Eoropean Jottrmd of Hmrnlacolo~,;*..... Moh,cular Phormacoh~gy Section, 22~ (It~92)367 ~,371 ~e~19~12Elsevier Science Publishers B.V. All righls reserved 0922-4106/92/$t~5.00
367
EJPMf?&. N}082 Short cotnrnunication
Nort ern blot and ribonuclease protection study of a -adr ;noceptor subtype,'s in cultured cell lines Lu Sun, S a t o s h i U m e m u r a , M e l a n i c C h u n a n d W i l l i a m A. P e t t i n g e r Midwest Itypertension Research Center, DtTmrtment of Medichle, Creighton Unit'ersity, School of Medicitw, Omaha, NE 68131, USA
Received 6 May 1992, accepted 2 June 1992
Northern blot and ribonucleasc protection assay were used to identify c~2-adrcnoceptor subtypes in haman colonic ade;mcarcinoma (!13"29), neuroblastoma × glioma rat-mouse hybrid NGI08-15 (NG1OS) and opossum kidney (OK) cell lines. Radioligand binding studies showed that the ~t~-adrenoceplor expressed in HT29, NG108 and OK cells represent the pharmacologicai ~e~^, ~zn and a~c subtypes respectively. In our Northern blot analysis, hybridization of poly(A) ~ RNA t'rom I! 1~.~3,~ ; G ~ ~Jn,~l t)t~ cells with human kidney a,-adrcnoccptor eDNA probe (a,-C4) identified a single band of 4.4, 4.2 and 4.4 kb respectively in each cell line. tlybridization with a human platelet aa-adrenoceptor genomic probe (a~oC10) resulted in two bands for tIT20 cells with the size of 4.4 kb and 3.9 kb. No bands were sccn for HT29, NGI08 and OK cells when hybridized with a third a~-adrcnoceptor human genomic DNA probe which is localized in chromosome 2 (a2-C2). For the HT29 cells, ',he 3.9 kb band was seen only when using the ot~-Cl0 probe. Thus, this band probably represents a2-Cl0 mRNA To further characterize the a2-adrenoccptor m R N A expressed in HT29, NG 108 and OK cells, the sensitive ribonuelease protection assay was pertbrmed. A single band about 900 bp was protected when the poly(A)+ RNA from NG108 and OK cells was hybridized with an az-C4 RNA probe and digested with RNAases. Hybridization of mRNA from HT29 cells with c,~-Cl0 RNA probe and digestion with RNAases protected a 500 bp fragment. No bands were seen when the poly(A)+ RNA of tl,ese three cell lines hybridized with lhe a2-C2 RNA probe and digested with RNAases. In conclusion, Northern blot and ribor~lcleasc protection assay in this study provided evidence that the ot,-C4 is related to the a2n pharmacological cQ-mlrenoceptor subtype and is actively transcribed in OK and NGI08 cells. The o¢2-C10 is related to the or,A pharmacological subtype and is actively ira,scribed in HT29 coifs.. ct2-Adrcnoccptor mRNA; HT29 cells; OK cells; NG 108 ~:clls
1. Introduction The existence of subtypes for a2-adrenoceptors was first suggested by Bylund (198P, on the basis of cornparison between [3H]clonidir, e and [3H]yohimbine binding in a variety of tissues and species. It was demonst,:aled that sore,- regions of human (Petrash ,and Byl-md, 1986) and rat (Bylund, 1985) brain contain two populations of a2-adrenoceptor sites which differ in their affinity for prazosin by 30-40-fold. On the basis of these and other studies, Bylund has defined three subtypes of a,-adrenoceptors which are designated as a2/,, a2u and a2c. (Bylund, 1985). The human p!atelet and ,the HT29 human colonic adrenocarcinoma (HT29) cell contain aZA st,~type which has a low
Correspondence to: Wii~iam X. Pettinger, M.D., Midwest Hypertension Research (_'enter, Delk~:tment of Medicir,e Creig.~.on !!.fiver.~;i~,School ~i Medicine, 6{11North 30flaStreet, Suite 6730, Omaha, NE 68131, USA. Tel. (4(}21280-4336; Fax (402) 28tl-4101.
affinity for prazosin. The neonatal rat lung and the NGI08-15 neuroblastoma x glioma rat-mouse hybrid (NGI08) cell contain the a , n subtype (Byhmd, 19881 which has a high affinity for prazosin. The ~2-adrenoceptor on O K cell, an opossum kidney derived cell line (Murpt:y and Bylund, 1988)has a unique pharmacological profile and represents a third subtype, a : c . Three human a2-adrenoceptor genes have been cloned and identified. Based on their chromosomal location, they have been termed a2-C10 (Kobilka et al., 1987), a2-C4 (Regan et al., 1988) and a2.-C2 (Lomasney et al., 1990). The pharmacological characterization of the txz-C10 receptor clearly identifies it as an O~2Aadrenoceptor, based on a correlation between Kobilka's data from the receptor expressed in COS-7 cells (Kobilka et al., 1987) and Bylund's data in both the platelet and ~hc HT29 cell (Bylund, 1988). The a~.-C'~ and oz,-C2 receptors are different pharmacologically from the a,-C10 recentoc and canno~ ho identified "'~ clearly as one or another of the present pharnlaco[og[cally defined a2-adrenoceptor subtypes.
|'here arc discrepancies m the ctassificatkm of c~.,adrenoccptors using phammcologicat and mt~lceular cloning mcthod~,,. ':'a,ace" m R N A is an infomw.tion carrim' fronl gc,,~e to protein, tile study of the stlbtypes of c~2-adren,~-'ept{~r ;~t the mRNA level n ight pr{widc some hnpo~tant info~ma~i.~r wific!, ::',a.7 ~.
367
EJPMf?&. N}082 Short cotnrnunication
Nort ern blot and ribonuclease protection study of a -adr ;noceptor subtype,'s in cultured cell lines Lu Sun, S a t o s h i U m e m u r a , M e l a n i c C h u n a n d W i l l i a m A. P e t t i n g e r Midwest Itypertension Research Center, DtTmrtment of Medichle, Creighton Unit'ersity, School of Medicitw, Omaha, NE 68131, USA
Received 6 May 1992, accepted 2 June 1992
Northern blot and ribonucleasc protection assay were used to identify c~2-adrcnoceptor subtypes in haman colonic ade;mcarcinoma (!13"29), neuroblastoma × glioma rat-mouse hybrid NGI08-15 (NG1OS) and opossum kidney (OK) cell lines. Radioligand binding studies showed that the ~t~-adrenoceplor expressed in HT29, NG108 and OK cells represent the pharmacologicai ~e~^, ~zn and a~c subtypes respectively. In our Northern blot analysis, hybridization of poly(A) ~ RNA t'rom I! 1~.~3,~ ; G ~ ~Jn,~l t)t~ cells with human kidney a,-adrcnoccptor eDNA probe (a,-C4) identified a single band of 4.4, 4.2 and 4.4 kb respectively in each cell line. tlybridization with a human platelet aa-adrenoceptor genomic probe (a~oC10) resulted in two bands for tIT20 cells with the size of 4.4 kb and 3.9 kb. No bands were sccn for HT29, NGI08 and OK cells when hybridized with a third a~-adrcnoceptor human genomic DNA probe which is localized in chromosome 2 (a2-C2). For the HT29 cells, ',he 3.9 kb band was seen only when using the ot~-Cl0 probe. Thus, this band probably represents a2-Cl0 mRNA To further characterize the a2-adrenoccptor m R N A expressed in HT29, NG 108 and OK cells, the sensitive ribonuelease protection assay was pertbrmed. A single band about 900 bp was protected when the poly(A)+ RNA from NG108 and OK cells was hybridized with an az-C4 RNA probe and digested with RNAases. Hybridization of mRNA from HT29 cells with c,~-Cl0 RNA probe and digestion with RNAases protected a 500 bp fragment. No bands were seen when the poly(A)+ RNA of tl,ese three cell lines hybridized with lhe a2-C2 RNA probe and digested with RNAases. In conclusion, Northern blot and ribor~lcleasc protection assay in this study provided evidence that the ot,-C4 is related to the a2n pharmacological cQ-mlrenoceptor subtype and is actively transcribed in OK and NGI08 cells. The o¢2-C10 is related to the or,A pharmacological subtype and is actively ira,scribed in HT29 coifs.. ct2-Adrcnoccptor mRNA; HT29 cells; OK cells; NG 108 ~:clls
1. Introduction The existence of subtypes for a2-adrenoceptors was first suggested by Bylund (198P, on the basis of cornparison between [3H]clonidir, e and [3H]yohimbine binding in a variety of tissues and species. It was demonst,:aled that sore,- regions of human (Petrash ,and Byl-md, 1986) and rat (Bylund, 1985) brain contain two populations of a2-adrenoceptor sites which differ in their affinity for prazosin by 30-40-fold. On the basis of these and other studies, Bylund has defined three subtypes of a,-adrenoceptors which are designated as a2/,, a2u and a2c. (Bylund, 1985). The human p!atelet and ,the HT29 human colonic adrenocarcinoma (HT29) cell contain aZA st,~type which has a low
Correspondence to: Wii~iam X. Pettinger, M.D., Midwest Hypertension Research (_'enter, Delk~:tment of Medicir,e Creig.~.on !!.fiver.~;i~,School ~i Medicine, 6{11North 30flaStreet, Suite 6730, Omaha, NE 68131, USA. Tel. (4(}21280-4336; Fax (402) 28tl-4101.
affinity for prazosin. The neonatal rat lung and the NGI08-15 neuroblastoma x glioma rat-mouse hybrid (NGI08) cell contain the a , n subtype (Byhmd, 19881 which has a high affinity for prazosin. The ~2-adrenoceptor on O K cell, an opossum kidney derived cell line (Murpt:y and Bylund, 1988)has a unique pharmacological profile and represents a third subtype, a : c . Three human a2-adrenoceptor genes have been cloned and identified. Based on their chromosomal location, they have been termed a2-C10 (Kobilka et al., 1987), a2-C4 (Regan et al., 1988) and a2.-C2 (Lomasney et al., 1990). The pharmacological characterization of the txz-C10 receptor clearly identifies it as an O~2Aadrenoceptor, based on a correlation between Kobilka's data from the receptor expressed in COS-7 cells (Kobilka et al., 1987) and Bylund's data in both the platelet and ~hc HT29 cell (Bylund, 1988). The a~.-C'~ and oz,-C2 receptors are different pharmacologically from the a,-C10 recentoc and canno~ ho identified "'~ clearly as one or another of the present pharnlaco[og[cally defined a2-adrenoceptor subtypes.
|'here arc discrepancies m the ctassificatkm of c~.,adrenoccptors using phammcologicat and mt~lceular cloning mcthod~,,. ':'a,ace" m R N A is an infomw.tion carrim' fronl gc,,~e to protein, tile study of the stlbtypes of c~2-adren,~-'ept{~r ;~t the mRNA level n ight pr{widc some hnpo~tant info~ma~i.~r wific!, ::',a.7 ~.
