Page 1Articles of 37 in PresS. Am J Physiol Endocrinol Metab (January 2, 2008). doi:10.1152/ajpendo.00657.2007
D4 Dopamine Receptor enhances angiotensin-II stimulated aldosterone secretion through PKC and calcium signaling
Hong-Wei Chang, Vin-Cent Wu, Chao-Yuan Huang*, Hong-Yu Huang, Yung-Ming Chen, Tzong-Shinn Chu, Kwan-Dun Wu, Bor-Shen Hsieh Nephrology Division, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan *, Department of Urology, National Taiwan University Hospital, Taipei, Taiwan Short Title: Dopamine Receptor in Aldosterone Secretion Keywords: Dopamine Receptor, Aldosterone producing adenoma, Protein Kinase C, Hypertension, Calcium Signaling. Disclosure statement: The authors have nothing to disclose Figures: 7; Abstract: 250 Correspondence: Kwan-Dun Wu, MD., PhD. Room 1419, Clinical Research Building, Department of Internal Medicine, National Taiwan University Hospital, 7 Chung-Sun South Road, Taipei, Taiwan 100
[email protected], Fax: +886-2-23934176
Copyright © 2008 by the American Physiological Society.
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ABSTRACT Background: 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 Cµ (PKCµ) and calcium-dependent signaling. The mechanism of D4R effect on angiotensin II (AII)-stimulated aldosterone secretion is explored in this study. Methods: 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). Results: The D4R agonist, PD168, 077, enhanced AII-stimulated aldosterone synthesis and secretion as early as 30 minutes following exposure, independent of the modulation of aldosterone synthase (CYP11B2) transcription. CYP11B2 mRNA level elevated by AII was augmented by D4R in later period. These effects were reversed by the D4R antagonist, L745, 870. AII activated PKC / II, PKC , and PKCµ, but not PKC ,
or / of H295R cells. The
D4R agonist selectively enhanced AII-stimulated PKC 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 translocation by the PKC -specific inhibitory
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peptide attenuated AII-stimulated aldosterone secretion, CYP11B2 mRNA expression, and elevation of intracellular IP3 and [Ca2+]i. Conclusion: D4R augmented aldosterone synthesis/secretion induced by AII. The mechanisms responsible for this augmentation are mediated through enhancing PKC phosphorylation and [Ca2+] i elevation.
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INTRODUCTION There is increasing evidence that aldosterone plays a direct role in the pathogenesis of chronic heart failure and vascular inflammation (1, 2). The vascular and perivascular inflammatory responses to angiotensin II (AII) infusion and salt-loading, both reported to increase cardiovascular aldosterone synthesis (3, 4), are completely prevented by adrenalectomy (5).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 is 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 (6). Dopamine D2-like receptors have been found to play a pivotal role in inhibiting aldosterone secretion (7, 8). We and other investigators 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 (9, 10), and exert opposite effects on aldosterone secretion (9). The D2R attenuates AII-induced secretion of aldosterone, whereas the D4R enhances that effect. An increase of intracellular Ca2+ ([Ca2+]i) plays as an important signal in AII-induced
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aldosterone secretion and CYP11B2 mRNA expression (11, 12), and involvement of protein kinase C (PKC) has also been noted in the regulation of adrenal steroidogenic genes (13-15). Our recent work has revealed that D2R modulates AII-stimulated aldosterone synthesis and secretion through attenuation of PKCµ phosphorylation and [Ca2+]i elevation (16). Paradoxically, constitutive activation of some subtypes of PKC has been shown to inhibit AII-stimulated CYP11B2 gene expression (17, 18). Therefore, the expression of the CYP11B2 gene may be differentially regulated by different PKC subtypes (17). In the current 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 , and [Ca2+]iele vation.
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MATERIAL AND METHODS Materials AII, PD168,077, L745,870 were purchased from Sigma Chemical Co. (St. Louis, MO, USA), and phospho-PKC-specific antibodies and PKC subtype-specific antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). PD168,077 and L745,870 are highly selective agonist and antagonist for D4R respectively (19, 20). PKC translocation inhibitor peptide and PKC translocation inhibitor peptide-negative controls were obtained from Calbiochem (Cambridge, MA, USA). An IP3 RRA (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, USA). AT1R antibody and its immunizing peptide were obtained from Santa Cruz (AT1N10 and sc1173, respectively; Santa Cruz, CA, USA). 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 pre-immunized 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 the American Type Culture Collection (ATCC, Rockville, MD, USA). The primary human adrenal
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cortical cells were prepared from surgical specimen of the patients without any adrenal disease, and the study was approved by the ethical committee of the National Taiwan University Hospital. The cell culture experiments have been described previously (9). The cells expressed D4R, and significantly secreted aldosterone after addition of AII. After testing the doses of the drugs that regulate aldosterone secretion/synthesis, AII 10 nmol/L, PD168,077 (PD) 10-6mol/L, and L745,870 (L) 10-6mol/L were used in all the experiments of 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.
Immunoblotting For PKC assays, H295R cells were scraped 5 minutes 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, USA).
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
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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, CA, USA), according to the manufacturer’s instructions. RNA was subjected to deoxyribonuclease (DNase) treatment using 1 U DNase I (amp grade)/µg RNA was incubated in DNase reaction buffer for 15 min at 37 °C (NEB, Beverly, MA, USA) to remove genomic DNA contamination. The reaction was stopped by heating to 99 °C for 5 min. RNA was reverse-transcribed by using a reverse transcription system kit (Promega, Madison, WI, USA), 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 Hs00609526_m1 and Hs01597732_m1; (Applied Biosystems, CA, USA). Assay Hs01597732_m1 with probe locating over 963 regions on NM 000498 did not detect CYP11B1 mRNA. GAPDH (assay ID Hs99999905_m1) was used as an endogenous control in a TaqMan human endogenous control plate. Sample dilutions were comprised of 100 ng 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 level of D4R and CYP11B2 were corrected with the mRNA level of GAPDH, and expressed as 2-
CT
, where
CT = ( CTx – CTGAPDH, X= D4R and CYP11B2). The mRNA level of the
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tumor portion was corrected with that of the non-tumor portion.
Isolation of membrane protein. Cells were scraped in ice cold 1x PBS and centrifuged at 600 x 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 1000 x g in 4 °C for 5 min to separate the nuclear portion. The supernatant was centrifuged again at 53,000 x g in 4 °C for 30 min. The pellet containing the membrane portion was resuspended in RIPA buffer (20mmol/L Tris-HCl, pH7.4; 150 mmol/L NaCl; 2mol/L EDTA; 0.1% Triton X100; 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 (22). In brief, cells were cultured in serum-free media for 24 hours. The cells were incubated and washed by PBS at room temperature and in an ice bath in two sequential 2-minute intervals. Thereafter, the cells were incubated with freshly prepared permeabilization buffer containing the desired peptides for 10 minutes in an ice bath. ATP was added just before adding the permeabilization buffer to cells. The cells were then gently washed four times on ice and recovered in PBS on ice for 20 minutes. The cells were placed in PBS at room temperature
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for 2 minutes. 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 hours 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, USA). The fluorescent ratio was calibrated by adding digitonin to a final concentration of 75 µg/ml and then adding 1mM EDTA at a 1:50 dilution and 10 N NaOH at a 1:700 dilution, and [Ca2+]i was calculated as described (23).
Determination of inositol 1, 4, 5 trisphosphate. Cells were washed twice and pre-incubated 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 sec, 250 µl ice cold perchloric acid (10% vol/vol) was added to
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terminate the response. After scraping, the cells were washed with 250 µl 10% perchloric acid and centrifuged at 12,000 x g for 5 min at 4°C. The supernatants were neutralized with 50 µl 1.5 M NaOH in the presence of universal indicator. IP3 levels were measured by a specific competitive binding assay kit (PerkinElmer Life Sciences, Inc.). Each incubation contained 500 µl receptor preparation/ [3 H]-IP3 tracer 1:15 (vol/vol), 100 µl of standard IP3 (0–120 pmol/0.1ml), or cell extract. The tubes were agitated and incubated for 45 min on ice. Incubations were terminated by centrifugation at 12,000 x 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 Utest, using the Stat View software package [Abacus Concepte, Inc., Berkeley, CA, USA]. Statistical significance was considered at the 5% level.
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RESULTS D4 receptor was expressed on both human adrenal cortex and H295R cells In the previous study (9), 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. D4 receptor modulated AII-stimulated CYP11B2 mRNA expression and aldosterone secretion The D4R-agonist, PD168,077 (10-6 mol/L), augmented the increase of AII-stimulated 24hr 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 (upper panels of Fig. 2A and 2B). Both the effects of PD168,077 and L745,870 were concentration-dependent from 10-9 to 10-6 mol/L (data not shown). Consistent with its effect on AII-stimulated aldosterone secretion, PD168,077 at 10-6 mol/L effectively augmented AII-stimulated CYP11B2 mRNA expression as early as 2 h after exposure and for up to 24 h (data not shown), and L745,870 reversed the effect of PD168,077. The CYP11B2 mRNA expression of primary human adrenal cortical cells and H295R cell 4 h after treatment was
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illustrated (Lower panels of Fig. 2A and 2B). The augmented effect of PD168,077 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 L745,870 (Fig. 2C and 2D). In addition to the elevation of aldosterone levels in the supernatant, the intracellular aldosterone level was also increased by AII, which was augmented by PD168,077, and L745,870 reversed the effect of PD168,077. 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 PKCa/ßII Thr638/641, PKC Ser729, PKCµ Ser744/748, and PKCµ Ser916 , but not of PKC Thr505,
Thr538, or / Thr410/403 on both primary human adrenal cortical cells and H295R
cells (16). Addition of PD168,077 did not alter the basal or AII-stimulated phosphorylation of PKC / II Thr638/641, PKCµ Ser916, and PKCµ Ser744/748, but significantly enhanced the AII-stimulated phosphorylation of PKC Ser729 ; L745,870 reversed this augmenting effect of PD168,077 (Fig. 3A and 3B). Immunoblotting with antibodies for total forms of PKC , PKC II, PKC , and PKCµ 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
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distributions (Fig. 3C). PD168,077 enhanced the AII-induced translocation of PKC to the membrane accompanied with a reciprocal changed PKC in the cytosol, and exerted no effect on other PKC isoforms. The enhancing effect was again reversed by L745,870. Therefore, D4R selectively augmented AII-stimulated PKC activation. PKC -specific peptide inhibited AII-induced aldosterone secretion and CYP11B2 mRNA expression Permeabilization of the synthetic PKC -specific inhibitory peptide into H295R cells selectively inhibited the AII-stimulated translocation of PKC 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. D4R and PKC modulated an AII-stimulated [Ca2+]i increase The AII-stimulated increase of [Ca2+]i was augmented by the D4R agonist, PD168,077. PD168,077 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 PD168, 077 on AII stimulated increased of [Ca2+]i was also observed on the similar experiments with the Ca2+-free medium (Fig 5B). Delivering the PKC inhibitory peptide attenuated the AII-stimulated peak [Ca2+]i, the increased rate of
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[Ca2+]I (slope), and the plateau of [Ca2+]i (Fig. 5C). 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 pre-treatment with PD168,077. The augmenting effect of PD168,077 was reversed by the D4R antagonist, L745,870 (Fig. 6). Additionally, the AII-stimulated elevation of intracellular IP3 was significantly attenuated by the PKC inhibitory peptide (Fig. 6).
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DISCUSSION As shown in our previous study (9), 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 showed that the inhibitory effect of D2R was exerted through attenuation of PKCµ phosphorylation and [Ca2+]i elevation induced by AII (16). The augmentation of aldosterone secretion/synthesis by D4R is mediated through another specific PKC isoform, PKC , 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 (24-25), the results lacked consensus. An increase of aldosterone secretion was observed when the cells were treated with PKC activator (24-25), and a PKC inhibitor reduced AII-stimulated aldosterone secretion (26-27). However, by transfection of H295 cells with constitutively active PKC isoforms, LeHoux et al. observed that PKC , , and
inhibited basal and stimulatory hamster CYP11B2 promoter
activity (17), whereas PKC 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 (28). Second, cells subjected to prolonged treatment with phorbol ester or transfected with
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constitutively active PKC may extensively down-regulate the PKC activities and the downstream signaling, rather than activate them (29-30). Finally, although kinase-inactive PKC mutants and PKC regulatory domains have been widely used as dominant-negative inhibitors, the high homology among PKC isoforms and the lack of knowledge about the intracellular targets of these mutants makes this approach questionable (28). H295R cells express various PKC isoforms, whereas AII only increased the activities of PKC , II, , and µ, but not of PKC , or . Among all PKC isoforms that were activated by AII, only the phosphorylation of PKC was altered by D4R. By inducting the PKC specific inhibitory peptide, we found that the activation of PKC contributed partially, at least 25%, to the aldosterone synthesis/secretion induced by AII. Recently, we found that inhibition of PKCµ activity by its specific shRNA could reduce AII-stimulated CYP11B2 mRNA level and aldosterone secretion by approximately 80% (16). Therefore, activations of both PKCµ and PKC account for most AII-induced aldosterone synthesis/secretion. In comparison, PKCµ may be more important in the regulation of aldosterone synthesis/secretion than PKC . This speculation is supported by our recent data that an increase in PKCµ phorsphorylation is ubiquitously present in aldosterone-producing adenomas (16). 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
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may involve several mechanisms: (1) transfer of free cholesterol to the outer mitochondrial membrane via a calmodulin-dependent kinase II (CaMKinase II), (2) activation of the StAR protein by a CaMKinase II-mediated process, and (3) increase in mitochondrial [Ca2+] concentration leading to activation of mitochondrial dehydrogenases of the tricarboxylic acid cycle and NAD(P)H production (30). D4R, like D2R, can modulate the [Ca2+]i induced by AII, and in turn aldosterone secretion. The modulation of [Ca2+]iby D4R is probably through its effect on intracellular IP3 production. Stimulation of the AT1 receptor 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 (DAG); the latter can activate protein kinase C (PKC). We noted that D4R can enhance AII-induced elevation of [Ca2+]i, even when the extracellular calcium was absent. This result suggests that D4R enhances AII-induced [Ca2+]ielevation via increasing [IP3] i level, which through IP3 receptors release calcium from the intracellular calcium stores. Interestingly, inhibition of PKC activity can attenuate AngII-induced elevation of intracellular IP3 and [Ca2+]i. Therefore, the effect of D4R on [Ca2+]ielevation can also be mediated by activation of PKC , which is calcium-independent (28). However, the augmentation of [Ca2+]i by D4R occurred much earlier (within seconds) than its effect on AII-induced PKC activation (within minutes) (data not shown). Therefore, the augmentation of AII-induced elevation of [Ca2+]i by D4R may be
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biphasic. How can D4R enhance the immediate AngII-stimulated [Ca2+]i increase? D2R has been reported to have a direct negative coupling with phospholipase C (PLC) by heterotrimeric Gi1/2 proteins in rat anterior pituitary cell membrane. Dopamine and D2 agonists inhibited TRH- and AngII-stimulated membrane PLC activities (31). Our recent report has shown D2 agonist, bromocriptine, can attenuate AII-stimulated intracellular IP3 accumulation that also suggested there is negative regulation of D2R on the PLC activity. Opposing to D2R, we found that D4R enhanced AII-stimulated intracellular IP3 accumulation. D4R has been proved to stimulate PLC activity in prefrontal cortex (32). It is possible that D4R could be able to enhance PLC activity through its coupling G protein components. By enhancing AngII-stimulated PLC activity, D4R could increase AngII-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 only occurred 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 down stream signaling (33). D4R has been reported to mediate inhibition of potassium current in neurophysial nerve terminals (34). D4R could also inhibit the potassium current of H25R cells and partially depolarize the membrane potential that enhances AII-induced T-type calcium channel opening. D2R may regulate the “negative-regulating protein”, such as
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phosphatases, through spinophilin, a protein phophatase-1 interacting protein (35). It is also possible that D4 inhibits the phosphatase which plays role in turning down the activated PKC from the AT1 receptor. In addition, we found that inhibiting activation of PKC attenuated both AII-induced intracellular free calcium increase and IP3 accumulation. Though this mechanism is still not clear, it is possible that PKC enhanced PLC activity in the adrenal cortical cells. By way of inhibition of PLC activity, PKC inhibitory peptide attenuated AII-induced IP3 accumulation and intracellular free calcium increase. We illustrate the possible signals of D4R-modulated effects in figure 7. In summary, similar 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 up-regulates CYP11B2 expression, but also enhances acute aldosterone synthesis. The modulation is mediated through enhancing PKC and of [Ca2+]i elevation.
Acknowledgement This work was supported by the grants from the National Science Council, NSC91-2314-B-002-340, 92-2314-B-002-190 and NSC93-2314-B-002-141 (to KD Wu), and the Mrs. Hsiu-Chin Lee Kidney Research Fund. We thank the staffs of the 2nd Core Lab, Department of Medical Research, National Taiwan University Hospital for technical supports.
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Figure 1.
Expression of D4R and AT1R in human adrenal cortex (lane 1), primary cultured
cells of human adrenal cortex (lane2) and H295R cells (lanes 3-5). Immunoblotting with specific antiserum to D4R or AT1R (lanes 1-3) revealed 46 kDa- and 42 kDa-sized bands, respectively. The bands were almost absent when neutralizing the primary antibodies with their respective immunizing peptides (lane 4) or using pre-immunized serum in place of the D4R antiserum (lane 5).
Figure 2.
Modulation of D4R on AII-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 (upper panel) and CYP11B2 mRNA expression 4 h after treatment (lower panel), * significantly different from AII, (p
