AJP - Endo Information on EB 2010
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 294: E622-E629, 2008. First published January 2, 2008; doi:10.1152/ajpendo.00657.2007
0193-1849/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/3/E622    most recent
00657.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chang, H.-W.
Right arrow Articles by Hsieh, B.-S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chang, H.-W.
Right arrow Articles by Hsieh, B.-S.

D4 dopamine receptor enhances angiotensin II-stimulated aldosterone secretion through PKC-{varepsilon} and calcium signaling

Hong-Wei Chang,1 Vin-Cent Wu,1 Chao-Yuan Huang,2 Hong-Yu Huang,1 Yung-Ming Chen,1 Tzong-Shinn Chu,1 Kwan-Dun Wu,1 and Bor-Shen Hsieh1

1Nephrology Division, Department of Internal Medicine; and 2Department of Urology, National Taiwan University Hospital, Taipei, Taiwan

Submitted 11 October 2007 ; accepted in final form 11 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Aldosterone secretion is subjected to dopaminergic regulation. Our previous study showed that both human D2 and D4 dopamine receptors (D2R and D4R) modulate aldosterone secretion, but in opposing directions. The inhibitory effect of D2R is mediated by attenuating protein kinase C-µ (PKC-µ) and calcium-dependent signaling. The mechanism of D4R effect on angiotensin II (AII)-stimulated aldosterone secretion is explored in this study. Experiments were done with primary human adrenal cortical cells and human adrenocarcinoma (NCI-H295R) cells. Activation of different PKC isoforms was detected by specific phospho-PKC antibodies and PKC translocation. The role of calcium-dependent signaling was examined by measuring the cytoplasmic inositol 1,4,5-triphosphate (IP3) and calcium ([Ca2+]i). The D4R agonist PD-168,077 enhanced AII-stimulated aldosterone synthesis and secretion as early as 30 min following exposure independently of the modulation of aldosterone synthase (CYP11B2) transcription. CYP11B2 mRNA level elevated by AII was augmented by D4R in the later period. These effects were reversed by the D4R antagonist L-745,870. AII activated PKC-{alpha}/βII, -{varepsilon}, and -µ but not PKC-{delta}, -{theta}, or -{zeta}/{lambda} of H295R cells. The D4R agonist selectively enhanced AII-stimulated PKC-{varepsilon} phosphorylation and its translocation to the cell membrane. Furthermore, the D4R agonist enhanced the AII-stimulated elevation of intracellular IP3 and [Ca2+]i. Inhibition of PKC-{varepsilon} translocation by the PKC-{varepsilon}-specific inhibitory peptide attenuated AII-stimulated aldosterone secretion, CYP11B2 mRNA expression, and elevation of intracellular IP3 and [Ca2+]i. We conclude that D4R augmented aldosterone synthesis/secretion induced by AII. The mechanisms responsible for this augmentation are mediated through enhancing PKC-{varepsilon} phosphorylation and [Ca2+]i elevation.

aldosterone-producing adenoma; protein kinase C-{varepsilon}; hypertension

THERE IS INCREASING EVIDENCE (8, 12) that aldosterone plays a direct role in the pathogenesis of chronic heart failure and vascular inflammation. The vascular and perivascular inflammatory responses to angiotensin II (AII) infusion and salt loading, both reported to increase cardiovascular aldosterone synthesis (28, 29), are completely prevented by adrenalectomy (25). This suggests that the regulation of adrenal aldosterone production is more important than that of local cardiac and/or vascular synthesis of aldosterone. Although the regulation of aldosterone production by AII is well established, the modulating factors affecting itself or its downstream signaling are controversial and far from being completely delineated. The presence of chromaffin cells originating from the medulla in the cortical layers is strong evidence supporting the concept of the neurohormonal control of zona glomerulosa cell secretion (5).

Dopamine D2-like receptors have been found (9, 19) to play a pivotal role in inhibiting aldosterone secretion. We (31) and other investigators (23) have demonstrated that two subtypes of dopamine receptors, D2 and D4 receptors (D2R and D4R), are expressed in the adrenal cortex, mainly in the zona glomerulosa, and exert opposite effects on aldosterone secretion. The D2R attenuates AII-induced secretion of aldosterone, whereas the D4R enhances that effect.

An increase of intracellular Ca2+ concentration ([Ca2+]i) plays as an important signal in AII-induced aldosterone secretion and CYP11B2 mRNA expression (21, 32), and involvement of protein kinase C (PKC) has also been noted (2, 3, 17) in the regulation of adrenal steroidogenic genes. Our recent work (6) has revealed that D2R modulates AII-stimulated aldosterone synthesis and secretion through attenuation of PKC-µ phosphorylation and [Ca2+]i elevation. Paradoxically, constitutive activation of some subtypes of PKC has been shown (15, 16) to inhibit AII-stimulated CYP11B2 gene expression. Therefore, the expression of the CYP11B2 gene may be differentially regulated by different PKC subtypes (16). In the present study, we explored the role of D4R and its signals in the regulation of CYP11B2 mRNA expression and aldosterone secretion of human adrenal cortical cells. We found that D4R could augment both acute and chronic phases of aldosterone secretion/synthesis by AII through specific activation of the novel PKC-{varepsilon} and [Ca2+]i elevation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. AII, PD-168,077, and L-745,870 were purchased from Sigma Chemical (St. Louis, MO), and phospho-PKC-specific antibodies and PKC subtype-specific antibodies were purchased from Cell Signaling Technology, (Beverly, MA). PD-168,077 and L-745,870 are the highly selective agonist and antagonist, respectively, for D4R (10, 20). PKC-{varepsilon} translocation inhibitor peptide and PKC-{varepsilon} translocation inhibitor peptide-negative controls were obtained from Calbiochem (Cambridge, MA). An inositol 1,4,5-triphosphate (IP3) radioreceptor assay (NEK064) kit was bought from PerkinElmer Life Sciences (DuPont-New England Nuclear, Boston, MA). Fura 2-AM, Pluronic F-127 was obtained from Molecular Probes (Eugene, OR). Angiotensin type 1 receptor (AT1R) antibody and its immunizing peptide (AT1N10 and sc1173, respectively) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). D4R antibody was designed to target the third intracellular domain. The antibody was purified from the serum of a rabbit immunized with the synthesized oligopeptide RAPRRPSGPGPPSPT at the aa233–247 position. The preimmunized serum of the same rabbit was also collected as a negative control. The antibody detected a single band, 46 kDa in size, in both the adrenal cortex, primary human adrenal cortical cells, and H295R cells.

Cell culture. The human adrenocortical carcinoma cell line H295R was obtained from American Type Culture Collection (Rockville, MD). The primary human adrenal cortical cells were prepared from surgical specimens of the patients without any adrenal disease. The primary culture was prepared as described previously (18). In brief, small pieces of normal tissues were dissociated with 0.3% collagenase IA and 20 mg/l deoxyribonuclease I (Sigma Chemical) in culture medium at 37°C. The digestion was carried out during two 2-h periods for normal, adjacent adrenals. The dispersed cells were washed once with medium and plated on six-well plastic cell culture dishes. The cells were maintained in Dulbecco's Modified Eagle's Medium-Ham's F-12 medium containing 10% fetal calf serum, 2 mmol/l glutamine (Gibco, Grand Island, NY), 1.25 x 105 IU/l penicillin, and 0.125 g/l streptomycin sulfate (Gibco). The cultures were maintained at 37°C in humidified 95% air-5% CO2, with replacement of medium every 3rd day until they had achieved subconfluency on days 5–10 of culture. The H295R cell culture experiments have been described previously (31). The cells expressed D4R and significantly secreted aldosterone after addition of AII. After the doses of the drugs that regulate aldosterone secretion/synthesis were tested, 10 nmol/l AII, 10–6 mol/l PD-168,077, and 10–6 mol/l L-745,870 were used in all of the experiments in the present study, unless otherwise indicated. All experiments were performed at least in triplicate; for each experiment the data for analysis was the mean of three measured samples. The study was approved by the Ethics Committee of the National Taiwan University Hospital.

Immunoblotting. For PKC assays, H295R cells were scraped 5 min after treatment and solubilized in lysis buffer. Equal amounts of protein (40 µg for H295R cells) were separated on a 10% polyacrylamide gel and transferred to Immobilon P. Blots were probed with different antibodies, followed by a horseradish peroxidase-coupled anti-rabbit secondary antibody. Immunoreactive proteins were visualized with enhanced chemiluminescence (Pierce, Rockford, IL).

Measurement of aldosterone. The culture supernatant and cell lysate were collected 30 min or 24 h after treatment, respectively. The aldosterone levels of the culture medium were measured by radioimmunoassay with commercial kits (Aldosterone Maia kit; Biochem Immunosystems, Bologna, Italy).

Quantitative real-time PCR. Total RNA was extracted from H295R using a Trizol RNA isolation reagent (Invitrogen) according to the manufacturer's instructions. RNA was subjected to deoxyribonuclease treatment using 1 U deoxyribonuclease I (amp grade)/µg RNA incubated in deoxyribonuclease reaction buffer for 15 min at 37°C (New England Bilolabs, Beverly, MA) to remove genomic DNA contamination. The reaction was stopped by heating to 90°C for 5 min. RNA was reverse transcribed by using a reverse transcription system kit (Promega, Madison, WI) as described in the manufacturer's protocol. The gene expression levels of D4R and CYP11B2 were then quantified using TaqMan technology on an ABI PRISM 7900 sequence detection system (assay ID nos. Hs00609526_m1 and Hs01597732_m1; Applied Biosystems). Assay no. Hs01597732_m1 with probe locating over 963 regions on NM 000498 did not detect CYP11B1 mRNA. GAPDH (assay ID no. Hs99999905_m1) was used as an endogenous control in a TaqMan human endogenous control plate. Sample dilutions were comprised of 100 ng of template cDNA. All samples were tested in a total volume of 20 µl in triplicate. The cycle-to-threshold (CT) value was recorded for statistical analysis. The mRNA levels of D4R and CYP11B2 were corrected with the mRNA level of GAPDH and expressed as 2–{Delta}{Delta}CT, where {Delta}{Delta}CT = ({Delta}CT x – {Delta}CT GAPDH, X = D4R and CYP11B2). The mRNA level of the tumor portion was corrected with that of the nontumor portion.

Isolation of membrane protein. Cells were scraped in ice cold 1x PBS and centrifuged at 600 g for 5 min. The pellet was resuspended and homogenized in solubilization buffer without detergent (20 mmol/l Tris·HCl, pH 7.5, 150 mmol/l NaCl, 5 mmol/l EDTA, 1 mmol/l PMSF, 10 mmol/l NaF, 25 µg/ml leupeptin, and 0.1 mg/ml aprotinin). This homogenate was then centrifuged at 1,000 g in 4°C for 5 min to separate the nuclear portion. The supernatant was centrifuged again at 53,000 g at 4°C for 30 min. The pellet containing the membrane portion was resuspended in RIPA buffer (20 mmol/l Tris·HCl, pH 7.4, 150 mmol/l NaCl, 2 mol/l EDTA, 0.1% Triton X-100, 2.5 mmol/l Na-pyrophosphate, 1 mmol/l Na3VO4, and 1 mmol/l PMSF). The supernatant contained cytosolic protein.

Peptide transfer. The conditions of peptide transfer were as described previously (13). In brief, cells were cultured in serum-free medium for 24 h. The cells were incubated and washed with PBS at room temperature and in an ice bath in two sequential 2-min intervals. Thereafter, the cells were incubated with freshly prepared permeabilization buffer containing the desired peptides for 10 min in an ice bath. ATP was added just before the permeabilization buffer was added to the cells. The cells were then gently washed four times on ice and recovered in PBS on ice for 20 min. The cells were placed in PBS at room temperature for 2 min. This step was repeated with PBS at 37°C, after which the original cell media (conditioned media) were added back to the cells at 37°C. The cells were further incubated for 2 h at 37°C before further experiments.

Cytoplasmic Ca2 ± measurement. The cells were suspended in PBS containing 2 mmol/l EDTA by periodic shaking, washed in a Ca2+-containing solution (140 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l MgCl2, 1 mmol/l CaCl2, 11 mmol/l glucose, 10 mmol/l HEPES, pH 7.4, and 0.1% BSA), and incubated in 4 µM fura 2-AM with 0.04% Pluronic F-127 for 35 min. Cells were then washed and resuspended in the Ca2+-containing solution for the following experiments. Intracellular Ca2+ was measured in cells suspended in Ca2+-containing solution at the ratio of fluorescence with 340- and 380-nm excitation and 510-nm emission (fluorolog 2; Spex Industries, Edison, NJ). The fluorescent ratio was calibrated by adding digitonin to a final concentration of 75 µg/ml and then adding 1 mM EDTA at a 1:50 dilution and 10 N NaOH at a 1:700 dilution, and [Ca2+]i was calculated as described (8).

Determination of IP3. Cells were washed twice and preincubated for 30 min at 37°C in an incubation buffer (145 mmol/l NaCl, 5.6 mmol/l KCl, 5.6 mmol/l glucose, 0.01% BSA, and 10 mmol/l HEPES, pH 7.4). After treatment in 1 ml of warmed incubation buffer for 10 s, 250 µl of ice-cold perchloric acid (10% vol/vol) was added to terminate the response. After scraping, the cells were washed with 250 µl of 10% perchloric acid and centrifuged at 12,000 g for 5 min at 4°C. The supernatants were neutralized with 50 µl of 1.5 M NaOH in the presence of universal indicator. IP3 levels were measured by a specific, competitive binding assay kit (PerkinElmer Life Sciences). Each incubation contained 500 µl of receptor preparation/[3H]IP3 tracer 1:15 (vol/vol), 100 µl of standard IP3 (0–120 pmol/0.1 ml), or cell extract. The tubes were agitated and incubated for 45 min on ice. Incubations were terminated by centrifugation at 12,000 g for 5 min at 4°C. The supernatant was removed by aspiration, and the pellet was dissolved in scintillation liquid and counted.

Statistics. Statistical analysis was performed with the Mann-Whitney U-test using the Statview software package (Abacus Concept, Berkeley, CA). Statistical significance was considered at the 5% level.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
D4 receptor was expressed on both human adrenal cortex and H295R cells. In a previous study (31), we demonstrated mRNA expression of human D2R and D4R in adrenal cortex and NCI-H295R (H295R) cells. Immunoblotting with the D4 receptor-specific antibody revealed a single band of 46 kDa in the human adrenal cortex, primary human adrenal cortical cells, and H295R cells (Fig. 1). The band was almost abolished by using preimmunized serum as the primary antibody or by pretreating the anti-D4R serum with the D4R-immunizing peptide.


Figure 1
View larger version (51K):
[in this window]
[in a new window]

  		Fig. 1. Expression of D4 receptor (D4R) and angiotensin type 1 receptor (AT1R) in human adrenal cortex (lane 1), primary cultured cells of human adrenal cortex (lane 2), and H295R cells (lanes 3–5). Immunoblotting with specific antiserum to D4R or AT1R (lanes 1–3) revealed 46- and 42-kDa-sized bands, respectively. The bands were almost absent when the primary antibodies were neutralized with their respective immunizing peptides (lane 4) or when preimmunized serum in place of the D4R antiserum was used (lane 5).

 
D4 receptor modulated AII-stimulated CYP11B2 mRNA expression and aldosterone secretion. The D4R agonist PD-168,077 (10–6 mol/l) augmented the increase of AII-stimulated 24-h aldosterone secretion of both primary human adrenal cortical cells and H295R cells. This enhancing effect of the D4R was reversed by the D4 antagonist L745,870 (Fig. 2, A and B, top). The effects of both PD-168,077 and L-745,870 were concentration dependent from 10–9 to 10–6 mol/l (data not shown). Consistent with its effect on AII-stimulated aldosterone secretion, PD-168,077 at 10–6 mol/l effectively augmented AII-stimulated CYP11B2 mRNA expression as early as 2 h after exposure and for ≤24 h (data not shown), and L-745,870 reversed the effect of PD-168,077. The CYP11B2 mRNA expression of primary human adrenal cortical cells and H295R cells 4 h after treatment was illustrated (Fig. 2, A and B, bottom).


Figure 2
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 2. Modulation of D4R on angiotensin II (AII; at 10–8 mol/l)-stimulated aldosterone secretion and expression of CYP11B2 mRNA from primary cultured cells of human adrenal cortex (A and C) and H295R cells (B and D). A and B: aldosterone secretion 24 h after treatment (top) and CYP11B2 mRNA expression 4 h after treatment (bottom). *Significantly different from AII (P < 0.05). C and D: aldosterone level of the supernatant (solid bars) and the intracellular compartment (hatched bars) 30 min after treatment. {dagger}Significantly different from AII (P < 0.05). Cont, control; PD, PD-168,077 at 10–6 mol/l; L, L-745,870 at 10–6 mol/l. All experiments were performed in at least triplicate; for each experiment the data for analysis were the mean of 3 measured samples. Values are shown as means ± SD.

 
The augmented effect of PD-168,077 on AII-stimulated aldosterone secretion of both primary human adrenal cortical cells and H295R cells was observed as early as 30 min after exposure and could be reversed by L-745,870 (Fig. 2, C and D). In addition to the elevation of aldosterone levels in the supernatant, the intracellular aldosterone level was also increased by AII, which was augmented by PD-168,077, and L-745,870 reversed the effect of PD-168,077. There was no significant change of CYP11B2 mRNA level 30 min after treatment with AII (data not shown).

D4R altered the AII-stimulated PKC response. Phospho-PKC-specific antibodies showed that AII stimulated phosphorylation of PKC-{alpha}/βII Thr638/641, PKC-{varepsilon} Ser729, PKC-µ Ser744/748, and PKC-µ Ser916, but not of PKC-{delta} Thr505, PKC-{theta} Thr538, or PKC-{zeta}/{lambda} Thr410/403, on both primary human adrenal cortical cells and H295R cells (6). Addition of PD-168,077 did not alter the basal or AII-stimulated phosphorylation of PKC-{alpha}/βII Thr638/641, PKC-µ Ser916, or PKC-µ Ser744/748 but significantly enhanced the AII-stimulated phosphorylation of PKC-{varepsilon} Ser729; L-745,870 reversed this augmenting effect of PD-168,077 (Fig. 3, A and B).


Figure 3
View larger version (40K):
[in this window]
[in a new window]

  		Fig. 3 Effect of D4R on AII-stimulated PKC activation of primary human adrenal cortical cells and H295R cells. Phosphorylation of PKC isoenzymes 5 min after treatment in primary human adrenal cortical cells (A) and H295R cells (B) was detected with phospho-PKC-specific antibodies. Quantification of phospho-PKC-{varepsilon} on immunoblotting was measured by densitometer. *Significantly different from AII (P < 0.05). C: membrane translocation of PKC isoenzymes 5 min after treatment in H295R cells. Both the membrane (M) and cytosol proteins (C) were immunoblotted with phospho-PKC-specific antibodies. {dagger}Significantly different from AII, (P < 0.05). The quantitative change of PKC-{varepsilon} in the membrane (solid bar) and cytosol (hatched bars) is shown in the bar graph. All experiments were performed in at least triplicate; for each experiment the data for analysis were the mean of 3 measured samples. Values are shown as means ± SD.

 
Immunoblotting with antibodies for total forms of PKC-{alpha}, PKC-βII, PKC-{varepsilon}, and PKC-µ of H295R cells showed a predominant cytosol fraction of these PKC isoforms. AII significantly translocated these PKC isoforms to the membrane with a reciprocal decrease of their cytosol distributions (Fig. 3C). PD-168,077 enhanced the AII-induced translocation of PKC-{varepsilon} to the membrane accompanied with a reciprocal, changed PKC-{varepsilon} in the cytosol and exerted no effect on other PKC isoforms. The enhancing effect was again reversed by L-745,870. Therefore, D4R selectively augmented AII-stimulated PKC-{varepsilon} activation.

PKC-{varepsilon}-specific peptide inhibited AII-induced aldosterone secretion and CYP11B2 mRNA expression. Permeabilization of the synthetic PKC-{varepsilon}-specific inhibitory peptide into H295R cells selectively inhibited the AII-stimulated translocation of PKC-{varepsilon} to the membrane (Fig. 4A) and reduced the elevations of aldosterone and CYP11B2 mRNA levels by AII (Fig. 4B); a 25% reduction of the aldosterone level was noted. These effects were not observed when the control peptide was inducted into the cells or when the cells were treated with the saponin-containing permeabilization buffer only.


Figure 4
View larger version (55K):
[in this window]
[in a new window]

  		Fig. 4. Effect of PKC-{varepsilon} inhibitory peptide (IPep) on AII-stimulated 5-min PKC translocation in H295R cells. A: cells were induced with IPep or control peptide (CPep). Top: both the M and C were immunoblotted with PKC subtype-specific antibodies. Bottom: quantification of membrane and cytosol-phosphorylated PKC-{varepsilon}. *P < 0.05 vs. AII. B: aldosterone secretion 24 h after treatment (solid bars) and CYP11B2 mRNA expression 4 h after treatment (hatched bars). {dagger}Significantly different from AII (P < 0.05). S, saponin. All experiments were performed in at least triplicate; for each experiment the data for analysis were the mean of 3 measured samples. Values are shown as means ± SD.

 
D4R and PKC-{varepsilon} modulated an AII-stimulated [Ca2+]i increase. The AII-stimulated increase of [Ca2+]i was augmented by the D4R agonist PD-168,077. PD-168,077 not only augmented the peak [Ca2+]i but also accelerated the increased rate of [Ca2+]i after AII stimulation; the plateau of [Ca2+]i after this initial surge was also augmented significantly (Fig. 5A). The effect of PD-168,077 on AII-stimulated increased of [Ca2+]i was also observed on the similar experiments with the Ca2+-free medium (Fig. 5B). Delivery of the PKC-{varepsilon} inhibitory peptide attenuated the AII-stimulated peak [Ca2+]i, the increased rate of [Ca2+]i (slope), and the plateau of [Ca2+]i (Fig. 5C).


Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 5. Intracellular Ca2+ concentration ([Ca2+]i)change in H295R cells induced by AII at 10–8 mol/l with or without pretreatment of PD at 10–6 mol/l for 30 min (A: under standard culture medium; B: under calcium-free culture medium). Peak-baseline, the difference between the peak level after AII and the basal level; slope, the peak-baseline divided by the interval from addition of AII to the time after reaching peak; plateau-baseline, the difference between plateau level 120 s after AII treatment and the basal level. The effect of PD on [Ca2+]i in terms of peak, slope, and plateau are analyzed. *Significantly different from AII (P < 0.05) C: effect of PKC-{varepsilon} on AII-stimulated [Ca2+]i. H295R cells were introduced with either IPep or CPep after perforation by S. The time-course difference of AII + S and AII + S + IPep was demonstrated. {dagger}Significantly different from AII + S (P < 0.05; also see the legend to Fig. 4). All experiments were performed in at least triplicate; for each experiment the data for analysis were the mean of 3 measured samples. Values are shown as means ± SD.

 
D4R modulated the intracellular IP3 level. A significant elevation of the intracellular IP3 of H295R cells was noted when AII was added, and a further 25% increase of IP3 was observed by pretreatment with PD-168,077. The augmenting effect of PD-168,077 was reversed by the D4R antagonist L745,870 (Fig. 6). Additionally, the AII-stimulated elevation of intracellular IP3 was significantly attenuated by the PKC-{varepsilon} inhibitory peptide (Fig. 6).


Figure 6
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 6. Effect of D4R and Ipep on AII-stimulated intracellular inositol 1,4,5-triphosphate ([IP3]i) increase. The [IP3]i level was measured 10 s after treatment. *Significantly different from AII (P < 0.05); {dagger}significantly different from AII, (P < 0.05). All experiments were performed in at least triplicate; for each experiment the data for analysis were the mean of 3 measured samples. Values are shown as means ± SD.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
As shown in our previous study (31), D4R exerts opposite effect on aldosterone secretion to D2R. In the present study, we further demonstrated that D4R modulates AII-induced aldosterone secretion/synthesis in both acute and chronic phases. Recently, we (6) showed that the inhibitory effect of D2R was exerted through attenuation of PKC-µ phosphorylation and [Ca2+]i elevation induced by AII. The augmentation of aldosterone secretion/synthesis by D4R is mediated through another specific PKC isoform, PKC-{varepsilon}, in addition to the modulation of [Ca2+]i. Therefore, two PKC isoforms are involved in dopaminergic regulation of aldosterone secretion/synthesis.

Although the role of PKC in regulating CYP11B2 transcriptional activity and aldosterone secretion has been studied in H295R cells (22, 35), the results lacked consensus. An increase of aldosterone secretion was observed when the cells were treated with PKC activator (22, 35), and a PKC inhibitor reduced AII-stimulated aldosterone secretion (4, 14). However, by transfection of H295 cells with constitutively active PKC isoforms, LeHoux and Lefebvre (16) observed that PKC-{alpha}, -{varepsilon}, and -{theta} inhibited basal and stimulatory hamster CYP11B2 promoter activity, whereas PKC-{zeta} increased it. Several problems may have contributed to the different results in those experiments. First, the PKC isoforms mediating AII-stimulated CYP11B2 mRNA expression and aldosterone secretion may be species dependent (33). Second, cells subjected to prolonged treatment with phorbol ester or transfected with constitutively active PKC may extensively downregulate the PKC activities and the downstream signaling rather than activate them (27, 34). Finally, although kinase-inactive PKC mutants and PKC regulatory domains have been widely used as dominant-negative inhibitors (33), the high homology among PKC isoforms and the lack of knowledge about the intracellular targets of these mutants make this approach questionable.

H295R cells express various PKC isoforms, whereas AII increased only the activities of PKC-{alpha}, -βII, -{varepsilon}, and -µ but not of PKC-{theta}, -{delta}, or -{zeta}. Among all PKC isoforms that were activated by AII, only the phosphorylation of PKC-{varepsilon} was altered by D4R. By inducting the PKC-{varepsilon}-specific inhibitory peptide, we found that the activation of PKC-{varepsilon} contributed partially, at least 25%, to the aldosterone synthesis/secretion induced by AII. Recently, we (6) found that inhibition of PKC-µ activity by its specific shRNA could reduce AII-stimulated CYP11B2 mRNA level and aldosterone secretion by ~80%. Therefore, activations of both PKC-µ and PKC-{varepsilon} account for most AII-induced aldosterone synthesis/secretion. In comparison, PKC-µ may be more important in the regulation of aldosterone synthesis/secretion than PKC-{varepsilon}. This speculation is supported by our recent data (6) that an increase in PKC-µ phorsphorylation is ubiquitously present in aldosterone-producing adenomas.

The parallel, rather than a reciprocal, change of intracellular and extracllular levels of aldosterone indicates that AII induces acute steroidogenesis instead of an exocytosis of secretory vesicles. This acute steroidogenesis is independent of CYP11B2 transcription and may involve the following mechanisms: 1) transfer of free cholesterol to the outer mitochondrial membrane via a calmodulin-dependent kinase II, 2) activation of the StAR protein by a calmodulin-dependent kinase II-mediated process, and 3) increase in mitochondrial [Ca2+] leading to activation of mitochondrial dehydrogenases of the tricarboxylic acid cycle and NAD(P)H production (27). D4R, like D2R, can modulate the [Ca2+]i induced by AII and, in turn, aldosterone secretion. The modulation of [Ca2+]i by D4R is probably through its effect on intracellular IP3 production.

Stimulation of the AT1R initiates a cascade of signaling events, including the activation of phosphoinositide-specific phospholipase C and the hydrolysis of phosphatidylinositol 4,5-bisphosphate to yield soluble IP3 and diacylglycerol; the latter can activate PKC. We noted that D4R could enhance AII-induced elevation of [Ca2+]i even when the extracellular calcium was absent. This result suggests that D4R enhances AII-induced [Ca2+]i elevation via increasing [IP3]i level, which through IP3 receptors releases calcium from the intracellular calcium stores. Interestingly, inhibition of PKC-{varepsilon} activity can attenuate AII-induced elevation of intracellular IP3 and [Ca2+]i. Therefore, the effect of D4R on [Ca2+]i elevation can also be mediated by activation of PKC-{varepsilon}, which is calcium independent (33). However, the augmentation of [Ca2+]i by D4R occurred much earlier (within seconds) than its effect on AII-induced PKC-{varepsilon} activation (within minutes; data not shown). Therefore, the augmentation of AII-induced elevation of [Ca2+]i by D4R may be biphasic.

How can D4R enhance the immediate AII-stimulated [Ca2+]i increase? D2R has been reported (24) to have a direct negative coupling with phospholipase C (PLC) by heterotrimeric Gi-1/2 proteins in rat anterior pituitary cell membrane. Dopamine and D2 agonists inhibited TRH- and AII-stimulated membrane PLC activities (11a). Our recent report has shown that the D2 agonist bromocriptine can attenuate AII-stimulated intracellular IP3 accumulation, a finding that has also suggested that there is negative regulation of D2R on the PLC activity. In contrast to D2R, we (11) found that D4R enhanced AII-stimulated intracellular IP3 accumulation. D4R has been proven to stimulate PLC activity in prefrontal cortex. It is possible that D4R could be able to enhance PLC activity through its coupling G protein components. By enhancing AII-stimulated PLC activity, D4R could increase AII-stimulated IP3 accumulation and thereafter Ca2+ release from intracellular stores through IP3 receptors.

Our experiments showed that the D4R-modulated effects on aldosterone secretion/synthesis occurred only in the presence of AII. It is also possible that the effects are mediated through a direct interaction of these two G protein-coupled receptors to enhance the downstream signaling (1). D4R has been reported (30) to mediate inhibition of potassium current in neurophysial nerve terminals. D4R could also inhibit the potassium current of H295R cells and partially depolarize the membrane potential that enhances AII-induced T-type calcium channel opening. D2R may regulate the "negative regulating proteins" such as phosphatases through spinophilin, a protein phophatase-1-interacting protein (26). It is also possible that D4 inhibits the phosphatase that plays role in turning down the activated PKC-{varepsilon} from the AT1R. In addition, we found that inhibiting activation of PKC-{varepsilon} attenuated both AII-induced intracellular free calcium increase and IP3 accumulation. Although this mechanism is still not clear, it is possible that PKC-{varepsilon} enhanced PLC ACTIVIty in the adrenal cortical cells. By way of inhibition of PLC activity, PKC-{varepsilon} inhibitory peptide attenuated AII-induced IP3 accumulation and intracellular free calcium increase. We illustrate the possible signals of D4R-modulated effects in Fig. 7.


Figure 7
View larger version (70K):
[in this window]
[in a new window]

  		Fig. 7. Mechanism of D4R effects on AII-stimulated aldosterone synthesis/secretion. The solid lines indicate the established pathways or effects, the dashed lines indicate the proposed ones, the blocked lines indicate the inhibitory effect, and the arrow lines are stimulatory. T-CC, T-type calcium channel; PLC-βI, phospholipase C-β1; PIP2, phosphatidylinositol 4,5-bisphosphate; DAG, diacylglycerol; IP3, inositol 1,4,5-triphosphate; IP3R, inositol 1,4,5-triphosphate receptor.

 
In summary, similarly to some other families of G protein-coupled receptors, among D2-like receptors, D4R has opposing action to D2R in AII-stimulated aldosterone secretion. D4R not only upregulates CYP11B2 expression but also enhances acute aldosterone synthesis. The modulation is mediated through enhancing of PKC-{varepsilon} and of [Ca2+]i elevation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Science Council Grants NSC-91-2314-B-002-340, 92-2314-B-002-190, and NSC93-2314-B-002-141 (to K.-D. Wu) and the Hsiu-Chin Lee Kidney Research Fund.


   ACKNOWLEDGMENTS
 
We thank the staffs of the 2nd Core Laboratory, Department of Medical Research, National Taiwan University Hospital, for technical support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K.-D. Wu, Rm. 1419, Clinical Research Bldg., Dept. of Internal Medicine, National Taiwan University Hospital, 7 Chung-Sun South Road, Taipei, Taiwan 100 (e-mail: kdw{at}ntumc.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. AbdAlla S, Lother H, el Massiery A, Quitterer U. Increased AT(1) receptor heterodimers in preeclampsia mediate enhanced angiotensin II responsiveness. Nat Med 7: 1003–1009, 2001.[CrossRef][Web of Science][Medline]
  2. Betancourt-Calle S, Calle RA, Isales CM, White S, Rasmussen H, Bollag WB. Differential effects of agonists of aldosterone secretion on steroidogenic acute regulatory phosphorylation. Mol Cell Endocrinol 173: 87–94, 2001.[CrossRef][Web of Science][Medline]
  3. Bodart V, Ong H, De Léan A. A role for protein tyrosine kinase in the steroidogenic pathway of angiotensin II in bovine zona glomerulosa cells. J Steroid Biochem Mol Biol 54: 55–62, 1995.[CrossRef][Web of Science][Medline]
  4. Bollag WB, Jung E, Calle RA. Mechanism of angiotensin II-induced phospholipase D activation in bovine adrenal glomerulosa cells. Mol Cell Endocrinol 192: 7–16, 2002.[CrossRef][Web of Science][Medline]
  5. Bornstein SR, Ehrhart-Bornstein M. Ultrastructural evidence for a paracrine regulation of the rat adrenal cortex mediated by the local release of catecholamines from chromaffin cells. Endocrinology 131: 3126–3128, 1992.[Abstract/Free Full Text]
  6. Chang HW, Chu TS, Huang HY, Chueh SC, Wu VC, Chen YM, Hsieh BS, Wu KD. Down-regulation of D2 dopamine receptor and increased protein kinase Cmu phosphorylation in aldosterone-producing adenoma play roles in aldosterone overproduction. J Clin Endocrinol Metab 92: 1863–1870, 2007.[Abstract/Free Full Text]
  7. Chu TS, Peng Y, Cano A, Yanagisawa M, Alpern RJ. Endothelin(B) receptor activates NHE-3 by a Ca2+-dependent pathway in OKP cells. J Clin Invest 97: 1454–1462, 1996.[Web of Science][Medline]
  8. Coca SG, Perazella MA. The role of aldosterone blockers in the management of chronic heart failure. Am J Med Sci 330: 176–183, 2005.[CrossRef][Web of Science][Medline]
  9. Ganguly A. Dopaminergic regulation of aldosterone secretion: how credible? Clin Sci (Lond) 66: 631–637, 1984.[Medline]
  10. Glase SA, Akunne HC, Georgic LM, Heffner TG, MacKenzie RG, Manley PJ, Pugsley TA, Wise LD. Substituted [(4-phenylpiperazinyl)-methyl]benzamides: selective dopamine D4 agonists. J Med Chem 40: 1771–1772, 1997.[CrossRef][Web of Science][Medline]
  11. Gu Z, Yan Z. Bidirectional regulation of Ca2+/calmodulin-dependent protein kinase II activity by dopamine D4 receptors in prefrontal cortex. Mol Pharmacol 66: 948–955, 2004.[Abstract/Free Full Text]
  12. Jarvis WD, Judd AM, MacLeod RM. Attenuation of anterior pituitary phosphoinositide phosphorylase activity by the dopamine receptor. Endocrinology 123: 2793–2799, 1988.[Abstract/Free Full Text]
  13. Joffe HV, Adler GK. Effect of aldosterone and mineralocorticoid receptor blockade on vascular inflammation. Heart Fail Rev 10: 31–37, 2005.[CrossRef][Web of Science][Medline]
  14. Johnson JA, Gray MO, Karliner JS, Chen CH, Mochly-Rosen D. An improved permeabilization protocol for the introduction of peptides into cardiac myocytes. Application to protein kinase C research. Circ Res 79: 1086–1099, 1996.[Abstract/Free Full Text]
  15. Kapas S, Purbrick A, Hinson JP. Role of tyrosine kinase and protein kinase C in the steroidogenic actions of angiotensin II, alpha-melanocyte-stimulating hormone and corticotropin in the rat adrenal cortex. Biochem J 305: 433–438, 1995.[Web of Science][Medline]
  16. LeHoux JG, Dupuis G, Lefebvre A. Control of CYP11B2 gene expression through differential regulation of its promoter by atypical and conventional protein kinase C isoforms. J Biol Chem 276: 8021–8028, 2001.[Abstract/Free Full Text]
  17. LeHoux JG, Lefebvre A. Transcriptional activity of the hamster CYP11B2 promoter in NCI-H295 cells stimulated by angiotensin II, potassium, forskolin, and bisindolylmaleimide. J Mol Endocrinol 20: 183–191, 1998.[Abstract]
  18. Li J, Feltzer RE, Dawson KL, Hudson EA, Clark BJ. Janus kinase 2 and calcium are required for angiotensin II-dependent activation of steroidogenic acute regulatory protein transcription in H295R human adrenocortical cells. J Biol Chem 278: 52355–52362, 2003.[Abstract/Free Full Text]
  19. Liu J, Kahri AI, Heikkilä P, Ilvesmäki V, Voutilainen R. H19 and insulin-like growth factor-II gene expression in adrenal tumors and cultured adrenal cells. J Clin Endocrinol Metab 80: 492–496, 1995.[Abstract]
  20. Missale C, Memo M, Liberini P, Spano P. Dopamine selectively inhibits angiotensin II-induced aldosterone secretion by interacting with D-2 receptors. J Pharmacol Exp Ther 246: 1137–1143, 1988.[Abstract/Free Full Text]
  21. Patel S, Freedman S, Chapman KL, Emms F, Fletcher AE, Knowles M, Marwood R, McAllister G, Myers J, Curtis N, Kulagowski JJ, Leeson PD, Ridgill M, Graham M, Matheson S, Rathbone D, Watt AP, Bristow LJ, Rupniak NM, Baskin E, Lynch JJ, Ragan CI. Biological profile of L-745,870, a selective antagonist with high affinity for the dopamine D4 receptor. J Pharmacol Exp Ther 283: 636–647, 1997.[Abstract/Free Full Text]
  22. Pezzi V, Clyne CD, Ando S, Mathis JM, Rainey WE. Ca(2+)-regulated expression of aldosterone synthase is mediated by calmodulin and calmodulin-dependent protein kinases. Endocrinology 138: 835–838, 1997.[Abstract/Free Full Text]
  23. Pilon A, Martin G, Bultel-Brienne S, Junquero D, Delhon A, Fruchart JC, Staels B, Clavey V. Regulation of the scavenger receptor BI and the LDL receptor by activators of aldosterone production, angiotensin II and PMA, in the human NCI-H295R adrenocortical cell line. Biochim Biophys Acta 1631: 218–228, 2003.[Medline]
  24. Pivonello R, Ferone D, de Herder WW, de Krijger RR, Waaijers M, Mooij DM, van Koetsveld PM, Barreca A, De Caro ML, Lombardi G, Colao A, Lamberts SW, Hofland LJ. Dopamine receptor expression and function in human normal adrenal gland and adrenal tumors. J Clin Endocrinol Metab 89: 4493–4502, 2004.[Abstract/Free Full Text]
  25. Rasolonjanahary R, Gerard C, Dufour MN, Homburger V, Enjalbert A, Guillon G. Evidence for a direct negative coupling between dopamine-D2 receptors and PLC by heterotrimeric Gi1/2 proteins in rat anterior pituitary cell membranes. Endocrinology 143: 747–754, 2002.[Abstract/Free Full Text]
  26. Rocha R, Martin-Berger CL, Yang P, Scherrer R, Delyani J, McMahon E. Selective aldosterone blockade prevents angiotensin II/salt-induced vascular inflammation in the rat heart. Endocrinology 143: 4828–4836, 2002.[Abstract/Free Full Text]
  27. Smith FD, Oxford GS, Milgram SL. Association of the D2 dopamine receptor third cytoplasmic loop with spinophilin, a protein phosphatase-1-interacting protein. J Biol Chem 274: 19894–19900, 1999.[Abstract/Free Full Text]
  28. Spat A, Hunyady L. Control of aldosterone secretion: a model for convergence in cellular signaling pathways. Physiol Rev 84: 489–539, 2004.[Abstract/Free Full Text]
  29. Takeda Y, Miyamori I, Yoneda T, Hatakeyama H, Inaba S, Furukawa K, Mabuchi H, Takeda R. Regulation of aldosterone synthase in human vascular endothelial cells by angiotensin II and adrenocorticotropin. J Clin Endocrinol Metab 81: 2797–2800, 1996.[Abstract/Free Full Text]
  30. Takeda Y, Yoneda T, Demura M, Miyamori I, Mabuchi H. Cardiac aldosterone production in genetically hypertensive rats. Hypertension 36: 495–500, 2000.[Abstract/Free Full Text]
  31. Wilke RA, Hsu SF, Jackson MB. Dopamine D4 receptor mediated inhibition of potassium current in neurohypophysial nerve terminals. J Pharmacol Exp Ther 284: 542–548, 1998.[Abstract/Free Full Text]
  32. Wu KD, Chen YM, Chu TS, Chueh SC, Wu MH, Bor-Shen H. Expression and localization of human dopamine D2 and D4 receptor mRNA in the adrenal gland, aldosterone-producing adenoma, and pheochromocytoma. J Clin Endocrinol Metab 86: 4460–4467, 2001.[Abstract/Free Full Text]
  33. Yagci A, Müller J. Induction of steroidogenic enzymes by potassium in cultured rat zona glomerulosa cells depends on calcium influx and intact protein synthesis. Endocrinology 137: 4331–4338, 1996.[Abstract]
  34. Yang C, Kazanietz MG. Divergence and complexities in DAG signaling: looking beyond PKC. Trends Pharmacol Sci 24: 602–608, 2003.[CrossRef][Medline]
  35. Yang SD, Chang HC, Lee SC. Okadaic acid, sphingosine, and phorbol ester reversibly modulate heat induction on protein kinase FA/GSK-3 alpha in A431 cells. J Cell Biochem 60: 218–225, 1996.[CrossRef][Web of Science][Medline]
  36. Zheng X, Bollag WB. AngII induces transient phospholipase D activity in the H295R glomerulosa cell model. Mol Cell Endocrinol 206: 113–122, 2003.[CrossRef][Web of Science][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/3/E622    most recent
00657.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chang, H.-W.
Right arrow Articles by Hsieh, B.-S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chang, H.-W.
Right arrow Articles by Hsieh, B.-S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.