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Activation of Angiotensin II Subtype 2 Receptor Induces Catecholamine Release in an Extracellular Ca²⁺-Dependent Manner through a Decrease of Cyclic Guanosine 3',5'-Monophosphate Production in Cultured Porcine Adrenal Medullary Chromaffin Cells¹

Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

Address all correspondence and requests for reprints to: Dr. Kazuhiro Takekoshi, Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8575, Japan. E-mail: k-takemd{at}md.tsukuba.ac.jp

	Abstract

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We have previously demonstrated that CGP 42112 (AT₂ agonist: 1 nM) markedly reduces catecholamine biosynthesis through AT₂, which is the major angiotensin II (AngII) receptor subtype in cultured porcine chromaffin cells. Also, we have shown that CGP 42112 (1 nM) induces a reduction in cGMP production in these cells. The present study showed that AngII reduced cGMP production via AT₂ in a manner similar to that found with CGP 42112. AngII (1 nM) significantly increased catecholamine secretion from cultured porcine adrenal medullary chromaffin cells. The stimulation was significantly inhibited by PD 123319 (AT₂ antagonist). The stimulation was moderately, but significantly, attenuated by CV-11974 (AT₁ antagonist, 10 nM), suggesting an involvement of AT₁. Moreover, CGP 42112 (10 nM) markedly increased catecholamine release from these cells. The stimulation by CGP 42112 was abolished by PD 123319, whereas CV-11974 had no effect, indicating that this response is also mediated by AT₂. We further examined whether extracellular Ca²⁺ is involved in the stimulatory effect of AT₂ on catecholamine secretion. Removal of external Ca²⁺ significantly suppressed either AngII plus CV-11974 (100 nM; which simulates specific AT₂ stimulation) or CGP 42112- induced catecholamine secretion. AngII plus CV-11974 or CGP 42112 caused a sustained increase in intracellular Ca²⁺ ([Ca²⁺]_i), as determined in fura-2-loaded chromaffin cells in an extracellular Ca²⁺-dependent manner. In the presence of EGTA, the subsequent addition of AngII with CV-11974 and CGP 42112 did not cause any increase in [Ca²⁺]_i levels. Consistent with this finding, CGP 42112 (10 nM to 1 µM) did not alter inositol triphosphate (IP₃) production, a messenger for mobilization of Ca²⁺ from intracellular storage sites. In addition, the intracellular Ca²⁺ chelator 1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'- tetraacetic acid acetoxymethylester (BAPTA) did not affect CGP 42112-induced catecholamine release. We tested whether a decrease in cGMP was the cause of the stimulatory effect of AT₂ on catecholamine secretion. Pretreatment with 8-bromo-cGMP (1 mM) prevented the stimulatory effect of AngII plus CV-11974 and CGP 42112 on both catecholamine secretion and [Ca²⁺]_i. When 8-bromo-cGMP was added after application of AngII plus CV-11974 or CGP 42112, [Ca²⁺]_i induced by these agents was gradually reduced toward the baseline values. Similarly, guanylin completely abolished the AngII- plus CV-11974-induced increase in both NE secretion and [Ca²⁺]_i. The Ca²⁺ channel blockers, nicardipine and -conotoxin G VIA, at 1 µM in both cases, were also effective in inhibiting AT₂ stimulation-induced secretion. On the other hand, neither T-type voltage-dependent Ca²⁺ channel blockers, flunarizine, nor Ni²⁺ affected catecholamine release caused by AT₂ stimulation. These findings demonstrate that AT₂ stimulation induces catecholamine secretion by mobilizing Ca²⁺ through voltage-dependent Ca²⁺ channels without affecting intracellular pools and that these effects could be mediated by a decrease in cGMP production.

	Introduction

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ANGIOTENSIN II (AngII) is known to exert its actions through two major subtypes of receptors, AngII subtype 1 and subtype 2 receptors (AT₁ and AT₂) (1, 2, 3). Most of AngII’s physiological effects, such as those exerted on the cardiovascular system and fluid volume homeostasis, are mediated by AT₁; these effects are linked to 1,4,5-inositol triphosphate (IP₃) production after phospholipase C activation, resulting in mobilization of intracellular Ca²⁺ (1).

In contrast to AT₁, little is known about the physiological role of and signal transduction pathways connected with AT₂. AT₂ was assumed to be involved in cell growth, differentiation, and apoptosis (4, 5, 6). AT₂ is highly expressed in the developing fetus (7). In contrast, in adults, AT₂ expression is restricted to only a few organs and cell lines, such as the brain, uterus, and cardiac myocytes (8, 9, 10). Indeed, AT₂ is abundantly expressed in the adrenal medulla of adult rats (11, 12). In addition, it was demonstrated that AT₂ participates in cGMP regulation. Stimulation of AT₂ induced a decrease in intracellular cGMP content in neuronal cells, including chromaffin cells (13, 14). However, the exact physiological role(s) of AT₂ in adrenal chromaffin cells remains to be clarified.

AngII is a secretogogue for catecholamine secretion that is believed to be mediated through IP₃ production by AT₁ (15, 16). Indeed, Wong et al. demonstrated that AngII-induced catecholamine release is mediated by AT₁ in the rat adrenal medulla (15). AT₁-mediated phospholipase C activation and subsequent IP₃ formation may increase cytosolic Ca²⁺ levels by releasing Ca²⁺ from intracellular storage, with subsequent activation of catecholamine release (14). Indeed, it has been shown that addition of IP₃ to permeabilized bovine chromaffin cells releases intracellular Ca²⁺ (17). Furthermore, addition of Ca²⁺ to permeabilized bovine chromaffin cells was reported to cause catecholamine secretion (18).

Another important mechanism for regulating the intracellular concentration of calcium ([Ca²⁺]_i) involves the use of voltage-dependent Ca²⁺ channels (VDCC) that mobilize Ca²⁺ entirely from extracellular Ca²⁺ pools localized on the outer surface of chromaffin cell membranes (19). It is still controversial whether the increase in Ca²⁺ entry through VDCC is involved in AngII-induced stimulation of catecholamine secretion. Several studies suggested that extracellular Ca²⁺ was also involved in the secretory mechanisms mediated by AngII in chromaffin cells (19, 20, 21, 22).

Belloni et al. recently demonstrated that stimulation of AT₂ induced catecholamine release from the adrenal medulla (23). Catecholamine secretion in response to AngII was markedly inhibited by PD 123319 (AT₂ antagonist) in the rat adrenal medulla. They also showed that the stimulation of catecholamine secretion caused by CGP 42112 (AT₂ agonist) was blocked by PD 123319 (AT₂ antagonist), but was not affected by DuP 753 (AT₁ antagonist), confirming that the stimulation by CGP 42112 is mediated by AT₂. However, these investigators did not study the detailed mechanism of AT₂-mediated catecholamine secretion.

It has been reported that the cGMP/cGMP-dependent protein kinase (PKG) pathway inhibits catecholamine release by inhibition of Ca²⁺ mobilization through VDCC in chromaffin cells (24, 25, 26, 27). Indeed, it has previously been shown that cGMP-elevating agents, such as nitric oxide (NO) and natriuretic peptides (NPs; atrial, brain, and C-type), inhibit catecholamine release through inhibition of VDCC in a cGMP/PKG-dependent manner (27).

Using cultured porcine chromaffin cells (28), we have previously shown that 1) AT₂ is predominantly expressed in these cells; 2) CGP 42112 (1 nM) significantly inhibited cGMP production, as measured from the basal level; 3) CGP 42112 ( BORDER="0">1 nM) significantly inhibited tyrosine hydroxylase (TH; the rate-limiting enzyme in the biosynthesis of catecholamine) enzyme activity along with TH messenger RNA and protein levels; 4) pretreatment with 8-Br-cGMP (a membrane-permeable cGMP analog) prevented the inhibitory effect of CGP 42112 on TH enzyme activity. These findings suggest that the AT₂ agonist, CGP 42112, inhibits catecholamine biosynthesis through a decrease in cGMP production in cultured porcine adrenal medullary cells.

The major aim of the present study, then, is to clarify the details of this intriguing mechanism of AT₂-induced catecholamine secretion in cultured porcine adrenal medullary cells.

	Materials and Methods

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Reagents
Unless otherwise noted, all reagents were purchased from Wako Seiyaku (Tokyo, Japan). The AT₂ agonist, CGP 42112, was purchased from the Peptide Institute (Osaka, Japan). The AT₂ antagonist, PD123319, was purchased from Funakoshi (Tokyo, Japan). The AT₁ antagonist, CV-11974, was provided by Takeda Chemical Industries Co., Ltd. (Osaka, Japan). 1,2-Bis(2-amino-phenoxy)ethane-N,N,N',N'- tetraacetic acid acetoxymethylester (BAPTA), N^G-nitro-L-arginine methyl ester (L-NAME), and guanylin were purchased from Sigma (St. Louis, MO).

Cell culture
Dissociated primary cells, derived from porcine adrenal medulla, were prepared and purified by the differential plating method as previously described (29, 30, 31). In brief, the cells (1 x 10⁶) were plated into 35-mm polystyrene dishes and maintained as a monolayer culture in DMEM (Life Technologies, Inc., Gaithersburg, MD) containing 10% FBS (Life Technologies, Inc.), 100 U/ml penicillin, 100 µg/ml streptomycin, and 1.3 µg/ml fungizone in a humidified atmosphere of 5% CO₂/95% O₂ at 37 C for 2–3 days before being used for experiments.

Measurement of cGMP production
Briefly, cells were washed twice with Eagle’s MEM (EM) and preincubated in EM containing 0.2 mM 3-isobutyl-1-methylxanthine (IBMX) for 5 min. Experiments were initiated by replacing the medium with HEPES-buffered Krebs buffer including the test substances and 0.2 mM IBMX, with the cells subsequently incubated at 37 C for 10 min. The reaction was terminated by adding 100 µl of 1 N HCl followed by incubation on ice for 30 min. The cGMP in the acid extract was then measured using a cGMP kit (Pharmacia Biotech, Piscataway, NJ).

Determination of catecholamine content
Catecholamine concentrations in media were determined as previously described (29), using a catecholamine autoanalyzer (H8030, TOSOH, Tokyo, Japan) with a built-in high performance liquid chromatograph and a spectrofluorometer.

Effect of removal of external Ca²⁺ on AngII with CV-11974- or CGP 42112-induced catecholamine release
Cells were treated with AngII with CV-11974 or CGP 42112 (100 nM) in Ca²⁺-free medium containing 0.1 mM EDTA for 30 min, and the resulting media were examined with a catecholamine analyzer as described above.

Measurement of [Ca²⁺]_i with fura-2
Cells were cultured on collagen-coated 96-well plates. The cells were incubated with 4 µM fura-2 acetoxymethylester at 37 C for 30 min and then washed twice with HEPES-buffered Krebs buffer. The level of [Ca²⁺]_i was measured in fura-2-loaded chromaffin cells using the Multi Cell-based Assay system (FDSS2000, Hamamatsu Photonics, Hamamatsu, Japan), using excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm.

Measurement of the production of IP₃
Production of inositol IP₃ was measured using a specific IP₃ binding assay kit (Pharmacia Biotech). Briefly, the cells were washed twice with EM containing 0.5% BSA, then stimulated with various concentrations (10 nM to 1 µM) of CGP 42112 or AngII (1 nM) with or without PD123319 (100 nM) or CV-11974 (100 nM) for 10 min. The reaction was quenched by removal of the medium, followed by rapid mixing of an equal volume of ice-cold 15% trichloroacetic acid. After sedimentation of the resulting precipitates, the supernatants were extracted three times with 10 vol H₂O₂-saturated diethyl ether and evaporated to dryness, and the pH was adjusted to 7.5 with NaHCO₃. The amount of IP₃ in the sample was determined according to the manufacturer’s protocol for the above-mentioned assay kit.

Effects of pretreatment with 8-bromo-cGMP (8-Br-cGMP) on AngII- plus CV-11974-induced or CGP 42112-induced catecholamine secretion and [Ca²⁺]_i
Cells were preincubated with 1 mM 8-Br-cGMP for 30 min (26). Thereafter, they were treated with AngII and CV-11974 or CGP 42112 (100 nM). Catecholamine and [Ca²⁺]_i levels were measured as described above.

Effects of pretreatment with guanylin on AngII with CV-11974-induced catecholamine secretion and [Ca²⁺]_i
Cells were preincubated with 10 nM guanylin for 30 min (32). Subsequently, identical experiments, as described above in the case of 8- Br-cGMP, were carried out.

Determination of NOx (NO^2-/NO^3-)
Briefly, cells were washed twice with EM. Experiments were initiated by replacing the medium with HEPES-buffered Krebs buffer along with the test substances, and the cells were subsequently incubated at 37 C for 1 min. The NOx in the extract was then determined according to the manufacturer’s protocol for the assay kit (Cayman Chemicals, Ann Arbor, MI).

Statistical analysis
Data were analyzed between groups by one-way ANOVA using means derived from the StatView computer software program (Abacus Concepts, Inc., Berkeley, CA). When ANOVA showed significant differences, post-hoc analysis was performed using Tukey’s test. P < 0.05 was considered significant. All data are expressed as the mean ± SD.

	Results

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Effect of AngII on cGMP level in cultured porcine adrenal medullary cells
It was demonstrated by us and others that stimulation of AT₂ by either AngII or CGP 42112 caused a decrease in intracellular cGMP content in neuronal cells (13, 14, 28). To further elaborate on these findings, we examined the effect of AngII on cGMP production. As shown in Fig. 1, AngII (1 nM) significantly inhibited cGMP production from the basal level. Also, the inhibitory effect of AngII on cGMP production was abolished by PD 123319 (AT₂ antagonist) in a dose-dependent manner. In contrast, CV-11974 addition (AT₁ antagonist: 1, 10, and 100 nM) did not affect this inhibitory effect. These findings confirmed that the effect of AngII on cGMP production was mediated by AT₂.

View larger version (52K): [in this window] [in a new window]   Figure 1. Effect of PD123319 and CV-11974 on AngII (1 nM)-induced cGMP inhibition in cultured porcine adrenal medullary cells. Cells (1 x 106) were incubated with AngII alone (1 nM) or with AngII (1 nM) and PD123319 (1, 10, or 100 nM) or CV-11974 (1, 10, or 100 nM) for 10 min at 37 C in the presence of IBMX. cGMP was measured according to the method described in Materials and Methods. The data shown are the mean ± SD (n = 4). *, Significantly different (P < 0.05) from the basal value; #, significantly different (P < 0.05) from the value obtained with AngII alone (1 nM). Data points are expressed as picomoles per 106 cells.

Effects of CV-11974 and PD 123319 on AngII (1 nM)-induced catecholamine release
The increase in epinephrine (E) was comparable to that in norepinephrine (NE). Hence, findings for NE are shown in Fig. 2. AngII (1 nM) alone significantly increased NE secretion from cultured porcine adrenal medullary chromaffin cells to approximately 2.0-fold over the basal value. The stimulatory effect of AngII on catecholamine secretion was significantly inhibited (by 80.5%) with 100 nM PD 123319 (AT₂ antagonist; IC₅₀, 10 nM). Thus, we demonstrate that the effect of AngII on catecholamine release was mainly mediated by AT₂. Also, the stimulation of catecholamine secretion by AngII was moderately, but significantly, attenuated (by 17.5%) by 100 nM CV-11974 (AT₁ antagonist; IC₅₀, 1 µM), suggesting an involvement of AT₁ in AngII-induced catecholamine release. To further confirm this finding, we examined the effect of AngII with PD 123319 (100 nM; which simulates specific AT₁ stimulation) on catecholamine release (Table 1). AngII with PD 123319 moderately induced catecholamine release (1.2-fold over the basal value), with this marginal increase showing significant suppression by BAPTA (10 µM; intracellular Ca²⁺ chelator) (34). These findings indicate that AT₁-mediated catecholamine release is dependent on intracellular Ca²⁺ release, in agreement with previous reports (1, 14).

View larger version (23K): [in this window] [in a new window]   Figure 2. Effect of PD123319 and CV-11974 on AngII (1 nM)-stimulated catecholamine secretion in cultured porcine adrenal medullary cells. The increase in epinephrine was comparable to that in norepinephrine. Hence, the data derived from the norepinephrine study areshown. Cells were incubated for 10 min with 1 nM AngII alone (control) or 1 nM of AngII with several concentrations of either PD123319 or CV-11974, as indicated, and the resulting media were examined with a catecholamine analyzer according to the method described in Materials and Methods. The data shown are the mean ± SD (n = 4–6). *, Significantly different (P < 0.05) from the basal value; #, significantly different (P < 0.05) from the value obtained with AngII alone (1 nM).

View larger version (28K): [in this window] [in a new window]   Figure 3. Effect of CGP 42112 on catecholamine secretion in cultured porcine adrenal medullary cells. Cells were incubated for 10 min with varied concentrations (10 pM to 10 µM) of CGP 42112 either with or without PD123319 (100 nM) or CV-11974 (100 nM). The medium was subsequently examined with a catecholamine analyzer as described in the text. The data shown are the mean ± SD (n = 4–6). *, Significantly different (P < 0.05) from the basal value.

View larger version (35K): [in this window] [in a new window]   Figure 4. Effects of removing external Ca2+ on AngII with CV-11974- and CGP 42112-induced catecholamine release. Cells were treated with AngII with CV-11974 (A) or CGP 42112 (100 nM; B) for 10 min with or without 2.2 mM Ca2+, and the medium was examined with a catecholamine analyzer. The data shown are the mean ± SD (n = 6). *, Significantly different (P < 0.05) from the basal value. #, Significantly different (P < 0.05) from value obtained from AngII with CV-11974 or CGP 42112 (100 nM) induction.

View larger version (20K): [in this window] [in a new window]   Figure 5. Effects of AngII with CV-11974 and CGP 42112 on [Ca2+]i. Time-course changes with respect to [Ca2+]i levels in fura-2-loaded chromaffin cells were measured using the system described in Materials and Methods.

View larger version (48K): [in this window] [in a new window]   Figure 6. Effects of AngII and CGP 42112 on IP3 production. Cells were incubated for 10 min with varied concentrations (10 nM to 1 µM) of CGP 42112 or AngII (1 nM) either with or without PD123319 (100 nM) or CV-11974 (100 nM). IP3 was measured according to the method described in Materials and Methods. Values are the mean ± SD (n = 4). *, Significantly different (P < 0.05) from the basal value. #, Significantly different (P < 0.05) from the value obtained with AngII alone (1 nM).

View larger version (78K): [in this window] [in a new window]   Figure 7. The stimulatory effects of AngII with CV-11974 and CGP 42112 on catecholamine release are dependent on a decrease in cGMP. A and E, Effects of pretreatment with 8-Br-cGMP (membrane-permeable cGMP analog) on AngII with CV-11974- or CGP 42112-induced catecholamine secretion. Cells were preincubated with 1 mM 8-Br-cGMP for 30 min, then treated with AngII plus CV-11974 or CGP 42112 (100 nM). The medium was subsequently examined with a catecholamine analyzer. Values are the mean ± SD (n = 7). As the results for E were comparable to those for NE, the findings for NE are presented. *, Significantly different (P < 0.05) from basal value; #, significantly different (P < 0.05) from the value obtained with AngII with CV-11974 or CGP 42112 (100 nM). B, C, F, and G, Effects of pretreatment with 8-Br-cGMP (membrane-permeable cGMP analog) on AngII with CV-11974- or CGP 42112-induced [Ca2+]i. Cells were preincubated with 1 mM 8-Br-cGMP for 30 min, then treated with AngII with CV-11974 or CGP 42112 (100 nM). Representative data are shown in B and F. The values are the mean ± SD (n = 12). #, Significantly different (P < 0.05) from value obtained from AngII with CV-11974 or CGP 42112 (100 nM) induction. D and H, Effects of 8-Br-cGMP on [Ca2+]i induced by AngII with CV-11974 or CGP 42112. The addition of AngII with CV-11974 or 1 µM CGP 42112 was followed by the addition of 1 mM 8-Br-cGMP after 5 min. Time-course changes in [Ca2+ ]i levels were measured as before.

View larger version (40K): [in this window] [in a new window]   Figure 8. Effect of guanylin (activator of guanylate cyclase) on AngII with CV-11974-induced catecholamine secretion. A, Effects of pretreatment with guanylin (activator of guanylate cyclase) on AngII with CV-11974-induced catecholamine secretion. Cells were preincubated with 10 nM guanylin for 30 min, then treated with AngII plus CV-11974. The medium was subsequently examined with a catecholamine analyzer. Values are the mean ± SD (n = 7). As the results for E were comparable to those for NE, the findings for NE are presented. *, Significantly different (P < 0.05) from basal value; #, significantly different (P < 0.05) from the value obtained with AngII with CV-11974. B and C, Effects of pretreatment with guanylin on AngII with CV-11974-induced [Ca2+]i. Cells were preincubated with 10 nM guanylin for 30 min, then treated with AngII with CV-11974. Representative data are shown in B. The values are the mean ± SD (n = 12). #, Significantly different (P < 0.05) from value obtained from AngII with CV-11974 induction. D, Effects of guanylin on [Ca2+]i induced by AngII with CV-11974. The addition of AngII with CV-11974 was followed by the addition of 10 nM guanylin after 5 min. Time-course changes in [Ca2+ ]i levels were measured as before.

View larger version (81K): [in this window] [in a new window]   Figure 9. Effects of the Ca2+ channel blocker on AngII with CV-11974- or CGP 42112-induced catecholamine release and [Ca2+]i. The cells were stimulated by AngII with CV-11974 or CGP 42112 (100 nM) either with or without nicardipine (L-type Ca2+ channel blocker, 1 µM) or -conotoxin VIA (N-type Ca2+ channel blocker, 1 µM). A and D, Catecholamine levels were analyzed as indicated. As the results obtained for E were comparable to those for NE, findings for NE are presented here. The data shown are the mean ± SD (n = 4). *, Significantly different (P < 0.05) from basal value; #, significantly different (P < 0.05) from the value obtained with AngII with CV-11974 or CGP 42112 (100 nM). B and E, Representative data with respect to [Ca2+]i levels are shown. C and F, The values are the mean ± SD (n = 12). #, Significantly different (P < 0.05) from the value induced by AngII with CV-11974 or CGP 42112 (100 M) as a control. [Ca2+]i levels were measured according to the method described in Materials and Methods.

	Discussion

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Belloni et al. recently demonstrated that stimulation of AT₂ evokes a novel secretagogic response for the adrenal medulla (23). In the present study we showed that the catecholamine response to AngII was markedly inhibited by PD 123319 (AT₂ antagonist); these data are in agreement with results obtained by Belloni et al. In addition, CGP 42112 significantly increased catecholamine secretion in cultured porcine chromaffin cells. Thus, we have further confirmed here that activation of AT₂ results in a novel secretagogic response for the adrenal medulla.

Although mounting evidence suggests that catecholamine release from adrenal medulla is related to AT₁ stimulation (1, 15, 16), Belloni et al. also reported that the catecholamine response to AngII in the rat adrenal medulla was only moderately attenuated by high concentrations of Dup 753 (AT₁ antagonist). This led these investigators to claim a marginal involvement of AT₁ in AngII-induced catecholamine release (23). Similarly, we showed that CV-11974 (10 nM) caused a moderate, but significant, inhibition of catecholamine release induced by AngII, also indicating that AT₁ could be involved in AngII-induced catecholamine release. Indeed, AT₁ stimulation moderately induced catecholamine release in an IP₃-mediated intracellular Ca²⁺-dependent manner (Fig. 6, Table 1), which is in agreement with a previous paper by Israel et al. (14). Therefore, catecholamine release evoked by AngII might be about 80% AT₂ mediated and 20% AT₁ mediated in cultured porcine chromaffin cells (Figs. 2 and 10).

View larger version (39K): [in this window] [in a new window]   Figure 10. Outline of proposed model for the effects of AT2 stimulation on catecholamine release in chromaffin cells. This outline is prepared on the basis of results suggested by the current experiments together with data from our previous report (28 ). AT2 is dominantly expressed in cultured porcine chromaffin cells. Binding of AngII to its receptor (AT2) causes a decrease in cGMP/PKG levels through an unknown mechanism, which, in turn, stimulates VDCC without affecting intracellular Ca2+ storage. Consequently, AT2-induced catecholamine secretion is indeed dependent on external Ca2+ via VDCC. In contrast, AT1-induced catecholamine release is dependent on IP3-mediated Ca2+ release from intracellular storage sites. Catecholamine release evoked by AngII was assessed to be about 80% AT2 mediated and 20% AT1 mediated in cultured porcine chromaffin cells. PLC, phospholipase C; ER, endoplasmic reticulum. Broad arrows, Signaling pathways suggested or confirmed by the current experiments.

Controversy exists about whether G proteins are involved in the effects of AT₂ (2, 3). In the present study we showed that PTX pretreatment did not affect the inhibitory effect of the AngII/CV-11974 combination on cGMP production, indicating that neither G_i nor G_o proteins are involved in the inhibitory mechanisms of AT₂ in our experimental system (data not shown). It is still possible, however, that G proteins other than G_i or G_o, as suggested by Buisson et al. (38), could be involved in the inhibitory mechanisms of AT₂ observed in this study.

AT₂ recently has been shown to play an important role in AngII-induced NO production in kidney and vascular cells (37). However, different from the results found with kidney and vascular cells, stimulation of AT₂ did not affect NOx production in the chromaffin cells (data not shown). Also, the NOS inhibitor L-NAME did not affect AT₂-induced catecholamine release (data not shown). These results indicate that NO may not be involved in AT₂-induced catecholamine release in chromaffin cells.

The precise mechanism by which AT₂ stimulation induces catecholamine secretion is unclear. Recently, the existence of a cGMP-mediated intracellular signaling pathway has been reported by several researchers in chromaffin cells (24, 25, 26, 27, 42, 43). Indeed, PKG plays a central role in mediating the action of cGMP, with PKG activity and PKG immunoreactivity having been clearly present in the aforementioned cells (24, 25, 26, 27, 42). Moreover, growing evidence suggests that the cGMP/PKG pathway modulates catecholamine release. It is noteworthy that both NO and NPs cause increases in intracellular cGMP levels, and both inhibit catecholamine release by inhibition of Ca²⁺ mobilization via VDCC, with this inhibitory effect being mediated by activation of PKG (24, 25, 26, 27). In contrast to NO and NPs, we showed that activation of AT₂ significantly induced a decrease in cGMP in adrenomedullay chromaffin cells (28), suggesting that this reduction in cGMP may be responsible for the simulative action on VDCC. Consistent with this hypothesis, we showed that pretreating chromaffin cells with either 8-Br-cGMP or guanylin completely abolished the stimulatory effect of AT₂ on both catecholamine secretion and [Ca²⁺]_i (Fig. 7, A–C, E–G; Fig. 8, A–C). Moreover, the addition of either 8-Br-cGMP or guanylin after application of AT₂ caused a gradual decrease in [Ca²⁺]_i toward baseline values (Fig. 7, D, H; Fig. 8D). Considering that NO may not be involved in AT₂-induced catecholamine release, the most likely explanation for this release might be that activation of AT₂ reduces PKG activity as a result of a decrease in cGMP production. This may, in turn, increase Ca²⁺ mobilization through VDCC, resulting in activation of catecholamine release from chromaffin cells (Fig. 10).

Although it has been shown that PKG modulates the activity of VDCC (24, 25, 26, 27, 42), the specific target remains obscure. As the AT₂-induced increase in [Ca²⁺]_i is entirely dependent on L- and N-type VDCC, the target for PKG may be a specific action on both dihydropyridine-sensitive and insensitive VDCC, comparable to what Desole et al. previously reported (25). Rodriguez-Pascual et al. (24), however, demonstrated that PKG may mediate its specific actions on dihydropyridine-insensitive VDCC. Also, it is unclear whether the action of PKG either 1) directly affects some phosphorylated molecule on VDCC, or 2) works through an indirect action mediated by another regulatory molecule whose function is modulated by phosphorylation.

Adrenal medullary catecholamines are known to enhance aldosterone release in a paracrine manner through activation of ß-adrenoreceptors localized in zona glomerulosa cells (44). Indeed, many regulatory peptides, including PACAP (29), that have been shown to evoke catecholamine release are able to enhance aldosterone release in a similar manner. It was recently demonstrated by Mazzocchi et al. (45) that activation of AT₂ in rat adrenal medullary chromaffin cells may cause the local release of catecholamines, which, in turn, potentiate aldosterone release in a paracrine manner through activation of ß-adrenoreceptors localized in zona glomerulosa cells.

Along with this line of evidence concerning the physiological relevance of AT₂ for catecholamine release, we previously reported that AT₂ stimulation inhibited catecholamine synthesis, a finding that is, at first glance, the opposite of the results presented here (28). In contrast to AT₂, cGMP-elevating agents, such as NO and NPs, have been reported to inhibit catecholamine release while also stimulating catecholamine biosynthesis. Consequently, the resulting net intracellular catecholamine content is increased (27, 42, 43). However, the precise physiological reason for why cGMP exerts opposing effects on catecholamine synthesis and release is unknown. It should be stated that we previously observed that an extended exposure to AT₂ causes significant and reproducible reduction of intracellular net catecholamine content (manuscript submitted), probably as a result of both stimulation of catecholamine secretion and inhibition of catecholamine synthesis. Thus, we speculate that AT₂ may play a role, at least in part, in reducing catecholamine net content.

With respect to regulation of cGMP levels in chromaffin cells, cGMP production under basal conditions is likely to be high enough to tonically activate PKG, perhaps by endogenous NO, thereby suppressing VDCC (38, 46). Thus, it can be suggested that the AT₂-mediated cGMP reduction observed here may negatively regulate or induce relaxation of this tonic suppression of VDCC and the subsequent secretory response.

To date, the precise functional significance of the stimulatory effect of AT₂ on catecholamine release and subsequent reduction of catecholamine net content in vivo remains to be determined. Also, the difference between cultured chromaffin cells and equivalent cells within the body should be considered when interpreting the data presented here (47).

	Acknowledgments

 
The authors thank Kohei Sawada, Ph.D. (Senior Scientist, Department of Drug Discovery, Tsukuba Research Laboratories, Eizai Co. Ltd.), for measurement of [Ca²⁺]_i mobilization.

	Footnotes

 
¹ This work was supported in part by Grant 11770624 from the Ministry of Education and by the University of Tsukuba Research Project.

Received September 14, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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