TY - JOUR
T1 - Heterologous expression of the cloned guinea pig α2A, α2B, and α2C adrenoceptor subtypes
T2 - Radioligand binding and functional coupling to a cAMP-responsive reporter gene
AU - Svensson, Samuel P.S.
AU - Bailey, Thomas J.
AU - Porter, Amy C.
AU - Richman, Jeremy G.
AU - Regan, John W.
N1 - Funding Information:
This work has been supported by grants from the Arizona Affiliate of the American Heart Association and by the R. W. Johnson Pharmaceutical Research Institute. Additional support has been prooided to J. W. R. from the National Institutes of Health (EY09355) and by Allergun Inc. S. P. S. Svensson is supported by a postdoctoral fellowship from the Swedish Society for Medical Research, The Swedish Medical Research Council, and Sandoz. We also would like to thank Dr. Robert R&r (R. W. Johnson) for his encouragement and support and Dr. J. P. Hiebk (Smith- Kline Beechum) and Dr. J. M. Saoola (Orion Corp./Funnos) interest in this work.
PY - 1996/2/9
Y1 - 1996/2/9
N2 - Functional studies have shown that 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4,5-tetrahydro-3- benzazepine (SKF 104078) has very low affinity for prejunctional α2-adrenoceptors (α2-AR) in the guinea pig atrium. In this study, we have cloned guinea pig homologues of the human α2-C10, α2-C2 and α2-C4 AR subtypes and have studied them in isolation by heterologous expression in cultured mammalian cells. Oligonucleotide primers, designed from conserved areas of the human α2-ARs were used in a polymerase chain reaction (PCR) with template cDNA synthesized from guinea pig atrial mRNA. Three PCR products were obtained that shared identity with the three human α2-AR subtypes. A guinea pig (gp) genomic library was screened with a cDNA clone encoding a portion of the gp-α2A, and genes containing the complete coding sequences of the guinea pig α2A, α2B, and α2C AR subtypes were obtained. These guinea pig genes were subcloned into a eukaryotic expression plasmid and were expressed transiently in COS-7 cells. The binding of the α2-selective antagonist [3H]MK-912 to membranes prepared from these cells was specific and of high affinity with Kd values of 810 pM for gp-α2A, 2700 pM for gp-α2B and 110 pM for gp-α2C. Competition for the binding of [3H]MK-912 by SKF 104078 indicated that it was of moderately high affinity (∼100 nM) but that it was not selective for any of the guinea pig α2-AR subtypes. Co-expression of guinea pig α2-AR subtypes with a cyclicAMP-responsive chloramphenicol acetyltransferase (CAT) reporter gene resulted in agonist-dependent modulation of CAT activity. For the gp-α2A, a biphasic response was obtained with low concentrations of noradrenaline (NE) decreasing forskolin-stimulated CAT activity and high concentrations causing a reversal. For the gp-α2B, NE produced mostly potentiation of forskolin-stimulated activity, and for the gp-α2C, NE caused mainly inhibition. Overall, the pharmacology of the cloned guinea pig α2-AR subtypes was in agreement with data obtained for the native guinea pig receptors and was functionally similar to that of the cloned human α2-AR subtypes.
AB - Functional studies have shown that 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4,5-tetrahydro-3- benzazepine (SKF 104078) has very low affinity for prejunctional α2-adrenoceptors (α2-AR) in the guinea pig atrium. In this study, we have cloned guinea pig homologues of the human α2-C10, α2-C2 and α2-C4 AR subtypes and have studied them in isolation by heterologous expression in cultured mammalian cells. Oligonucleotide primers, designed from conserved areas of the human α2-ARs were used in a polymerase chain reaction (PCR) with template cDNA synthesized from guinea pig atrial mRNA. Three PCR products were obtained that shared identity with the three human α2-AR subtypes. A guinea pig (gp) genomic library was screened with a cDNA clone encoding a portion of the gp-α2A, and genes containing the complete coding sequences of the guinea pig α2A, α2B, and α2C AR subtypes were obtained. These guinea pig genes were subcloned into a eukaryotic expression plasmid and were expressed transiently in COS-7 cells. The binding of the α2-selective antagonist [3H]MK-912 to membranes prepared from these cells was specific and of high affinity with Kd values of 810 pM for gp-α2A, 2700 pM for gp-α2B and 110 pM for gp-α2C. Competition for the binding of [3H]MK-912 by SKF 104078 indicated that it was of moderately high affinity (∼100 nM) but that it was not selective for any of the guinea pig α2-AR subtypes. Co-expression of guinea pig α2-AR subtypes with a cyclicAMP-responsive chloramphenicol acetyltransferase (CAT) reporter gene resulted in agonist-dependent modulation of CAT activity. For the gp-α2A, a biphasic response was obtained with low concentrations of noradrenaline (NE) decreasing forskolin-stimulated CAT activity and high concentrations causing a reversal. For the gp-α2B, NE produced mostly potentiation of forskolin-stimulated activity, and for the gp-α2C, NE caused mainly inhibition. Overall, the pharmacology of the cloned guinea pig α2-AR subtypes was in agreement with data obtained for the native guinea pig receptors and was functionally similar to that of the cloned human α2-AR subtypes.
KW - Adenylyl cyclase
KW - G-protein coupled
KW - Pharmacology
KW - Reporter gene
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UR - http://www.scopus.com/inward/citedby.url?scp=0030040778&partnerID=8YFLogxK
U2 - 10.1016/0006-2952(95)02179-5
DO - 10.1016/0006-2952(95)02179-5
M3 - Article
C2 - 8573196
AN - SCOPUS:0030040778
SN - 0006-2952
VL - 51
SP - 291
EP - 300
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 3
ER -