Expression and Localization of Human Dopamine D2 and D4 ...

0013-7227/01/$03.00/0 Printed in U.S.A.

The Journal of Clinical Endocrinology & Metabolism 86(9):4460 – 4467 Copyright © 2001 by The Endocrine Society

Expression and Localization of Human Dopamine D2 and D4 Receptor mRNA in the Adrenal Gland, Aldosterone-Producing Adenoma, and Pheochromocytoma KWAN-DUN WU, YUNG-MING CHEN, TZONG-SHINN CHU, SHIH-CHIEH CHUEH, MING-HSIOW WU, AND HSIEH BOR-SHEN Departments of Internal Medicine (K.-D.W., Y.-M.C., T.-S.C., M.-H.W., H.B.-S.) and Urology (S.-C.C.), National Taiwan University Hospital, Taipei 100, Taiwan Aldosterone secretion is evidently regulated by a dopaminergic inhibitory mechanism. Pharmacological characterization and autoradiographic studies revealed D2-like receptors in the adrenal cortex, especially in the zona glomerulosa. However, the subtype of the dopamine receptors involving this regulation has not been elucidated. To investigate which subtype of receptors expresses in the adrenal cortex, we examined the messages of D2-like receptors, D2, D3, and D4, by RT-PCR and in situ hybridization of adrenal glands and adrenal neoplasm. Both D2 and D4 receptors were expressed in normal adrenal glands, pheochromocytoma, and aldosteroneproducing adenoma. However, the D2 receptors were not universally expressed, in contrast with the D4 receptors that were detected in all cases of aldosterone-producing adenoma and adrenal remnant. No D3 receptor message was detected by RT-PCR in any adrenal sample. Both D2 and D4 receptors were expressed in significant amounts in the adrenal medulla and pheochromocytoma. In the adrenal cortex, the expression of the D2 receptors was in the zona glomerulosa and zona reticularis, with no different signal intensities between the two zones. D4 receptors were mainly localized in the zona

T

HERE IS EVIDENCE that aldosterone secretion is subjected to a dopaminergic inhibitory mechanism (1– 4). In several animal species as well as in humans, administration of dopaminergic antagonists, such as metoclopramide (MCP), causes a rise in plasma aldosterone levels, but not plasma cortisol levels (2, 5–7). Although there is disagreement over the mechanism of MCP-induced aldosterone secretion (8), dopamine-binding sites in the adrenal zona glomerulosa (ZG) have been well recognized (9 –12). Additionally, a substantial amount of dopamine (DA) has been found around the ZG (13, 14). The adrenocortical dopamine is suggested to be uptaken and released by local noradrenergic endings limited to the area around the ZG (14). Therefore, dopaminergic modulation of steroid secretion and/or biosynthesis seems limited to the ZG. As MCP also increases plasma aldosterone concentration in patients with aldosterone-producing adenoma (APA), we speculate Abbreviations: A-II, Angiotensin II; APA, aldosterone-producing adenoma; DA, dopamine; DR, dopamine receptor; MCP, metoclopramide; poly(A)⫹, polyadenylated; ZF, zona fasciculata; ZG, zona glomerulosa; ZR, zona reticularis.

glomerulosa and, to a lesser extent, in the zona reticularis. Both receptors were expressed at low levels in the zona fasciculata. In aldosterone-producing adenoma, the expression of D2 and D4 was especially found in nonzona fasciculata-like cells. To elucidate which dopamine receptor regulates aldosterone secretion, the effects of specific D2 and D4 antagonists, raclopride and clozapine, respectively, were examined in cultured NCI-H295 cells. Dopamine further increased angiotensin II-induced aldosterone secretion by 20%. In the presence of 1 ␮M dopamine and angiotensin II, 10ⴚ5–10ⴚ7 M clozapine decreased aldosterone levels by 40 –55%. The decrease in aldosterone secretion by clozapine was completely reversed when raclopride was added simultaneously. These data suggest that dopamine exerts dual effects on aldosterone secretion in NCI-H295 cells. Activation of D4 receptors can increase aldosterone secretion, whereas an inhibitory effect is mediated via D2 receptors. In summary, we demonstrated the existence of both D2 and D4 receptors in the human adrenal gland and adrenal neoplasm. Both receptors play significant roles in the modulation of aldosterone secretion, but in opposite directions. (J Clin Endocrinol Metab 86: 4460 – 4467, 2001)

that DA receptor (DR), which regulates aldosterone secretion, is present in APA (15). Based on pharmacological and molecular biological studies, there are five subtypes of human DR, D1–D5 (16). The subtype of DR regulating aldosterone secretion is speculated to be D2 or D2-like receptors (17–20). However, at the molecular level, the presence of this DR has not been proven. The mRNA of the D2 receptors has been detected in adrenal medullae and pheochromocytoma (21–23), but has not been observed in the adrenal cortex. Different characteristics of D2-like receptors between the adrenal cortex and adrenal medulla had been proposed (24, 25). Autoradiography showed that DR in the adrenal cortex, mainly in the ZG, possessed the highest affinity to clozapine (12, 26), which binds to D4 receptors with an affinity 10 times higher than to D2 and D3 receptors (27). In speculation of the D4 receptors expressing in the adrenal cortex, the transcript of the D4 receptors was not demonstrated in rat adrenal glands, however (28). In contrast, D4 receptor mRNA was observed in mouse adrenal gland (29), but the localization was not studied. Accordingly, the subtypes of DR expressing in adrenal cortex may be species dependent.

4460

Wu et al. • Dopamine Receptors in the Adrenal Gland

The Journal of Clinical Endocrinology & Metabolism, September 2001, 86(9):4460 – 4467 4461

To investigate which subtype of human DR expresses in the adrenal cortex, we used RT-PCR and in situ hybridization to examine the expression of human D2-like receptors, i.e. D2, D3, and D4, in the adrenal gland and adrenal neoplasm. The roles of these receptors in the regulation of aldosterone secretion were also examined pharmacologically in cultured adrenocortical carcinoma cells. Materials and Methods A human D4 receptor cDNA clone was donated by Dr. Mitsuyuki Matsumoto (Ibaraki, Japan). The cDNA contains two tandem repeats in exon 3. The probes used in this study were derived from this clone.

Isolation of polyadenylated [poly(A)⫹] RNA and RT-PCR amplification Poly(A)⫹ RNA was isolated from APA (six cases) and the remnant adrenal (three cases), pheochromocytoma (two cases), normal adrenal glands from radical nephrectomy of renal cell carcinoma (three cases), human kidney, and human brain tissue (from CLONTECH Laboratories, Inc., Palo Alto, CA). Five micrograms of poly(A)⫹ RNA from each sample were reverse transcribed by Moloney murine leukemia virus reverse transcriptase as described previously (15). The RT mixture was finally diluted to 100 ␮l. The primers for amplification of human D2 receptor were 5⬘-GGTCTACATCAAGATCTACATTGTCCTCC-3⬘ and 5⬘-TGGCGAGCATCTGAGTGGCTTTCTTCTCC-3⬘, corresponding to nucleotides 654 – 682 and 1161–1133, respectively. The primers amplify the short and long splicing variants, 420 and 507 bp, respectively. No product will be amplified from genomic DNA because the primers flank a large intron (13 kb). The primers for amplification of human D3 receptor were 5⬘CAGAATCTATGTGGTGCTGA-3⬘ (nucleotides 627– 646) and 5⬘-GGGAGAAGAAGGCAACCCAA-3⬘ (nucleotides 968 –987). The names and sequences of primers for human D4 receptor, M1 and M3, were identical to those reported by Matsumoto et al. (30): M1 (nucleotides 204 –223), 5⬘-CACCAACTCCTTCATCGTGA-3⬘; and M3 (nucleotides 602–583), 5⬘-AAGGAGCACACGGACGAGTA-3⬘. For examination of tandem repeat polymorphism of D4 receptor, D4I3a and D4I3b primers were used. The corresponding nucleotides were 5⬘-CTCTACTGGGCCACGTTCCGCGGCCTGC-3⬘ (nucleotides 634 – 661) and 5⬘-CCCTCATGGCCTTGCGCTCCCGGCCGGT-3⬘ (nucleotides 919 –946). The amplified products will be 313 and 409 bp for twoand four-repeat variants, respectively. The conditions for PCR amplification were as follows. Three microliters of the diluted RT mixture or 1 pg human D4 receptor cDNA clone were added in the presence of 10 pmol primers, 200 ␮m deoxy-NTPs, 1 mm MgCl2, 2.5 U Taq DNA polymerase (Life Technologies, Inc., Gaithersburg, MD), and 1 ⫻ pfx DNA polymerase buffer with 1 ⫻ Enhancer Solution (Life Technologies, Inc.). After denaturation at 96 C for 3 min, 35 cycles of amplification (96 C for 30 sec, 60 C for 30 sec, and 72 C for 1 min) were performed.

Southern blots Digoxigenin-labeled cDNA probes were synthesized with Klenow enzyme according to the manufacturer’s protocol. The probe for the D2 receptor was the long fragment from RT-PCR described above. The probe for the D4 receptor was the fragment (nucleotides 234 –538) derived from the human D4 receptor clone digested by NotI. The products of PCR were electrophoresed in 5% polyacrylamide gel of 0.5 ⫻ TBE and then transferred to positively charged nylon membrane (Roche Molecular Biochemicals, Indianapolis, IN) electrically. The membrane was denatured in 0.4 n NaOH for 10 min, followed by rinsing in 2 ⫻ SSCP for 5 min and UV cross-linking. Hybridization with digoxigenin-labeled cDNA probe was performed according to the manufacturer’s protocol (Roche Molecular Biochemicals). The signal was detected using alkaline phosphatase-conjugated antibody and disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2⬘-(5⬘chloro)tricyclo[3,3,1,1,3,7]decan}-4-yl)phenyl phosphate.

Synthesis of cRNA probes The antisense and sense digoxigenin-labeled cRNA probes for in situ hybridization of human D2 and D4 receptors were synthesized with RNA polymerase from the corresponding clones. For the D4 receptor, a fragment (663–1081 nucleotides) digested by HincII and PstI was ligated to KS-Bluescript plasmid (Stratagene, La Jolla, CA); for the D2 receptor, the PCR product amplified by primers 5⬘-AATGGGCATGCCAAAGACCA-3⬘ (nucleotides 1147–1166) and 5⬘-CATCCTGAACATACACTGTG-3⬘ (nucleotides 1344 –1363) was ligated to pGEM-T (Promega Corp., Madison, WI). Riboprobes used for in situ hybridization were filtered through Chroma Spin-100 columns (CLONTECH Laboratories, Inc.) to separate unincorporated digoxigenin-11-UTPs from digoxigenin-11-UTP-labeled riboprobes.

In situ hybridization The procedures were modified from previous methods (31). Eightmicron cryosections of 4% paraformaldehyde-immersed tissues were cut and mounted on siliconized glass slides. Sections were then permeabilized with proteinase K at 1 ␮g/ml for 20 min at room temperature. After permeabilization, sections were treated sequentially with 0.2% Triton-100 and 0.2 m HCl and acetylated with 0.1 m triethanolamine containing 0.25% acetic anhydride for 10 min at each step at room temperature. Sections were subsequently incubated with prehybridization buffer containing 4 ⫻ SSC (standard saline citrate) with 50% formamide at 48 C for at least 1 h. After prehybridization, sections were incubated with hybridization buffers containing 50% formamide, 10% dextran sulfate, 1 ⫻ Denhardt’s solution, 4 ⫻ SSC, 1 mg/ml sperm DNA, and 7 ␮g/ml digoxigeninlabeled RNA probe at 48 C overnight in a humid chamber. The sections were then washed three times for 10 min each time with 0.1 ⫻ SSC at 50 C and subjected to immunological detection. Blocking was performed with 10% skim milk for 30 min at room temperature, followed by antidigitoxin antibody conjugated with alkaline phosphatase for 1 h at room temperature. The sections are visualized with the enzyme substrate containing 4-nitro blue tetrazolium chloride and 5-bromo-4chloro-3-indolyl-phosphate 4-toluidine and were counterstained with methylene blue.

Cell culture Human NCI-H295 adrenocortical carcinoma cells were purchased from American Type Culture Collection (Manassas, VA). The conditions of cell culture followed the procedures described by Bird et al. (32). Briefly, cells were maintained in DMEM-Ham’s F-12 medium supplemented with insulin (1 ␮g/ml), transferrin (1 ␮g/ml), selenium (1 ng/ ml), linoleic acid (1 ␮g/ml) and antibiotics (100 U/ml penicillin and 100 ␮g/ml streptomycin) and were grown on six-well microplates at 37 C under an atmosphere of 5% CO2-95% air. Each well was loaded with 2 ⫻ 106 cells. After 48 h, the medium was changed to serum-free medium, and the cells were cultured for another 24 h before experiments. Angiotensin II (A-II), dopamine, clozapine and S(⫹)-raclopride were purchased from Sigma (St. Louis, MO).

Measurements of protein and aldosterone The cells were washed and solubilized in 50 mm Tris-HCl (pH 7.4) containing 0.25% deoxycholate, 1% IGEPAL CA-630 (Sigma), 1 mm EDTA, 0.5 mm MgCl2, 1 mm phenylmethylsulfonylfluoride, 1 ␮g/ml leupeptin, 1 ␮g/ml pepstatin, and 1 g/ml aprotinin. The protein content was determined using a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). The concentration of aldosterone was measured by RIA with commercial kits (Aldosterone Maia Kit, Biochem Immunosystems, Bologna, Italy) as described previously (15). The concentrations of aldosterone were expressed as the mean ⫾ sem (nanograms per dl/mg protein). Statistical analysis was performed with paired and unpaired t tests, using the StatView software package (Abacus Concepte, Inc., Berkeley, CA). Statistical significance was recognized at the 5% level.

4462

The Journal of Clinical Endocrinology & Metabolism, September 2001, 86(9):4460 – 4467


Results Detection of D2 and D4 receptors by RT-PCR

With M1/M3 primers, a product with the expected size of 399 bp was observed when a human D4 receptor cDNA clone was used as a template. This fragment was also detected in normal adrenal gland, brain tissue, kidney, APA, and pheochromocytoma (Fig. 1). We examined the specificity of this amplification by digesting the PCR products with BglI. Two fragments with expected sizes of 131 and 267 bp were observed (Fig. 1). Therefore, the human D4 receptors were expressed in cells derived from the adrenal cortex and adrenal medullae. There was no signal observed for the human D3 receptor of all adrenal samples (data not shown). Two fragments were amplified by the D2 primers, which indicated the long and short splicing variants of the D2 receptor. The short variant was of much less abundance in all samples. The expression of the D2 receptor message was observed in the human brain, normal adrenal gland, APA, and pheochromocytoma, but not in the kidney. The expression of D2 in APA was not universal, however. Figure 2 shows the results of RT-PCR of the D2 and D4 receptors in all samples of APA and three remnant adrenal glands. The D4 receptors were detected in all samples. In contrast, the amounts of D2 receptor mRNA were more various in either the APA or remnant adrenal glands. Only two cases of APA expressed the D2 receptors with much weaker signals compared with those in their respective remnant adrenals. Polymorphism of the D4 receptor was observed in APA. Three cases of APA were homozygotes of four-tandem repeat alleles (D4(4/4)) and the other three cases were heterozygotes of two- and four-tandem repeat alleles (D4(2/4)). As both two- and four-tandem repeat alleles were expressed in heterozygotes, the D4 receptor polymorphism is codominant.

FIG. 1. Southern blots of the products of RT-PCR amplified with the primers of D4 and D2 receptors. The products of RT-PCR by primers D4 receptor (upper panel) was digested with BglI into two fragments, 267 and 132 bp (middle panel). The two fragments in the D2 receptor are the short and long splicing variants (lower panel). Lane 1, The template is 1 pg cDNA of the human D4 receptor; lane 2, no template; lane 3, brain; lanes 4 and 6, remnant adrenal glands; lanes 5, 7, and 8, APA; lane 9, pheochromocytoma; lane 10, kidney.

FIG. 2. RT-PCR amplified with primers of D2 (upper panel) and D4 (lower panel) receptors of six cases of APA and their remnant adrenal glands (three cases). In D4 receptor, two- and four-tandem repeat variants are of 310 and 410 bp, respectively. Lane 1, The 100-bp DNA marker; lanes 2, 4, 6, 8, 9, and 10, APA; lanes 3, 5, and 7, the adrenal remnant of APA from cases of lanes 2, 4, and 6, respectively.

In situ hybridization

To investigate the localization of D2 and D4 receptors in the adrenal gland, we performed in situ hybridization. The expression of the D4 receptor mRNA in the adrenal cortex was significant (Fig. 3a), although the signals appeared later than those in the medulla (Fig. 3c). The receptors were mainly expressed in the ZG (Fig. 3a). The D4 receptors were also expressed, in a lesser amount, in the zona reticularis (ZR). Although D2 receptors were observed in ZG, the signals in this layer were not more significant than those in the ZR (Fig. 3d). Most of the zona fasciculata (ZF) cells had low signals of both receptors. However, some clustered cells with scanty cytoplasma in ZF expressed both receptors (Fig. 3, b and e). The adrenal medullae expressed both D2 and D4 receptors abundantly (Fig. 3, c and f). More than 50% of medullar cells expressed these DR. Accordingly, significant portions of cells expressed both DR. The cells expressing these receptors were evenly distributed in the medulla. Because of universal expression of the D4 receptors compared with that the D2 receptors, only the results of in situ hybridization with the D4 receptors in pheochromocytoma and APA are shown in Fig. 4. In the previous report we have described different histological patterns of APA (15). Three cases with different histology are demonstrated in Fig. 4, a– c; Fig. 4d is the result of hybridization with D4 sense probe in the same case of Fig. 4a. The percentages of ZF-like cells in these three cases were 55%, 30%, and 65%, respectively. The ZF-like cells were not evenly distributed in tumors. Not all tumor cells of APA expressed D4 receptors. The messages were mainly detected in the ZG-like cells (Fig. 4, a and c) and hybrid cells (Fig. 4, b and c). In contrast, signal was hardly detected in ZF-like cells. In those APA cases, in which D2 receptors were detected by RT-PCR, in situ hybridization showed a similar expression to that of D4 receptors, i.e. expression of D2 receptors also in non-ZF-like cells (data not shown). Pheochromocytoma expressed both D2 and D4 receptors. As observed in normal adrenal medulla, more than 50% of the tumor cells expressed both D2 and D4 receptors. However, the tumor cells expressed less than normal medullar cells (Fig. 4, e and f, vs. Fig. 3, c and f).



FIG. 3. In situ hybridization of normal adrenal gland with human D4 (a– c) and D2 (d–f) DR riboprobes. a, c, d, and f are from the same individual; b and e are from another two subjects, respectively. Arrows in b and e indicate cells clustering in ZF have expression of both receptors. The brown-purple color in the cytoplasma is a positive signal of mRNA. The slides were counterstained with methylene blue. Md, Medulla. Magnification, ⫻115.

Regulation of aldosterone secretion by DA via D2 and D4 receptors

H259R cells expressed both human D2 and D4 receptors (Fig. 5A). The D4 receptor expressing in this cell line is a homozygote of the four-tandem repeat allele. Two specific D2 and D4 antagonists, raclopride and clozapine, respectively, were tested to elucidate which DR plays a more significant role in the modulation of aldosterone secretion. After adding A-II (1 ␮m) for 24 h, the secretion of aldosterone increased significantly by 5-fold (0.82 ⫾ 0.07 vs.4.49 ⫾ 0.45 ng/dl䡠mg protein; n ⫽ 6; P ⬍ 0.001; Fig. 5B). There was no effect of DA alone on aldosterone secretion (data not shown). However, the elevation of aldosterone secretion by A-II was further increased by 20 –30% when 10⫺4–10⫺7 m DA was added (Fig. 5B). The elevation of aldosterone secretion by DA was not different among these concentrations. This augmentation of aldosterone secretion by DA was abolished by clozapine. In the presence of 1 ␮m A-II and DA, clozapine (10⫺5–10⫺7 m) decreased aldosterone levels by 40 –55% compared with A-II alone (Fig. 6). The reduction tended to be dose dependent, although no significant difference in the reduction was observed among these concentrations of clozapine. The inhibition was absent when the concentration of clozapine was 10⫺8 m. Addition of 10⫺5 m raclopride in the presence of 1 ␮m A-II and DA resulted in a 20% lower aldosterone level than that

stimulated by 1 ␮m A-II alone (P ⬍ 0.05; n ⫽ 5; Fig. 6). At higher concentrations of raclopride (10⫺6–10⫺8 m), aldosterone levels were greater than that stimulated by A-II alone, by 6%, 19%, and 29%, respectively (Fig. 6). However, the increased magnitude was only significant at the concentration of 10⫺8 m, but was not different from that induced by 1 ␮m A-II and DA. The increments in aldosterone secretion produced by raclopride were significantly augmented when 1 ␮m clozapine was added simultaneously. The increments were 15%, 55%, 77%, and 90%, respectively, in the presence of 10⫺5–10⫺8 m raclopride (Fig. 6). Neither clozapine nor raclopride alone had an effect on aldosterone secretion. Discussion

In pharmacological and autoradiographic studies, D2-like receptors in the adrenal cortex have been well demonstrated (10 –12, 18). However, the subtype of DR has not been determined at the molecular level. Pharmacological characterization suggested that the D4 receptors were the predominant subtype of D2-like receptors expressing in the adrenal cortex. Amenta et al. (12) demonstrated that clozapine, a D4-specific antagonist, was the most powerful displacer of spiroperidol. Their result cannot, however, exclude the presence of other D2-like receptors in the adrenal cortex. In the present study we demonstrated that both D2 and D4 receptors are expressed in the adrenal cortex as well as human

4464



FIG. 4. In situ hybridization with D4 receptor riboprobe in APA (a– d) and pheochromocytoma (e and f). Three cases of APA with different histologies are shown in a– c, with 55%, 30%, and 65% of ZF-like cells, respectively. d, Hybridization with D4 sense probe in the same case in a. D4 receptor mRNA detected in ZG-like cells (arrow, a and c), and hybrid cells (double arrows, b and c), but not in ZF-like cells (arrowhead). Magnification: a– e, ⫻115; f, ⫻ 225.

FIG. 5. Expression of D2 and D4 receptor mRNA in NCI-H295 adrenocortical carcinoma cells was examined by RT-PCR (A). Lanes 1 and 2, Amplified with D4 primers; lanes 3 and 4, amplified with D2 primers (see text); lanes 1 and 3, no template added. M, The 100-bp marker. The H295 cells expressed D4 receptors of four tandem repeats (409 bp; lane 2) and both the short and the long-splicing variants of D2 receptor (420 and 507 bp, respectively; lane 4). Addition of 1 ␮M A-II increased aldosterone secretion from these cells by 5-fold over the basal level (䡺; B). Addition of DA (o; 10⫺4–10⫺7 M) further increased aldosterone levels. Data represent the mean and SEM from five independent experiments. *, P ⬍ 0.01 vs. basal levels; †, P ⬍ 0.05 vs. A-II alone (u).

NCI-H295 adrenocortical carcinoma cells, which secrete aldosterone. In situ hybridization showed that D4 receptors were more specifically expressed in ZG, with a lesser extent in the ZR and even much less in the ZF. This distribution pattern of D4 receptors is compatible with the observation in autoradio-

graphic study (12). It has been shown that a substantial proportion of enzymes responsible for DA metabolism are present around ZG (14). The predominant localization of D4 receptors in ZG, therefore, indicates that D4 receptors may play an important role in the regulation of aldosterone secretion. In addition to D4, D2 receptors are also present in the



FIG. 6. The changes in aldosterone levels produced by different concentrations (10⫺5–10⫺8 M) of clozapine (Cloz; u) or raclopride (Raclo; p) in the presence of 1 ␮M A-II and DA (䡺). The aldosterone increments were calculated using the levels stimulated by 1 ␮M A-II alone as the baseline. f, Increments when raclopride (10⫺5–10⫺8 M) and 1 ␮M clozapine were added simultaneously. Data are the mean and SEM from five independent experiments. *, P ⬍ 0.01; †, P ⬍ 0.05 (vs. A-II alone).

adrenal cortex. Both ZG and ZR expressed D2 receptors, with not much differential expression between these two zones. Our previous study showed that the increments in the plasma aldosterone concentration by MCP in patients with APA correlated positively with the percentage of non-ZF-like cells of adenoma (15). We speculate that non-ZF-like cells of APA express more D2-like receptors than ZF-like cells and are, therefore, inhibited to a greater extent by DA. This speculation is supported by the present findings that both D2 and D4 receptors in APA are mainly expressed in non-ZF-like cells. However, the expression of D2 receptors is not ubiquitous in all adrenal samples. Low expression of D2 receptors was especially observed in APA. These results may suggest that D2 receptors are down-regulated in the presence of high plasma aldosterone. The mechanisms of the regulation of expression of D2 and D4 receptors in the adrenal cortex require further investigation. There are controversial results from in vitro studies of DA on AII-stimulating aldosterone secretion (1, 18, 20). Dual effects of DA on aldosterone secretion have been demonstrated in rat glomerulosa cells (33). The inhibitory effect of DA on aldosterone secretion was thought to act via D2-like receptors, whereas a stimulatory effect was speculated via the D1 subclass (20, 33). The D1 receptor has been found in the rat ZG (34); however, its significance in the human adrenal cortex is not understood (12, 35). The present study of H295 cells did demonstrate the dual effects of DA on aldosterone secretion. In the presence of AII, DA further increased aldosterone secretion mildly by 20 –30%. It is suggested that DA has a stimulatory effect on aldosterone secretion in H295 cells. To distinguish D2 and D4 receptors, clozapine, a specific antagonist of D4 receptors, was added (36). After the addition of clozapine, aldosterone secretion was reduced significantly, with levels 40 –55% lower than

those stimulated by A-II alone, instead of returning to the levels achieved by A-II alone. It is thought that if DA had only exerted its effect via D4 receptors on aldosterone secretion, the aldosterone levels, after adding clozapine, would be similar to the levels stimulated by A-II alone. It is thus proposed that the stimulatory effect of DA on aldosterone secretion may be mediated via D4 receptors in addition to D1 receptors, and that DA also had an inhibitory effect on aldosterone secretion. Raclopride is a more selective D2 antagonist, with 100-fold greater affinity for D2 than for D4 receptors (36, 37). The dissociation constant is nanomolar for D2 receptors and micromolar for D4 receptors. Our study showed that in the presence of DA and A-II, a high concentration (10⫺5 m) of raclopride decreased, whereas a low concentration (10⫺8 m) increased, aldosterone secretion. The data led to the speculation that DA had an inhibitory effect on aldosterone secretion through D2 receptors. The decreased aldosterone levels caused by a high concentration of raclopride might be due to its less specific effect on blocking D4 receptors. However, raclopride at a low concentration antagonized D2 receptors more specifically and resulted in elevation of aldosterone secretion. The data lead to the speculation that DA has an inhibitory effect on aldosterone secretion through D2 receptors. The speculation is supported by further experiments of simultaneous administrations of raclopride and clozapine. The decrease in aldosterone levels by clozapine was completely reversed when raclopride was added. These results demonstrate that DA modulates aldosterone secretion in opposite directions through two D2-like receptors, D2 and D4 receptors. Both D2 and D4 receptors are classified as D2-like receptors because they possess a general property of inhibiting adenylyl cyclase via coupling to the Gi class of G proteins (for review, see Ref. 37). The signal pathways of D2 and D4 on aldosterone secretion require further studies. The expression of DR in different tissues is discrepant among species. D4 receptor mRNA was detected in human adrenal glands (in the present study) as well as in mice (29), but not in rats (28). In the kidney, D2-like receptors have been identified by radioligand binding studies (38). By RT-PCR, different subtypes of D2-like receptors were detected in the kidney, including D2L (39) and D3 (40) in rats and D4 in humans (30). The present study shows that the human kidney expresses D4 receptors, but neither D2 nor D3 receptors. The human D4 receptors exist with different insertions in the third intracellular loop, which contains repeat sequences of 16 amino acids (41). The clinical significance of the polymorphism of the human D4 receptors is not clear, although different properties between shorter and longer variants were observed with respect to clozapine binding (41). In our 6 cases of APA, there are 3 cases each for D4(4/4) and D4(2/4) polymorphisms. In a study of 118 normal adult Chinese subjects, we demonstrated that these 2 variants are the most common variants, with 68% of D4(4/4) and 29% of D4(2/4), respectively (unpublished data). Although the case number in the present study is limited, APA probably does not express specific variants of D4 receptors. Further studies will elucidate whether different responsiveness of aldosterone secretion to MCP in APA results from different variants of D4 receptors.

4466


Several studies have demonstrated the expression of D2 receptors in the adrenal medulla and pheochromocytoma (21–23). The present study revealed that D2 as well as D4 receptors were expressed in these chromaffin cells. In situ hybridization did not show obvious difference of the distribution and abundance between these two D2-like receptors in either the adrenal medulla or pheochromocytoma. The lesser expression of these DR in pheochromocytoma compared with normal adrenal glands may be due to downregulation of the receptors by increased levels of catecholamines. Activation of D2-like receptors can inhibit catecholamine release from chromaffin cells of the adrenal gland (42). Administration of MCP in patients with pheochromocytoma may induce a hypertension crisis due to the surge of catecholamines (43, 44). The presence of both D2 and D4 receptors in the medulla and pheochromocytoma makes it difficult to clarify which receptor is responsible for the regulation of catecholamine secretion. In summary, this is the first study, to our knowledge, to demonstrate the existence of both D2 and D4 receptors in the human adrenal gland and adrenal neoplasm. Both DR were expressed in chromaffin cells equally. In the adrenal cortex, expression of D4 receptors is ubiquitous and more specifically localized in the ZG, whereas expression of D2 receptors is various. The study of NCI-H295 cells shows that both receptors play significant roles in the modulation of aldosterone secretion, but in opposite directions. Acknowledgments We are very grateful to Dr. Mitsuyuki Matsumoto (Ibaraki, Japan) for his generous donation of the human D4 receptor cDNA. We are indebted to Bin-Hong Chen and Fen-Fang Hsieh for their excellent technical assistance.

9. 10. 11. 12. 13. 14.

15.

16. 17. 18. 19. 20.

21.

22. 23.

Received April 25, 2001. Accepted May 3, 2001. Address all correspondence to: Bor-Shen Hsieh, M.D., Department of Internal Medicine, National Taiwan University Hospital, 7 Chung Sun South Road, Taipei, Taiwan. Address requests for reprints to: Kwan-Dun Wu, M.D., Ph.D., Department of Internal Medicine, National Taiwan University Hospital, 7 Chung Sun South Road, Taipei 100, Taiwan. E-mail: [email protected]. This work was supported by grants from the National Science Council (NSC-88-2314-B002-234, to K.D.W.), the Mrs. Hsiu-Chin Lee Kidney Research Fund, and the Asian Nephrology Forum.

References 1. McKenna TJ, Island DP, Nicholson WE, Liddle GW 1979 Dopamine inhibits angiotensin stimulated aldosterone biosynthesis in bovine adrenal cells. J Clin Invest 64:287–291 2. Carey RM, Thorner MO, Ortt EM 1980 Dopaminergic inhibition of metoclopramide-induced aldosterone secretion in man. J Clin Invest 66:10 –18 3. Ganguly A 1984 Dopaminergic regulation of aldosterone secretion: how credible? Clin Sci 66:631– 637 4. Porter ID, Whitehouse BJ, Price GM, Hinson JP, Vinson GP 1992 Effects of dopamine, high potassium concentration and field stimulation on the secretion of aldosterone by the perfused rat adrenal gland. J Endocrinol 133:275–282 5. Carey RM 1982 Acute dopaminergic inhibition of aldosterone secretion is independent of angiotensin II and adrenocorticotropin. J Clin Endocrinol Metab 54:463– 469 6. Kuo YM, Wu KD, Hsieh BS, Chen WY, Yen TS, Tsai TJ 1986 Dopaminergic regulation of plasma aldosterone secretion in hypertensive patients. J Formosan Med Assoc 84:1222–1232 7. Fraser R, Connell JMC, Inglis G, Kenyon CJ, Tree M 1989 The role of dopamine in the control of corticosteroid secretion and metabolism. J Steroid Biochem 32:217–222 8. Rizzi CA, Mierau J, Landinsky H 1997 Regulation of plasma aldosterone

24. 25. 26. 27. 28. 29. 30. 31.

32. 33. 34. 35.


levels by metoclopramide: a reappraisal of its mechanism from dopaminergic antagonist to serotonergic agonism. Neuropharmacology 36:763–768 Bevilacqua M, Vago T, Sorza D, Norbiato G 1982 Characterization of dopamine receptors by 3H-ADNT binding in calf zona glomerulosa. Biochem Biophys Res Commun 108:1669 –1669 Stern N, Ozaki L, Tuck ML 1986 Evidence for dopaminergic binding and inhibitory sites in the human adrenal cortex. Metabolism 35:1154 –1158 Missale C, Liberini P, Memo N, Carrura MO, Spano P 1986 Characterization of dopamine receptors associated with aldosterone secretion in rat adrenal glomerulosa. Endocrinology 119:2227–2232 Amenta F, Chiandussi L, Mancini M, Ricci A, Schena M, Veglio F 1994 Pharmacological characterization and autographic localization of dopamine receptors in the human adrenal cortex. Eur J Endocrinol 131:91–96 Pratt JH, Turner DA, Bowsher RR, Henry DP 1987 Dopamine in rat adrenal glomerulosa. Life Sci 40:811– 816 Vizi ES, Toth IE, Orso E, et al. 1993 Dopamine is taken up from the circulation by, and released from, local noradrenergic varicose axon terminals in zona glomerulosa of the rat: a neurochemical and immunocytochemical study. J Endocrinol 139:213–226 Wu K-D, Chen Y-M, Chu J-S, Hsieh T-S, Hsieh B-S 1995 Zona fasciculata-like cells determine the response of plasma aldosterone to metoclopramide and aldosterone synthase mRNA level in aldosterone-producing adenoma. J Clin Endocrinol Metab 80:783–789 Civelli O, Bunzow JR, Grandy DK, Zhou QY, Van Tol HHM 1991 Molecular biology of the dopamine receptors. Eur J Pharmacol 207:277–286 Barrett RJ, Wright KF, Taylor DR, Proakis AG 1987 Involvement of dopamine receptor subtypes in dopaminergic modulation of aldosterone secretion in rats. Life Sci 40:1499 –1506 Missale C, Memo M, Liberini P, Spano P 1988 Dopamine selectively inhibits angiotensin II-induced aldosterone secretion by interacting with D-2 receptors. J Pharmacol Exp Ther 246:1137–1143 Lombardi C, Missale C, De Cotiis R, et al. 1988 Inhibition of the aldosterone response to sodium depletion in man by stimulation of dopamine DA2 receptors. Eur J Clin Pharmacol 35:323–326 Gallo-Payet N, Chouinard L, Balestre M-N, Guillon G 1991 Mechanisms involved in the interaction of dopamine with angiotensin II on aldosterone secretion in isolated and cultured rat adrenal glomerulosa cells. Mol Cell Endocrinol 81:11–23 Schalling M, Dagerlind A, Goldstein M, Ehrlich M, Greengard P, Hokfelt T 1990 Comparison of gene expression of the dopamine D-2 receptor and DARPP-32 in rat brain, pituitary and adrenal gland. Eur J Pharmacol 188: 277–281 Pupilli C, Lanzillotti R, Fiorelli G, et al. 1994 Dopamine D2 receptor gene expression and binding sites in adrenal medulla and pheochromocytoma. J Clin Endocrinol Metab 79:56 – 61 Barili P, Zaccheo D, Amenta F 1996 Pharmacological characterization and autoradiographic localization of dopamine receptors in the rat adrenal medulla. Eur J Pharmacol 310:129 –135 Dunn MG, Bosmann HB 1981 Peripheral dopamine receptor identification: properties of a specific dopamine receptor in the rat adrenal zona glomerulosa. Biochem Biophys Res Commun 99:1081–1087 Quik M, Bergeron L, Mount H, Philie J 1987 Dopamine D2 receptor binding in adrenal medulla: characterization using 3H-spiperone. Biochem Pharmacol 36:3707–3713 Amenta F, Ricci A 1995 Autoradiographic localization of dopamine D2-like receptors in the rat adrenal gland. Clin Exp Hypertension 17:669 – 688 Van Tol HHM, Bunzow JR, Guan H-C, et al. 1991 Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine. Nature 350:610 – 614 O’Malley Kl, Harmon S, Tang L, Todd RD 1992 The rat dopamine D4 receptor: sequence, gene structure, and demonstration of expression in the cardiovascular system. New Biol 4:137–146 Suzuki T, Kobayashi K, Nagatsu T 1995 Genomic structure and tissue distribution of the mouse dopamine D4 receptor. Neurosci Lett 199:69 –72 Matsumoto M. Hidaka K. Tada S. Tasaki Y. Yamaguchi T 1995 Full-length cDNA cloning and distribution of human dopamine D4 receptor. Mol Brain Res 29:157–162 Chen YM, Wu KD, Hu-Tsai MI, Chu JS, Lai MK, Hsieh BS 1999 Differential expression of type 1 angiotensin II receptor mRNA and aldosterone responsiveness to angiotensin in aldosterone-producing adenoma. Mol Cell Endocrinol 152:47–55 Bird IM, Hanley NA, Word RA, et al. 1993 Human NCI-H295 adrenocortical carcinoma cells: a model for angiotensin-II-responsive aldosterone secretion. Endocrinology 133:1555–1561 Gallo-Payet N, Chouinard L, Balestre MN, Guillon G 1990 Dual effects of dopamine in rat adrenal glomerulosa cells. Biochem Biophys Res Commun 172:1100 –1108 Aherne AM, Vaughan CJ, Carey RM, O’Connell DP 1997 Localization of dopamine D1A receptor protein and messenger ribonucleic acid in rat adrenal cortex. Endocrinology 138:1282–1288 Hughes JM, Beck TR, Rose Jr CE, Carey RM 1988 The effect of selective


36. 37. 38. 39.


dopamine-1 receptor stimulation on renal and adrenal function in man. J Clin Endocrinol Metab 66:518 –525 Van Tol HH, Bunzow JR, Guan HC, et al. 1991 Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine. Nature 350:610 – 614 Missale C, Nash SR, Robinson SW, Jaber M, Caron MG 1998 Dopamine receptors: from structure to function. Physiol Rev 78:189 –225 Felder CC, McKelvey AM, Gitler MS, Eisner GM, Jose PA 1989 Dopamine receptor subtypes in renal brush border and basolateral membranes. Kidney Int 36:183–193 Gao DQ, Canessa LM, Mouradian MM, Jose PA 1994 Expression of the D2 subfamily of dopamine receptor genes in kidney. Am J Physiol 266:F646 – F650

40. Sokoloff PB, Giros B, Martres MP, Barthenet ML, Schwartz JC 1990 Molecular cloning and characterization of a novel dopamine receptor (D-3) as a target for neuroleptics. Nature 347:146 –151 41. Van Tol HHM, Wu CM, Guan H-C, et al. 1992 Multiple dopamine D4 receptor variants in the human population. Nature 358:149 –152 42. Mannelli P, Pupilli C, Fabbri G, et al. 1988 Endogenous dopamine and DA2 receptors: a mechanism limiting excessive sympathetic-adrenal discharge in humans. J Clin Endocrinol Metab 66:626 – 631 43. Kuchel O, Buu NT, Hamet P, Larochelle P 1985 Effect of metoclopramide on plasma catecholamine release in essential hypertension. Clin Pharmacol Ther 37:372–375 44. Hsu TS, Lee CP, Kuo CT 1993 Diagnostic use of metoclopramide in hypertension caused by pheochromocytoma. Int J Cardiol 42:79 – 86