5-hydroxytryptamine type 2A receptors regulate cyclic AMP accumulation in a neuronal cell line by protein kinase C-dependent and calcium/calmodulin-dependent mechanisms

Kelly A Berg, William P Clarke, Yibang Chen, Barbara J. Ebersole, Ronald D G McKay, Saul Maayani

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

The effects of 5-hydroxytryptamine (5-HT)2A receptor activation on cAMP formation were studied in a cell line derived from embryonic rat cortex (A1A1). 5-HT (EC50 = 0.87 μM) amplified the amount of cAMP formed in response to 5′-N-ethylcarboxamidoadenosine (an adenosine A2 receptor agonist), cholera toxin, and forskolin after 15 min of coincubation in the presence of the phosphodiesterase inhibitor rolipram. This effect of 5-HT was blocked by 10 nM ketanserin as well as by 10 nM spiperone, indicating a response mediated by the 5-HT2A receptor subtype. Similarly, cAMP accumulation was enhanced by coincubation with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore A23187. After exposure to PMA for 24 hr (PKC-depleted cells), 5-HT and A23187 still enhanced cAMP formed in response to forskolin and 5′-N-ethylcarboxamidoadenosine, whereas the amplifying effects of PMA were abolished. Analysis by Western blots and PKC activity measurements revealed that, of three PKC isoforms detected in A1A1 cells (α, δ, and ∈), only the calcium-independent isoform PKC-∈ remained in membrane fractions after long term PMA treatment. In PKC-depleted cells, 5-HT-mediated amplification was greatly reduced after treatment with the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl)-ester or the calmodulin antagonists calmidazolium and N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide hydrochloride. In addition, 5-HT-mediated amplification of cAMP accumulation was reduced by the PKC inhibitor staurosporine in normal cells but was unaffected in PKC-depleted cells. In conclusion, these data suggest that 5-HT2A receptor activation can amplify cAMP formation in A1A1 cells by two distinct pathways coupled to the hydrolysis of inositol phosphates, i.e., PKC and calcium/calmodulin.

Original languageEnglish (US)
Pages (from-to)826-836
Number of pages11
JournalMolecular Pharmacology
Volume45
Issue number5
StatePublished - May 1994
Externally publishedYes

Fingerprint

Serotonin Receptors
Calmodulin
Cyclic AMP
Protein Kinase C
Calcium
Cell Line
Serotonin
Acetates
Adenosine-5'-(N-ethylcarboxamide)
Receptor, Serotonin, 5-HT2A
Calcimycin
Colforsin
calmidazolium
Protein Isoforms
Adenosine A2 Receptor Agonists
Rolipram
Spiperone
Ketanserin
Ethane
Inositol Phosphates

ASJC Scopus subject areas

  • Pharmacology

Cite this

5-hydroxytryptamine type 2A receptors regulate cyclic AMP accumulation in a neuronal cell line by protein kinase C-dependent and calcium/calmodulin-dependent mechanisms. / Berg, Kelly A; Clarke, William P; Chen, Yibang; Ebersole, Barbara J.; McKay, Ronald D G; Maayani, Saul.

In: Molecular Pharmacology, Vol. 45, No. 5, 05.1994, p. 826-836.

Research output: Contribution to journalArticle

@article{c479e8b89e1b46c1b5026f9e3e1d0c66,
title = "5-hydroxytryptamine type 2A receptors regulate cyclic AMP accumulation in a neuronal cell line by protein kinase C-dependent and calcium/calmodulin-dependent mechanisms",
abstract = "The effects of 5-hydroxytryptamine (5-HT)2A receptor activation on cAMP formation were studied in a cell line derived from embryonic rat cortex (A1A1). 5-HT (EC50 = 0.87 μM) amplified the amount of cAMP formed in response to 5′-N-ethylcarboxamidoadenosine (an adenosine A2 receptor agonist), cholera toxin, and forskolin after 15 min of coincubation in the presence of the phosphodiesterase inhibitor rolipram. This effect of 5-HT was blocked by 10 nM ketanserin as well as by 10 nM spiperone, indicating a response mediated by the 5-HT2A receptor subtype. Similarly, cAMP accumulation was enhanced by coincubation with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore A23187. After exposure to PMA for 24 hr (PKC-depleted cells), 5-HT and A23187 still enhanced cAMP formed in response to forskolin and 5′-N-ethylcarboxamidoadenosine, whereas the amplifying effects of PMA were abolished. Analysis by Western blots and PKC activity measurements revealed that, of three PKC isoforms detected in A1A1 cells (α, δ, and ∈), only the calcium-independent isoform PKC-∈ remained in membrane fractions after long term PMA treatment. In PKC-depleted cells, 5-HT-mediated amplification was greatly reduced after treatment with the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl)-ester or the calmodulin antagonists calmidazolium and N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide hydrochloride. In addition, 5-HT-mediated amplification of cAMP accumulation was reduced by the PKC inhibitor staurosporine in normal cells but was unaffected in PKC-depleted cells. In conclusion, these data suggest that 5-HT2A receptor activation can amplify cAMP formation in A1A1 cells by two distinct pathways coupled to the hydrolysis of inositol phosphates, i.e., PKC and calcium/calmodulin.",
author = "Berg, {Kelly A} and Clarke, {William P} and Yibang Chen and Ebersole, {Barbara J.} and McKay, {Ronald D G} and Saul Maayani",
year = "1994",
month = "5",
language = "English (US)",
volume = "45",
pages = "826--836",
journal = "Molecular Pharmacology",
issn = "0026-895X",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
number = "5",

}

TY - JOUR

T1 - 5-hydroxytryptamine type 2A receptors regulate cyclic AMP accumulation in a neuronal cell line by protein kinase C-dependent and calcium/calmodulin-dependent mechanisms

AU - Berg, Kelly A

AU - Clarke, William P

AU - Chen, Yibang

AU - Ebersole, Barbara J.

AU - McKay, Ronald D G

AU - Maayani, Saul

PY - 1994/5

Y1 - 1994/5

N2 - The effects of 5-hydroxytryptamine (5-HT)2A receptor activation on cAMP formation were studied in a cell line derived from embryonic rat cortex (A1A1). 5-HT (EC50 = 0.87 μM) amplified the amount of cAMP formed in response to 5′-N-ethylcarboxamidoadenosine (an adenosine A2 receptor agonist), cholera toxin, and forskolin after 15 min of coincubation in the presence of the phosphodiesterase inhibitor rolipram. This effect of 5-HT was blocked by 10 nM ketanserin as well as by 10 nM spiperone, indicating a response mediated by the 5-HT2A receptor subtype. Similarly, cAMP accumulation was enhanced by coincubation with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore A23187. After exposure to PMA for 24 hr (PKC-depleted cells), 5-HT and A23187 still enhanced cAMP formed in response to forskolin and 5′-N-ethylcarboxamidoadenosine, whereas the amplifying effects of PMA were abolished. Analysis by Western blots and PKC activity measurements revealed that, of three PKC isoforms detected in A1A1 cells (α, δ, and ∈), only the calcium-independent isoform PKC-∈ remained in membrane fractions after long term PMA treatment. In PKC-depleted cells, 5-HT-mediated amplification was greatly reduced after treatment with the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl)-ester or the calmodulin antagonists calmidazolium and N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide hydrochloride. In addition, 5-HT-mediated amplification of cAMP accumulation was reduced by the PKC inhibitor staurosporine in normal cells but was unaffected in PKC-depleted cells. In conclusion, these data suggest that 5-HT2A receptor activation can amplify cAMP formation in A1A1 cells by two distinct pathways coupled to the hydrolysis of inositol phosphates, i.e., PKC and calcium/calmodulin.

AB - The effects of 5-hydroxytryptamine (5-HT)2A receptor activation on cAMP formation were studied in a cell line derived from embryonic rat cortex (A1A1). 5-HT (EC50 = 0.87 μM) amplified the amount of cAMP formed in response to 5′-N-ethylcarboxamidoadenosine (an adenosine A2 receptor agonist), cholera toxin, and forskolin after 15 min of coincubation in the presence of the phosphodiesterase inhibitor rolipram. This effect of 5-HT was blocked by 10 nM ketanserin as well as by 10 nM spiperone, indicating a response mediated by the 5-HT2A receptor subtype. Similarly, cAMP accumulation was enhanced by coincubation with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore A23187. After exposure to PMA for 24 hr (PKC-depleted cells), 5-HT and A23187 still enhanced cAMP formed in response to forskolin and 5′-N-ethylcarboxamidoadenosine, whereas the amplifying effects of PMA were abolished. Analysis by Western blots and PKC activity measurements revealed that, of three PKC isoforms detected in A1A1 cells (α, δ, and ∈), only the calcium-independent isoform PKC-∈ remained in membrane fractions after long term PMA treatment. In PKC-depleted cells, 5-HT-mediated amplification was greatly reduced after treatment with the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl)-ester or the calmodulin antagonists calmidazolium and N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide hydrochloride. In addition, 5-HT-mediated amplification of cAMP accumulation was reduced by the PKC inhibitor staurosporine in normal cells but was unaffected in PKC-depleted cells. In conclusion, these data suggest that 5-HT2A receptor activation can amplify cAMP formation in A1A1 cells by two distinct pathways coupled to the hydrolysis of inositol phosphates, i.e., PKC and calcium/calmodulin.

UR - http://www.scopus.com/inward/record.url?scp=0028235629&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028235629&partnerID=8YFLogxK

M3 - Article

C2 - 8190100

AN - SCOPUS:0028235629

VL - 45

SP - 826

EP - 836

JO - Molecular Pharmacology

JF - Molecular Pharmacology

SN - 0026-895X

IS - 5

ER -