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A newly found angiotensin II receptor subtype mediates cyclic GMP formation in differentiated Neuro-2A cells
European Journal of Pharmacology Molecular Pharmacology Section, 225 (1992) 355-356 © 1992 Elsevier Science Publishers B.V. All rights reserved 0922-4106/92/$05.00
355
-
EJPMOL 0109R Rapid communication
A newly found angiotensin II receptor subtype mediates cyclic GMP formation in differentiated Neuro-2A cells Shigeyuki Chaki and Tadashi Inagami Department of Biochemistry, Vanderbih UniversitySchool of Medicine, Naslwille, TN 37232, USA
Received 20 Februa6' 1992; accepted 28 February 1992
In search of the functional role of the newly found angiotensin II (Ang II) binding site which is expressed in differentiated Neuro-2A cells, we found that Ang II causes a marked stimulation of cGMP formation dose-dependently. The stimulation was blocked by the nonselective Ang II receptor antagonist [SarZ,IleS]Ang II but not by the AT~ antagonist DuP 753 or the AT 2 antagonist PD 123319. These results suggest that Ang II increased cGMP level via a new Ang It receptor subtype in differentiated Neuro-2A cells. Angiotensin II receptor; cGMP; Neuro-2A cells
T o date it has b e e n well accepted that angiotensin I1 (Ang II) stimulates phosphatidylinosi~ol turnover and inhibits cyclic A M P formation in several tissues and cells (Bauer et al., 1991). O n the o t h e r hand, A n g II has b e e n r e p o r t e d to decrease cyclic G M P ( c G M P ) levels in neuronal cultures from rat brain (Sumners and Myers, 1991). W e have recently found that differentiated N e u r o - 2 A cells possess a new type of binding site for A n g II (Chaki and Inagami, 1992) w ~ c h is distinct from either of the two receptor subtypes, AT~ or A T 2, defined recently (Chin et al., 1989; Whiteb~ead et al., 1989). The functional role of this binding site in the signal transduction m e c h a n i s m has not been clear. T h e regulatory, functions of A n g II particularly in neuronal tissues are not yet complete. In search of a possible new functional role for the newly found binding site as a receptor, we investigated the effect of A n g II on c G M P formation, which has b e e n implicated in intracellular transduction processes in many types of cells. Mouse N e u r o - 2 A neuroblastoma cells were cultured as previously described (Chaki and Inagami, 1992). For differentiation, the cells w e r e grown for 2 days in the culture m e d i u m containing 1% fetal calf serum, c G M P formation was d e t e r m i n e d as follows. T h e culture m e d i u m was r e m o v e d from the cells, and H E P E S buffered saline (20 m M H E P E S , 150 m M NaC1, 5.4
Correspondence to: Tadashi Inagami, Ph.D., Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Tel. 615 (322) 4347; Fro; 615 (343) 0704.
m M KC!, t.8 m M CaCt 2, 0.8 m M MgCt 2 and 25 m M glucose, p H 7.4) was added. After preincubation with A n g II receptor antagonist or vehicle in H E P E S buffered saline for 5 rain, the cells were further incubated with A n g II at 37°C for 30 s. The reaction was
600
:~
q-**I i~::7/ [i ": ?i~
0 Angtl(nM) Antagonist 0 ~M)
0
1
i0
100 -
I .... 1000 1000 1 0 0 0 1 0 0 0 DuP
PD
Sar
Fig. 1. Ang l~-induced cGMP elevation in differentiated Neuro-2A cells: dose dependence and effect of Ang 11 receptor antagonists. The assays were performed as described in the text. Each value represents the mean+_S.E, obtained from three separate experiments. Statistical significance of the data was assessed by Student's t-test. ** P<0.01 compared with absence of Ang II. #~P<0.01 compared with Ang l~ (10 - 6 M). DuP. DuP 753; PD, PD t;,~319; Sar, ISar1.11eS]Angti.
356 t e r m i n a t e d by adding an equal volume of absolute ethanol, a n d the c G M P c o n t e n t in the s u p e r n a t a n t was d e t e r m i n e d using a commercially available r a d i o i m munoassay kit ( A m e r s h a m Corp.). A s s h o w n in fig. 1, A n g II increased t h e c G M P level significantly a n d d o s e - d e p e n d e n t l y in the d i f f e r e n t i a t e d N e u r o - 2 A cells, A n g II stimulated c G M P f o r m a t i o n 8.1-fold at 10 -6 M. T h e subtype nonspecific a n t a g o n i s t against A n g II receptor, [SarJ,IleS]Ang II (10 -6 M), markedly a t t e n u a t e d c G M P elevation s t i m u l a t e d by 10 - 6 M A n g II. However, n e i t h e r D u P 753 ( A T I r e c e p t o r subtype specific a n t a g o n i s t ) n o r P D 123319 ( A T 2 r e c e p t o r subtype specific a n t a g o n i s t ) affected the A n g lI-stimulated c G M P elevation at respective conc e n t r a t i o n s of i 0 -6 M (fig. I). T h e s e a n t a g o n i s t s a l o n e did not alter t h e basal c G M P level (data not shown). T h e s e results indicate t h a t A n g II increased c G M P level specifically via the new A n g II r e c e p t o r subtype in differentiated N e u r o - 2 A cells, a n d t h a t this r e c e p t o r subtype is functional in m e d i a t i n g c h a n g e s in a n intracellular messenger. A l t h o u g h t h e d e t a i l e d intracellular m e c h a n i s m of t h e A n g I I - s t i m u l a t e d c G M P elevation r e m a i n s to be d e t e r m i n e d , o u r results d e m o n s t r a t e d t h a t the new A n g II receptor, which does not b e l o n g to the known subtypes, AT~ or ATe, h a s a new functional role in a n e u r o b l a s t o m a cell line a n d possibly in neuronal tissues.
Acknowledgements We thank Ms. Trinita Fitzgerald for her technical assistance, E.I. duPont de Nemours & Company for the supply of DuP 753 and Park Davis Co. for PD 123319. Supported by Research Grants HL-14192 and HL-35323 from the National Institutes of Health, USA.
References Bauer, P., A.T. Chiu and J.C. Garrison, 1991, DuP 753 can antagonize the effects of angiotensis II in rat liver, Mol. Pharmacol. 39, 579. Chaki, S. and T. Inagami, 1992, Identification and characterization of a new binding site for angiotensin 11 in mouse neuroblastoma Neuro-2A cells, Biochem. Biophys. Res. Commun. 182, 388. Chiu+ A.T.. W.F. Herblin, D.E. McCall, R.J. Ardecky, D.J. Carini, J.V. Duncia, L.J. Pease, P.C. Wong, R.R. Wexler+ A.L. Johnson and P.B.M.W.M. Timmermans, 1989, Identification of angiotensin II receptor subtypes, Biochem. Biophys. Res. Commun. 165+ 196. Sumners, C. and L.M. Myers, 1991, Angiotensin I1 decreases cGMP levels in neuronal cultures from rat brain, Am. J+ Physiol. 260. C79. Whitebread, S.. M. Mele, B. Kamber and M. de Gasparo, 1989, Preliminary biochemical characterization ef two angiotensin II receptor subtypes, Biochcm. Biophys. Res. Commun+ 163, 284.
355
-
EJPMOL 0109R Rapid communication
A newly found angiotensin II receptor subtype mediates cyclic GMP formation in differentiated Neuro-2A cells Shigeyuki Chaki and Tadashi Inagami Department of Biochemistry, Vanderbih UniversitySchool of Medicine, Naslwille, TN 37232, USA
Received 20 Februa6' 1992; accepted 28 February 1992
In search of the functional role of the newly found angiotensin II (Ang II) binding site which is expressed in differentiated Neuro-2A cells, we found that Ang II causes a marked stimulation of cGMP formation dose-dependently. The stimulation was blocked by the nonselective Ang II receptor antagonist [SarZ,IleS]Ang II but not by the AT~ antagonist DuP 753 or the AT 2 antagonist PD 123319. These results suggest that Ang II increased cGMP level via a new Ang It receptor subtype in differentiated Neuro-2A cells. Angiotensin II receptor; cGMP; Neuro-2A cells
T o date it has b e e n well accepted that angiotensin I1 (Ang II) stimulates phosphatidylinosi~ol turnover and inhibits cyclic A M P formation in several tissues and cells (Bauer et al., 1991). O n the o t h e r hand, A n g II has b e e n r e p o r t e d to decrease cyclic G M P ( c G M P ) levels in neuronal cultures from rat brain (Sumners and Myers, 1991). W e have recently found that differentiated N e u r o - 2 A cells possess a new type of binding site for A n g II (Chaki and Inagami, 1992) w ~ c h is distinct from either of the two receptor subtypes, AT~ or A T 2, defined recently (Chin et al., 1989; Whiteb~ead et al., 1989). The functional role of this binding site in the signal transduction m e c h a n i s m has not been clear. T h e regulatory, functions of A n g II particularly in neuronal tissues are not yet complete. In search of a possible new functional role for the newly found binding site as a receptor, we investigated the effect of A n g II on c G M P formation, which has b e e n implicated in intracellular transduction processes in many types of cells. Mouse N e u r o - 2 A neuroblastoma cells were cultured as previously described (Chaki and Inagami, 1992). For differentiation, the cells w e r e grown for 2 days in the culture m e d i u m containing 1% fetal calf serum, c G M P formation was d e t e r m i n e d as follows. T h e culture m e d i u m was r e m o v e d from the cells, and H E P E S buffered saline (20 m M H E P E S , 150 m M NaC1, 5.4
Correspondence to: Tadashi Inagami, Ph.D., Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Tel. 615 (322) 4347; Fro; 615 (343) 0704.
m M KC!, t.8 m M CaCt 2, 0.8 m M MgCt 2 and 25 m M glucose, p H 7.4) was added. After preincubation with A n g II receptor antagonist or vehicle in H E P E S buffered saline for 5 rain, the cells were further incubated with A n g II at 37°C for 30 s. The reaction was
600
:~
q-**I i~::7/ [i ": ?i~
0 Angtl(nM) Antagonist 0 ~M)
0
1
i0
100 -
I .... 1000 1000 1 0 0 0 1 0 0 0 DuP
PD
Sar
Fig. 1. Ang l~-induced cGMP elevation in differentiated Neuro-2A cells: dose dependence and effect of Ang 11 receptor antagonists. The assays were performed as described in the text. Each value represents the mean+_S.E, obtained from three separate experiments. Statistical significance of the data was assessed by Student's t-test. ** P<0.01 compared with absence of Ang II. #~P<0.01 compared with Ang l~ (10 - 6 M). DuP. DuP 753; PD, PD t;,~319; Sar, ISar1.11eS]Angti.
356 t e r m i n a t e d by adding an equal volume of absolute ethanol, a n d the c G M P c o n t e n t in the s u p e r n a t a n t was d e t e r m i n e d using a commercially available r a d i o i m munoassay kit ( A m e r s h a m Corp.). A s s h o w n in fig. 1, A n g II increased t h e c G M P level significantly a n d d o s e - d e p e n d e n t l y in the d i f f e r e n t i a t e d N e u r o - 2 A cells, A n g II stimulated c G M P f o r m a t i o n 8.1-fold at 10 -6 M. T h e subtype nonspecific a n t a g o n i s t against A n g II receptor, [SarJ,IleS]Ang II (10 -6 M), markedly a t t e n u a t e d c G M P elevation s t i m u l a t e d by 10 - 6 M A n g II. However, n e i t h e r D u P 753 ( A T I r e c e p t o r subtype specific a n t a g o n i s t ) n o r P D 123319 ( A T 2 r e c e p t o r subtype specific a n t a g o n i s t ) affected the A n g lI-stimulated c G M P elevation at respective conc e n t r a t i o n s of i 0 -6 M (fig. I). T h e s e a n t a g o n i s t s a l o n e did not alter t h e basal c G M P level (data not shown). T h e s e results indicate t h a t A n g II increased c G M P level specifically via the new A n g II r e c e p t o r subtype in differentiated N e u r o - 2 A cells, a n d t h a t this r e c e p t o r subtype is functional in m e d i a t i n g c h a n g e s in a n intracellular messenger. A l t h o u g h t h e d e t a i l e d intracellular m e c h a n i s m of t h e A n g I I - s t i m u l a t e d c G M P elevation r e m a i n s to be d e t e r m i n e d , o u r results d e m o n s t r a t e d t h a t the new A n g II receptor, which does not b e l o n g to the known subtypes, AT~ or ATe, h a s a new functional role in a n e u r o b l a s t o m a cell line a n d possibly in neuronal tissues.
Acknowledgements We thank Ms. Trinita Fitzgerald for her technical assistance, E.I. duPont de Nemours & Company for the supply of DuP 753 and Park Davis Co. for PD 123319. Supported by Research Grants HL-14192 and HL-35323 from the National Institutes of Health, USA.
References Bauer, P., A.T. Chiu and J.C. Garrison, 1991, DuP 753 can antagonize the effects of angiotensis II in rat liver, Mol. Pharmacol. 39, 579. Chaki, S. and T. Inagami, 1992, Identification and characterization of a new binding site for angiotensin 11 in mouse neuroblastoma Neuro-2A cells, Biochem. Biophys. Res. Commun. 182, 388. Chiu+ A.T.. W.F. Herblin, D.E. McCall, R.J. Ardecky, D.J. Carini, J.V. Duncia, L.J. Pease, P.C. Wong, R.R. Wexler+ A.L. Johnson and P.B.M.W.M. Timmermans, 1989, Identification of angiotensin II receptor subtypes, Biochem. Biophys. Res. Commun. 165+ 196. Sumners, C. and L.M. Myers, 1991, Angiotensin I1 decreases cGMP levels in neuronal cultures from rat brain, Am. J+ Physiol. 260. C79. Whitebread, S.. M. Mele, B. Kamber and M. de Gasparo, 1989, Preliminary biochemical characterization ef two angiotensin II receptor subtypes, Biochcm. Biophys. Res. Commun+ 163, 284.