase A hydroxytryptamine& x" u/ ^: c7 y9 i/ B6 ?
   INTRODUCTION

THE BIOGENIC AMINE DOPAMINE plays a key role in the regulation of a variety ofphysiological functions in the central nervous system ( 27 )and in peripheral organs ( 13, 33 ). Dopamine effects dependon the occupancy of specific G-coupled, seven-transmembrane domainreceptors, which were divided into two subfamilies, and on thesubsequent generation or inhibition of intracellular messengers ( 20 ). To date, the two families of dopamine receptors aredesignated as D 1 -like and D 2 -like. TheD 1 -like family is composed of two distinct receptors(D 1 and D 5 ). The D 2 -like familyincludes three distinct receptors (D 2 -D 4 ).The amount of dopamine available for receptor stimulation is one of thefactors affecting the extent of the cell effects elicited by thisamine. The tissue concentration of dopamine depends, in part, on theactivity of the amine-synthesizing or -degrading enzymes. Themitochondrial enzyme monoamine oxidases (MAOs) represent one of themajor metabolic pathways for biogenic amine degradation. These enzymesare subdivided into two major forms, A and B, encoded by two distinctgenes with an identical intron/exon structure ( 28 ) anddifferentiated on the basis of substrate specificity and inhibition bysynthetic compounds ( 37 ).$ h4 z6 Y7 Z$ @  ?

MAO-A and -B are widely distributed in renal cortex and medulla. Mostof the studies on renal MAOs concerned the proximal tubule. In thissegment of the nephron, MAO-A is the predominant isoform, and it isinvolved in regulation of the amount of dopamine and serotoninsynthesized and released by the epithelial tubular cells ( 8, 30 ). We have recently shown that hydrogen peroxide, which isproduced by MAO during dopamine degradation, may also mediate thereceptor-independent effect of dopamine. Indeed, we demonstrated thatdopamine, independently of receptor activation, induces sequential ERKactivation and renal epithelial cells proliferation that are fullydependent on hydrogen peroxide generation by MAOs ( 34, 35 ).
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MAO-A is also the predominant isoenzyme expressed in rat mesangialcells ( 24 ). These cells, which are a major constituent ofrenal glomerulus, play a critical role in the regulation of glomerularfiltration rate ( 26 ) and participate in the development offunctional and morphological glomerular abnormalities in inflammatory processes ( 9 ). In these cells, MAO-A regulates theextracellular concentrations and the proliferative effects ofserotonin, a biogenic amine involved in the development ofglomerulonephritis ( 24 ).2 H, q9 x5 f1 a& b; r, K# K; F

If the key role of MAOs in regulation of tissue concentrations andreceptor-mediated effects of dopamine has been largely demonstrated( 22, 28, 36, 37 ), the existence of a back-regulation ofMAO activity and expression by dopamine has not been investigated. Incontrast, some studies suggested that dopamine may regulate enzymesinvolved in catecholamine synthesis. Indeed, it has been shown thatdopamine agonists decrease tyrosine hydroxylase expression in ratmelanotrophs ( 21 ) and nigrostrial dopaminergic neurons ( 11 ) and rat brain DOPA-decarboxylase ( 7 ). Inaddition, it has been reported that cAMP and PKA, which areintracellular second messengers of dopamine receptors( 27 ), regulate the expression of tyrosine hydroxylase( 10, 17 ), dopamine -hydroxylase and phenylethanolamine N -methyltransferase ( 10 ), threecatecholamine-synthesizing enzymes. Although there are not directdemonstrations that dopamine could also regulate expression and/orfunction of MAO, such a possibility is supported by the followingobservations: 1 ) L -DOPA administration, whichincreases dopamine synthesis, also increases MAO activity ( 6, 18 ); 2 ) a concomitant modification of brain dopaminecontent and MAO activity is observed during ontogenesis ( 31 ) and aging ( 23, 37 ); and 3 )human MAO-A and MAO-B promoters possess a putative cAMP responsiveelement ( 39, 40 ) suggesting a potential genetranscriptional regulation by receptors coupled to adenylate cyclase.. }6 S3 c& e+ Z

On the basis of these observations, we investigated, in the presentstudy, whether dopamine receptor activation could mediate changes inMAO expression and function in proximal tubule and glomerular mesangialcells of rat kidney that contain MAO-A ( 24 ) as well asD 1 - and D 2 -like dopamine receptors ( 1, 2, 12, 29 ).' |/ c. C( Y' W3 s7 d6 ^
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Our results show for the first time that D 2 -like receptorsare responsible for the transcriptional regulation of MAO-A expression and the consequent increase in the enzyme activity. This effect wasobserved in mesangial cells but not in proximal tubule cells, indicating that dopamine-dependent MAO-A regulation may be cell specific.5 ?$ x8 D$ t" @7 B2 O

EXPERIMENTAL PROCEDURES
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Materials. RPMI 1640 (10×) and fetal calf serum were purchased from GIBCOBRL/Life Technologies (Cergy Pontoise, France). Bromocriptine mesylateand domperidone were obtained from RBI/Bioblock Scientific (Illkirch,France). Dopamine, H89 dihydrochloride, and pargyline were purchasedfrom Sigma (St. Louis, MO). SKF-38393, sulpiride, and quinpirole werepurchased from Tocris. [ 14 C]serotonin was obtained fromDuPont NEN Life Science Products (Boston, MA). Rabbit -ATPaseantibody was a generous gift from Dr. J. Lunardi (Grenoble, France).. V  n) ~" o$ Z  g$ c

Cultured mesangial cells. All animal experiments were carried out according to the principles ofLaboratory Animal Care formulated by the National Society for MedicalResearch and the Guide for the Care and Use of Laboratory Animals (Washington, DC: National Academy Press, 1996;authorization 00577, 1989, Paris, France). Rat mesangial cells werecultured from isolated renal glomeruli of male Sprague-Dawley rats (150 g body wt; Harlan-Gannat) as previously described ( 24 ). Inbrief, glomeruli were isolated by a conventional sieving method andused for primary culture in RPMI 1640 medium supplemented with 100 U/mlof penicillin/streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, and15% fetal bovine serum. Outgrowing cells were subcultured andmaintained in the same medium supplemented with 15% SVF. The cellsbetween passages 4 and 10 were used for experiments.6 K8 ?3 h3 {# J( U
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Rat proximal tubule cell culture. Rat proximal tubule epithelial cells were isolated from Sprague-Dawleyrats (40 g), as previously described ( 35 ). Briefly, kidneys were removed aseptically, decapsulated, and minced coarsely inHBSS supplemented with 10 mM HEPES and 5 mM D -glucose, pH7.4. Cortex was separated from medulla and incubated in HBSSsupplemented with 0.48 U/ml collagenase and 0.1% BSA in a flask undergentle stirring for 40 min at 37°C in a 5% CO 2 atmosphere. To separate homogeneous populations of nephron segments,the mixture of tubules was suspended in 42% Percoll that was madeisotonic with 10× concentrated Krebs-Henseleit buffer (1.18 M NaCl, 47 mM KCl, 100 mM HEPES, 200 mM cyclamic acid, 1.26 mM MgSO 4,11.4 mM KH 2 PO 4, and 50 mM glucose) and wascentrifuged (17,000 rpm, 30 min, 4°C). The F4 layer, composed ofproximal tubules, was suspended in culture medium (DMEM/Ham's F-12medium supplemented with 25 mM HEPES, 25 mM NaHCO 3, 4 mMglutamine, 20 nM sodium selenite, 10 ml/l of a 100× nonessential aminoacid mixture, 50 U/ml penicillin, 50 µg/ml streptomycin, 10 µg/mlinsulin, 5 nM transferrin, 0.1 nM dexamethasone, 10 ng/ml EGF, and 5 µg/ml triodothyronine) and plated at 6, 1, and 0.6 mg protein/150-and 60-mm Petri dish and six multiwell plates, respectively, that hadbeen coated with collagen Type I from calf skin. Fetal calf serum (5%)was added in the culture medium until the first change (2 days afterseeding). The experiments were performed at day 5.
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RNA isolation and RT-PCR. Total RNA was extracted from confluent mesangial cell culture by usingthe acid guanidinium thiocyanate-phenol-chloroform method as describedby Chomczynski and Sacchi ( 5 ).

First-strand cDNA was synthesized from 0.5 µg of total RNA by reversetranscription for 60 min at 42°C in a final volume of 20 µl of RTbuffer with 100 U of Superscript II (GIBCO BRL/Life Technologies) 0.25 µg oligo(dT) 12-18, 0.5 mM dNTPs, 5 mM DTT, and 32 U RNaseinhibitor. First strand cDNA (5 µl) was then used to amplify MAO-Aand GAPDH fragments by PCR. Reaction mix containing PCR buffer with 1.5 mM MgCl 2, 0.2 mM dNTPs, 60 nM of primers, 2 U Taq polymerase, and reverse transcription reaction wasdenatured at 93°C for 2 min 30 s, and consequently MAO-A wasamplified by 30 cycles with a DNA thermal cycler (TRIO-Thermoblock,Biometra, Göttingen, Germany). To evaluate the PCR productscomparatively, we amplified at the same time and for 20 cycles theGAPDH product. A cycle was composed of a denaturation step at 95°Cfor 1 min, a primers annealing step at 56°C, and an extension step at72°C for 1 min. The final extension step was prolonged to 10 min.
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The absence of contaminants was routinely checked by RT-PCR assays ofnegative control samples in which the Supersript was omitted.
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Primers used. Primers for MAO-A were defined by bases 1537-15565'-GTGGCTCTTCTCTGCTTTGT-3' (forward) and 2037-20165'-AGTGCCAAGGGTAGTGTGTATCA-3' (reverse); and for GAPDH by bases510-529 5'-AATGCATCCTGCACCACCAA-3' (forward) and 980-9605'-GTCATTGAGAGCAATGCCAGC-3' (reverse) ( 3 ). The expectedsize of the amplification product was 500 and 470 bp for MAO-A andGAPDH, respectively.- J# G8 H% W- \  e2 W! _6 k( k5 @

MAO activity. Mesangial cells were harvested by scraping in sodium phosphate buffer(50 mM, pH 7.5) supplemented by protease inhibitors [0.1 mMphenylmethylsulfonylfluoride, 10 µg/ml bacitracin, 2 µg/ml soybeantrypsin inhibitor (Sigma)]. Crude extracts of proteins (10-20µg) were incubated at 37°C for 20 min in sodium phosphate bufferwith 10-400 µM of [ 14 C]serotonin (AS: 52.3 mCi/mmol; NEN Life Science Products). The reaction was ended by theaddition of 0.1 ml of HCl, 4 N at 4°C. The reaction product wasextracted (efficiency 92%) with 1 ml of ethyl acetate/toluene(vol/vol), and the radioactivity contained in the organic phase wascounted in a liquid scintillation spectrometer at 97% efficiency.Nonspecific MAO-A activity was defined by activity remaining in thepresence of 10 5 M pargyline, an MAO inhibitor. Proteinswere measured according to the Lowry method (Bio-Rad) using bovinealbumin as standard.
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Western blot analysis. Crude protein extracts were solubilized in loading buffer (60 mMTris · HCl, pH 6.8, containing 2% SDS, 10% glycerol, 1% -mercaptoethanol, and 0.05% bromophenol blue) in a boiling waterbath for 5 min. Total proteins (50 µg) were loaded onto 10% SDS-PAGEgel. Resolved proteins were electrophoretically transferred to apolyvinyldiene difluoride membrane (NEN Life Science Products) bysemidry electroblotting (Trans-blot SD, Bio-Rad). Membranes wereblocked overnight at 4°C with 1% BSA (Amersham Pharmacia Biotech,Buckinghamshire, UK) dissolved in wash buffer (phosphate buffer saline,pH 7.5, and 0.1% Tween 20). The membranes were washed twice andincubated for 1 h at room temperature with rabbit polyclonalantisera to MAO-A and MAO-B ( 24 ) or -ATPase antibody.After two washes, membranes were incubated with peroxidase-labeledanti-rabbit IgG for 40 min (TEBU, Santa Cruz Biotechnology, Santa Cruz,CA). Bound antibodies were detected with enhanced chemiluminescence(Amersham Pharmacia Biotech) and exposure to Amersham Hyperfilm.
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cAMP production. The cells were incubated with D 1 - or D 2 -likedopaminergic receptor agonists for 15 min or pretreated with forskolinfor 10 min before bromocriptine addition and then washed twice with PBS and scraped. The cAMP contents were measured by using the cAMP [ 125 I] assay system (dual range) with magnetic separation(BIOTRAK, Amersham Pharmacia Biotech). The sensitivity of the assay was 2.7 pmol/ml. The cAMP values obtained were normalized with total protein contents in each dish as determined by the Lowry method.
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Statistical analysis. MAO steady-state kinetic parameters were evaluated by using nonlinearsquare curve-fitting procedures (Prism GraphPad, San Diego, CA).Results are expressed as means ± SE. The statistical significanceof difference between two experimental groups was evaluated byStudent's t -test. A P value significant.

RESULTS
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Dopamine increased MAO-A activity in rat mesangial cells viaD 2 -like dopamine receptors. MAO-A is the predominant isoform expressed in rat proximal tubule andmesangial cells ( 24 ). To define whether dopamine receptor stimulation is able to regulate MAO-A activity, we first incubated proximal tubule and mesangial cells with 10 µM dopamine, 10 µM SKF-38393 (D 1 -like agonist), and 5 µM bromocriptine(D 2 -like agonist). As shown in Fig. 1 A, incubation of proximaltubule cells with dopamine, SKF-38393, or bromocriptine for 48 hdid not modify MAO-A activity. In contrast, MAO-A activity wasincreased after incubation of mesangial cells with bromocriptine orquinpirole for 48 h (Fig. 1, A and B ). Nomodifications of the enzyme activity were observed at incubation timesshorter than 24 h, whichever D 2 -like agonists wereused. A significant increase in MAO-A activity was also observed byusing the endogenous receptor agonist dopamine. In contrast, the enzymeactivity was unaffected by cell treatment with the D 1 -likeagonist SKF-38393 (Fig. 1, A and B ). The effect of 1 µM bromocriptine was fully prevented by the preincubation ofmesangial cells with the D 2 -like receptor antagonistssulpiride and domperidone, indicating that D 2 -likereceptors are the dopamine receptor subtypes responsible for regulationof MAO-A activity (Fig. 1 C ). As shown in Fig. 2, the effect of bromocriptine was dose( A ) and time ( B ) dependent, reaching the maximumbetween 1 and 10 µM bromocriptine and 48 h, respectively.

Fig. 1. Effect of dopamine, D 1 - andD 2 -like receptor agonists and antagonists on monoamineoxidase (MAO)-A activity. A : proximal tubule and mesangialcells were treated for 48 h with dopamine (10 µM), theD 1 -like receptor agonist SKF-38393 (10 µM), or theD 2 -like receptor agonist bromocriptine (5 µM). Values arenormalized by setting basal MAO-A activity at 100% and are expressedas the mean ± SE of 4 independent experiments. B : ratmesangial cells were treated with dopamine (10 µM), theD 1 -like receptor agonist SKF-38393 (10 µM), or theD 2 -like receptor agonist bromocriptine (5 µM) andquinpirole (1 µM) for 24 and 48 h. The specific MAO-A activitywas assessed by oxidation of MAO-A substrate[ 14 C]5-hydroxytryptamine (5HT) (400 µM) in the absenceand presence of the irreversible MAO inhibitor pargyline(10 5 M). Values are expressed as the mean ± SE of 3 to 4 independent experiments. C : rat mesangial cells weretreated with 1 µM of bromocriptine in the presence or in the absenceof the D 2 -like receptor antagonists sulpiride (10 µM) ordomperidone (10 µM). Antagonists were preincubated for 15 min beforebromocriptine addition. MAO-A was determined in cell lysates bymeasuring [ 14 C]5HT oxidation after 48 h incubation.* P P$ m3 A  K. p, B9 I( d- [5 f: h
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Fig. 2. Dose and time dependency of MAO-A stimulation bybromocriptine. A : oxidation of [ 14 C]5HT in ratmesangial cell cultures was measured after 48 h of stimulationwith increasing bromocriptine concentrations. Values represent themean ± SE of 4 independent experiments. B : cells weretreated for the indicated time periods with 5 µM of bromocriptine andoxidation of 400 µM [ 14 C]5HT was measured in celllysates. Values were normalized by setting basal MAO-A activity at100% and are expressed as the mean ± SE of 4 independentexperiments. * P1 ~1 E5 D8 |6 j# Q; i& N8 X1 q6 D
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To determine whether the increase in MAO-A activity by bromocriptinewas related to a change in the amount of MAO-A and/or to a modificationof its affinity for the substrate, we performed velocity vs.substrate concentration experiments in treated and untreated mesangialcells. As shown in Fig. 3 A,incubation of mesangial cells with bromocriptine for 48 hincreased the V max of [ 14 C]5HToxidation (control cells: V max 99 ± 12 pmol · min 1 · mg 1 ofprotein; bromocriptine-treated cells: V max 215 ± 15 pmol · min 1 · mg 1 ofprotein, n = 3, p of the K m (controlcells: K m 55 ± 12 µM;bromocriptine-treated cells: K m 68 ± 7 µM). These results suggest that the augmentation of MAO-A bybromocriptine is related to the increase in the number of the enzymemolecules. This possibility was further supported by Western blotanalysis showing that the intensity of the immunoreactive bandcorresponding to MAO-A was stronger in lysates from bromocriptine thanin those from untreated cells (Fig. 3 B ). As we have reported above for the regulation of the enzyme activity (Fig. 1 ), the effect ofbromocriptine was mimicked by the endogenous amine dopamine.( \. ~. {  `9 ]5 |8 Y

Fig. 3. Effect of bromocriptine on MAO-A activity and proteinexpression in mesangial cells. A : enzyme assays wereperformed in cell lysates from mesangial cells treated or not treatedwith 5 µM bromocriptine for 48 h. V max and K m of MAO-A for [ 14 C]5HToxidation were determined for control cells and bromocriptine-treatedcells. Values are representative of 3 independent experiments. B : Western blot analyses were performed on cell lysates frommesangial cells untreated or treated with bromocriptine (5 µM) ordopamine (10 µM) for 48 h. Crude extract (50 µg) was loadedonto gels for electrophoresis and processed as described in EXPERIMENTAL PROCEDURES. The histogram represents the ratioof optical densities corresponding to MAO-A and -ATPase bands. Thefigure is representative of 4 independent experiments.* P
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D 2 -like dopamine receptors induced MAO-A mRNAexpression. To determine whether modifications in MAO-A activity by bromocriptinewere related to changes of mRNA turnover, we measured the expression ofMAO-A mRNA by semiquantitative RT-PCR. Mesangial cell incubation withbromocriptine (5 µM) for 24 h led to a significant increase inthe intensity of the RT-PCR product corresponding to MAO-A (Fig. 4 ). This effect was not maintained at48-h incubation, when the mRNA level of MAO-A returned to the controlvalue. To investigate whether the effect of bromocriptine involved amodification of mRNA synthesis and/or degradation, we determined theinfluence of the transcriptional inhibitor actinomycin D. As shown inFig. 5, incubation of mesangial cellswith actinomycin D did not modify the intensity of the MAO-A band attimes up to 24 h. This indicates that as previously reported forthe GADPH mRNA ( 15 ), which we used here as the referenceRT-PCR product, MAO-A mRNA belongs to the "long half-life" mRNAs.At all times tested, bromocriptine, in the presence of actinomycin D,did not affect the expression of MAO-A RT-PCR product, indicating thatthis D 2 -like receptor agonist does not modify mRNAdegradation. Compared with the results presented in Fig. 4, the datashowed in Fig. 5 reveal that actinomycin D prevented the increase inMAO-A expression by bromocriptine observed at 24 h. Theseresults indicate that bromocriptine increases MAO-A expression by atranscriptional mechanism. This possibility is further supported by thedemonstration that actinomycin D also prevented thebromocriptine-dependent increase in MAO-A activity (Fig. 5 B ).
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Fig. 4. Effect of bromocriptine on MAO-A mRNA expression inmesangial cells. Semiquantitative RT-PCR was performed as described in EXPERIMENTAL PROCEDURES from isolated total RNA ofbromocriptine untreated or treated cells for 24 or 48 h. Therelative expression of MAO-A mRNA was determined as the ratio ofoptical densities of MAO-A and GAPDH bands. The histogram isrepresentative of the average of 3 independent experiments.* P& x4 m, v& V9 q- p) s, b# C: F
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Fig. 5. Effect of actinomycin D on MAO-A mRNA and activityinduced by bromocriptine. A : inhibition of RNA synthesis wasachieved by exposing cell cultures to actinomycin D (10 µg/ml) at time 0 in the presence or absence of 5 µM bromocriptine.Total RNA from treated mesangial cells were submitted to RT-PCR, andthe amplified products corresponding to MAO-A and GAPDH were revealedby ethidium bromide staining. Graphs represent the ratio ofdensitometric values (mean ± SE) of MAO-A and GAPDH bands of 3 independent experiments. B : cells were treated withbromocriptine (5 µM) in presence or absence of actinomycin D (10 µg/ml). After 48 h, MAO-A activity was evaluated by measuring[ 14 C]5HT (400 µM) oxidation. Values are represented asmean ± SE of 4 independent experiments. * P; l7 {2 L2 h- w5 o
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Stimulation of MAO-A activity by bromocriptine was linked to aninhibition of adenylyl cyclase activity. In mesangial cells, previous studies have reported a stimulation ofcAMP generation by D 1 -like dopamine receptors, whereas thesecond messengers mediating the effects of D 2 -likereceptors have not been described. In a first series of experiments, we investigated the role of D 2 -like receptors on cAMPgeneration, the most widespread signal transduction pathway linked tothese receptors ( 32 ). Incubation of mesangial cells withbromocriptine strongly inhibited forskolin-stimulated generation ofcAMP, indicating that as previously reported for others tissues,D 2 -like receptors are linked to an inhibition of cAMPproduction (Fig. 6 ). To determine whethera decrease in intracellular cAMP is involved in the pathway mediatingthe D 2 -like receptor-dependent MAO-A regulation, we testedthe effect of the PKA inhibitor H89 on MAO-A activity. As shown in Fig. 7, H89 increased MAO-A activity to anextent similar to that observed with bromocriptine. In contrast,8-bromo-cAMP, a cell-permeable and stable analog of endogenouslygenerated cAMP, suppressed the bromocriptine-stimulated mesangial cellMAO-A activity. These results suggest that inhibition of cAMPgeneration and a consequent decrease in PKA activity are involved inthe regulation of MAO-A expression by D 2 -like receptors.
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Fig. 6. Inhibition of forskolin-stimulated cAMP by bromocriptine.Cells were pretreated or not with 2 µM forskolin and then wereincubated with bromocriptine (1, 5, or 10 µM) or 10 µM SKF-38393for 15 min before collecting cell lysates. The results were normalizedby setting basal cAMP levels at 100% and represent the mean ± SEof 4-7 independent experiments done in duplicate. ## P P P6 `% [0 p, g' D& u' e* a

Fig. 7. Effect of 8-bromo-cAMP (8-BrcAMP) and H89 on MAO-Aactivity. Mesangial cells were treated with the PKA inhibitor H89 orthe cell-permeable cAMP analog 8-BrcAMP in the presence or absence ofbromocriptine (5 µM) for 48 h. MAO-A activity was measured oncrude cell extracts and expressed as the percentage over basalactivity. Values are expressed as mean ± SE of 3 independentexperiments. # P P8 P. ]; g5 d4 U+ [- R

DISCUSSION6 J8 V7 Y. S5 \& C6 ^
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MAO expression and activity are regulated in various situations,including development ( 3, 14, 16 ), aging( 25 ), and pathologies ( 28, 37 ). In some ofthese cases, changes in MAO activity depends on the hormonalenvironment. Several studies have identified steroid hormones asfactors contributing to the hormonal MAO regulation ( 4, 19, 38 ). As far as we know, to date, the potential role of hormonesacting on seven-transmembrane domain receptors, and particularly ofcatecholamine receptors, in regulation of MAO expression has not been investigated., {( c- x7 c4 _: E

In the present study, we show that an MAO substrate, dopamine, canupregulate MAO-A in rat mesangial cells. These cells contain D 1 -like receptors positively linked to adenylate cyclase( 29 ), and we have shown that they also expressD 2 -like receptors responsible for inhibition of cAMPgeneration. Our results indicate that D 2 - but notD 1 -like receptors are responsible for MAO-A regulation. Indeed, we showed that MAO-A activity was increased by theD 2 -like receptor agonists bromocriptine and quinpirole andthis effect was fully prevented by the D 2 -like receptorantagonists sulpiride and domperidone. The fact that bromocriptineeffect was mimicked by quinpirole, a preferential D 3 agonist, suggests that dopamine-mediated regulation of MAO-A expressioninvolved this D 2 -like receptor subtype. This hypothesis issupported by radioligand binding and autoradiography studies, whichhave shown the expression of D 3 receptors in glomerularmesangial cells ( 2 ). In contrast, MAO-A activity wasunaffected by the D 1 -like receptor agonist SKF-38393. According to the intracellular messenger linked to two dopamine receptor subtypes, we found that the effect of D 2 -likereceptor stimulation was mimicked by the PKA inhibitor H89 but not by8-bromo-cAMP, an activator of the cAMP/PKA pathway. The effect ofD 2 -like receptor stimulation on MAO-A activity wasundetectable after 24 h and reached the maximum after 48 h,suggesting that MAO-A regulation may require an ex novo enzymesynthesis. This possibility was confirmed by enzyme assays and Westernblots that showed a concomitant increase in V max of [ 14 C]5HT oxidation and in the detection of the MAO-Aimmunoreactive band. The increase in MAO-A activity was preceded by asignificant augmentation of the MAO-A mRNA that was maximal after24 h and returned to the control values after 48 h. Theeffects on MAO-A mRNA and activity were fully prevented by actinomycinD, indicating that D 2 -like receptor stimulation increasesthe transcription of the MAO-A gene. It is interesting to note thatregulation of MAO-A expression was not observed in proximal tubulesthat as mesangial cells express D 1 - and D 2 -likereceptors. This suggests that MAO-A regulation by D 2 -likereceptors is not a generalized phenomenon but could be rather dependenton the cell type. Although the expression of D 2 -likereceptors in glomerular mesangial cells has been provided byradioligand binding and autoradiography studies ( 2 ), theirintracellular messengers as well as their functional properties werestill not defined. Our recent studies have shown that these receptorsare not involved in mesangial cell contraction or proliferation(Pizzinat N, Marchal-Victorion S, Maurel A, and Parini A, unpublishedobservations). In contrast, the demonstration thatD 2 -like receptors regulate MAO-A expression supplies the first evidence for a functional activity of these receptors in mesangial cells. It is conceivable that in vivo, long-term regulation of MAO-A expression by dopamine may participate in the control ofbiogenic amine availability and effects in glomeruli as well as in theregulation of the amount of hydrogen peroxide generated by MAO-A duringsubstrate degradation.
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If this is the first demonstration that dopamine D 2 -likereceptors are involved in regulation of an enzyme responsible for catecholamine degradation, another report showed that these receptors modulate the expression of the catecholamine-synthesizing enzyme tyrosine hydroxylase ( 21 ). That study, performed in ratmelanotroph, demonstrated that D 2 -like receptor stimulationinduced an effect on tyrosine hydroxylase expression that was oppositeto that observed for MAO-A in mesangial cells. However, even ifopposite, the effects of D 2 -like receptors on MAO-A andtyrosine hydroxylase expression share some common features,particularly the requirement of long incubation times and the ex novoenzyme synthesis. In addition, regulation of both MAO-A and tyrosinehydroxylase seems to depend on the decrease in the basal level ofintracellular cAMP. Indeed, we found that the increase in MAO-Aexpression by bromocriptine was mimicked by the PKA inhibitor H89 andwas prevented by the PKA activator 8-BrcAMP. Concerning tyrosinehydroxylase, it has been shown that as observed afterD 2 -like receptor stimulation in rat melanotroph( 21 ), H89 induced a decrease in the enzyme activity inbovine chromaffin cells ( 10 ). Interestingly, the decreasein basal intracellular cAMP also reduced the expression of two othercatecholamine-synthesizing enzymes, dopamine -hydroxylase andphenylethanolamine N -methyltransferase ( 10 ).Taken together, these results suggest that basal cAMP levels maycontribute to maintain the equilibrium between the expression ofcatecholamine-synthesizing and -degrading enzymes and, consequently,the normal tissue levels of catecholamines. It is conceivable thattonic stimulation of dopaminergic D 2 -like receptors may beone of the factors controlling the steady-state intracellular cAMPconcentration and the basal activity of the catecholamine synthetic andmetabolic pathway.' n: z# }2 \1 ~  G# L. X

In conclusion, this work revealed a previously unknown mechanism ofregulation of MAO-A expression by one of its substrates, dopamine.These results could be the first step for the further characterizationof the relationships between dopamine, their synthesizing and degradingsystems not only in kidney but also in other organs in which this amineplays a physiological or pathological role.
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ACKNOWLEDGEMENTS, o, W0 R" R9 y3 o% H

The authors wish to thank Jacqueline Mechi and MarieHélène Seguelas for assistance with the cell culture.9 s$ H8 p, F9 H  O# X8 X
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