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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

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.

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