Stable expression and heterologous coupling of the kappa opioid receptor in cell lines of neural and nonneural origin

P. Banerjee, B. A. Chromy, E. Berry-Kravis, D. Hammond, J. K. Singh, G. Dawson

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Signal transduction cascades initiated by the neuronal κ opioid receptor were studied following transfection of a neuronal (hippocampal) line, HN2, and the non-neural CHOs. Retinoic-acid mediated differentiation resulted in intense staining of the HN2 cells with a neurofilament protein antibody SMI 33 but not with an antibody to GFAP, thus establishing neuronal characteristics of the HN2 cell line. The κ opioid receptor was stably expressed in the two cell lines by electroporation mediated transfer of a Cytomegalovirus-promoter driven construct, pCMV-κ, harboring the κ-opioid receptor cDNA. Positive clones (HN2κ24 and CHOκ18) from both lines showed high expression of the κ opioid receptor, as identified by [3H] U-69,593 binding to membranes prepared from HN2κ24 and CHOκ18. Scatchard analysis revealed the presence of high affinity κ opioid receptors in both engineered cell lines (K(D) = 1.3 nM for HN2κ24 and 2.1 nM for CHOκ18). Functional coupling to adenylate cyclase was displayed by 1 μM U-69,593 mediated inhibition (55-63%) of prostaglandin E1-stimulated intracellular cAMP levels. A major difference between the two clones was observed in functional coupling of the expressed κ opioid receptor to phospholipases C (PL-C) and D (PL-D). U-69,593 (1 μM) treatment stimulated PL-C, but not PL-D, in HN2κ24 cells, whereas PL-D, but not PL-C, was stimulated following such treatment of CHOκ18 cells. Our results using the model neuronal system, HN2κ24, demonstrate cell-type specific, positive coupling of the kappa opioid receptor to the major Ca2+ mobilizing system, the PL-C cascade, which regulates neuronal firing.

Original languageEnglish (US)
Pages (from-to)1277-1284
Number of pages8
JournalLife Sciences
Volume58
Issue number15
DOIs
StatePublished - Mar 8 1996
Externally publishedYes

Fingerprint

kappa Opioid Receptor
Opioid Receptors
Cells
Type C Phospholipases
Cell Line
Clone Cells
Neurofilament Proteins
Phospholipase D
Signal transduction
Electroporation
Antibodies
Alprostadil
Tretinoin
Cytomegalovirus
Adenylyl Cyclases
Transfection
Signal Transduction
Complementary DNA
Staining and Labeling
Membranes

Keywords

  • Kappa opioid receptor
  • PL-C
  • PL-D
  • Signal transduction
  • Transfection

ASJC Scopus subject areas

  • Pharmacology

Cite this

Banerjee, P., Chromy, B. A., Berry-Kravis, E., Hammond, D., Singh, J. K., & Dawson, G. (1996). Stable expression and heterologous coupling of the kappa opioid receptor in cell lines of neural and nonneural origin. Life Sciences, 58(15), 1277-1284. https://doi.org/10.1016/0024-3205(96)00089-6

Stable expression and heterologous coupling of the kappa opioid receptor in cell lines of neural and nonneural origin. / Banerjee, P.; Chromy, B. A.; Berry-Kravis, E.; Hammond, D.; Singh, J. K.; Dawson, G.

In: Life Sciences, Vol. 58, No. 15, 08.03.1996, p. 1277-1284.

Research output: Contribution to journalArticle

Banerjee, P, Chromy, BA, Berry-Kravis, E, Hammond, D, Singh, JK & Dawson, G 1996, 'Stable expression and heterologous coupling of the kappa opioid receptor in cell lines of neural and nonneural origin', Life Sciences, vol. 58, no. 15, pp. 1277-1284. https://doi.org/10.1016/0024-3205(96)00089-6
Banerjee, P. ; Chromy, B. A. ; Berry-Kravis, E. ; Hammond, D. ; Singh, J. K. ; Dawson, G. / Stable expression and heterologous coupling of the kappa opioid receptor in cell lines of neural and nonneural origin. In: Life Sciences. 1996 ; Vol. 58, No. 15. pp. 1277-1284.
@article{460f116f9486484c984b2e323df52da4,
title = "Stable expression and heterologous coupling of the kappa opioid receptor in cell lines of neural and nonneural origin",
abstract = "Signal transduction cascades initiated by the neuronal κ opioid receptor were studied following transfection of a neuronal (hippocampal) line, HN2, and the non-neural CHOs. Retinoic-acid mediated differentiation resulted in intense staining of the HN2 cells with a neurofilament protein antibody SMI 33 but not with an antibody to GFAP, thus establishing neuronal characteristics of the HN2 cell line. The κ opioid receptor was stably expressed in the two cell lines by electroporation mediated transfer of a Cytomegalovirus-promoter driven construct, pCMV-κ, harboring the κ-opioid receptor cDNA. Positive clones (HN2κ24 and CHOκ18) from both lines showed high expression of the κ opioid receptor, as identified by [3H] U-69,593 binding to membranes prepared from HN2κ24 and CHOκ18. Scatchard analysis revealed the presence of high affinity κ opioid receptors in both engineered cell lines (K(D) = 1.3 nM for HN2κ24 and 2.1 nM for CHOκ18). Functional coupling to adenylate cyclase was displayed by 1 μM U-69,593 mediated inhibition (55-63{\%}) of prostaglandin E1-stimulated intracellular cAMP levels. A major difference between the two clones was observed in functional coupling of the expressed κ opioid receptor to phospholipases C (PL-C) and D (PL-D). U-69,593 (1 μM) treatment stimulated PL-C, but not PL-D, in HN2κ24 cells, whereas PL-D, but not PL-C, was stimulated following such treatment of CHOκ18 cells. Our results using the model neuronal system, HN2κ24, demonstrate cell-type specific, positive coupling of the kappa opioid receptor to the major Ca2+ mobilizing system, the PL-C cascade, which regulates neuronal firing.",
keywords = "Kappa opioid receptor, PL-C, PL-D, Signal transduction, Transfection",
author = "P. Banerjee and Chromy, {B. A.} and E. Berry-Kravis and D. Hammond and Singh, {J. K.} and G. Dawson",
year = "1996",
month = "3",
day = "8",
doi = "10.1016/0024-3205(96)00089-6",
language = "English (US)",
volume = "58",
pages = "1277--1284",
journal = "Life Sciences",
issn = "0024-3205",
publisher = "Elsevier Inc.",
number = "15",

}

TY - JOUR

T1 - Stable expression and heterologous coupling of the kappa opioid receptor in cell lines of neural and nonneural origin

AU - Banerjee, P.

AU - Chromy, B. A.

AU - Berry-Kravis, E.

AU - Hammond, D.

AU - Singh, J. K.

AU - Dawson, G.

PY - 1996/3/8

Y1 - 1996/3/8

N2 - Signal transduction cascades initiated by the neuronal κ opioid receptor were studied following transfection of a neuronal (hippocampal) line, HN2, and the non-neural CHOs. Retinoic-acid mediated differentiation resulted in intense staining of the HN2 cells with a neurofilament protein antibody SMI 33 but not with an antibody to GFAP, thus establishing neuronal characteristics of the HN2 cell line. The κ opioid receptor was stably expressed in the two cell lines by electroporation mediated transfer of a Cytomegalovirus-promoter driven construct, pCMV-κ, harboring the κ-opioid receptor cDNA. Positive clones (HN2κ24 and CHOκ18) from both lines showed high expression of the κ opioid receptor, as identified by [3H] U-69,593 binding to membranes prepared from HN2κ24 and CHOκ18. Scatchard analysis revealed the presence of high affinity κ opioid receptors in both engineered cell lines (K(D) = 1.3 nM for HN2κ24 and 2.1 nM for CHOκ18). Functional coupling to adenylate cyclase was displayed by 1 μM U-69,593 mediated inhibition (55-63%) of prostaglandin E1-stimulated intracellular cAMP levels. A major difference between the two clones was observed in functional coupling of the expressed κ opioid receptor to phospholipases C (PL-C) and D (PL-D). U-69,593 (1 μM) treatment stimulated PL-C, but not PL-D, in HN2κ24 cells, whereas PL-D, but not PL-C, was stimulated following such treatment of CHOκ18 cells. Our results using the model neuronal system, HN2κ24, demonstrate cell-type specific, positive coupling of the kappa opioid receptor to the major Ca2+ mobilizing system, the PL-C cascade, which regulates neuronal firing.

AB - Signal transduction cascades initiated by the neuronal κ opioid receptor were studied following transfection of a neuronal (hippocampal) line, HN2, and the non-neural CHOs. Retinoic-acid mediated differentiation resulted in intense staining of the HN2 cells with a neurofilament protein antibody SMI 33 but not with an antibody to GFAP, thus establishing neuronal characteristics of the HN2 cell line. The κ opioid receptor was stably expressed in the two cell lines by electroporation mediated transfer of a Cytomegalovirus-promoter driven construct, pCMV-κ, harboring the κ-opioid receptor cDNA. Positive clones (HN2κ24 and CHOκ18) from both lines showed high expression of the κ opioid receptor, as identified by [3H] U-69,593 binding to membranes prepared from HN2κ24 and CHOκ18. Scatchard analysis revealed the presence of high affinity κ opioid receptors in both engineered cell lines (K(D) = 1.3 nM for HN2κ24 and 2.1 nM for CHOκ18). Functional coupling to adenylate cyclase was displayed by 1 μM U-69,593 mediated inhibition (55-63%) of prostaglandin E1-stimulated intracellular cAMP levels. A major difference between the two clones was observed in functional coupling of the expressed κ opioid receptor to phospholipases C (PL-C) and D (PL-D). U-69,593 (1 μM) treatment stimulated PL-C, but not PL-D, in HN2κ24 cells, whereas PL-D, but not PL-C, was stimulated following such treatment of CHOκ18 cells. Our results using the model neuronal system, HN2κ24, demonstrate cell-type specific, positive coupling of the kappa opioid receptor to the major Ca2+ mobilizing system, the PL-C cascade, which regulates neuronal firing.

KW - Kappa opioid receptor

KW - PL-C

KW - PL-D

KW - Signal transduction

KW - Transfection

UR - http://www.scopus.com/inward/record.url?scp=0029997257&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029997257&partnerID=8YFLogxK

U2 - 10.1016/0024-3205(96)00089-6

DO - 10.1016/0024-3205(96)00089-6

M3 - Article

C2 - 8614281

AN - SCOPUS:0029997257

VL - 58

SP - 1277

EP - 1284

JO - Life Sciences

JF - Life Sciences

SN - 0024-3205

IS - 15

ER -