- Email: [email protected]
Muscarinic receptor subtypes in subpopulations of human blood mononuclear cells as analyzed by RT-PCR technique
N Jmrrnal nf”Neul-{)ir,lrnurlc,lugy 68 ( I’W6) 139-144
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Received6 October 1995; revised 30 April 1996: accepted I MrIy 1996
A this study we have analysed the expression of mRNA encoding the ml –m5 mAChR subtypes in humrm blood mononuclear cells and subpopulations of lymphocytes u reverse transcriptase–poly merase chain reaction (RT-PCR) technique. Total RNA was extracted from human blood mononuclear cells, T c mmuxytes, EB virus transformed B cells and from two leukemic cell lines and analysed by RT-PCR. Our results indicate that mRNAs for the m3, m4 and m5 muscarinic subtypes are expressed in mononuclear cells and purified T cells while m I and m2 mRNAs were not detected in these cells. No m I-m5 subtype mRNA was detected in B cells and mmrocytes. indicating absence of muscarinic receptors in these cells. The expression of muscarinic subtypes in the leukemic T cell line, Peer, and the promyelocytic leukemic cell line HL-60 was different from peripheral mononuclear cells. Both m3 and m5 subtypes were expressed in Peer cells but not the m4 subtype, whereas the m4 and m5 subtypes were detected in HL-60 cells. K#yMvrd.s: Reverse transcriptase-polymerase reaction; mRNA: Muscarinic receptors: Peripheral blood mononuclear cells: Lymphrrcytcs: Leukemic cells
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‘ Corresponding author. Tel.: +46-8-7465208; fax: +46-8-6899210. 0165-5728/96/$15.00
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Copyright 0 1996 Elsevier Science B.V. All rights reserved.
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Table 1 Primers used for PCR amplification of the ml–m5 subtypes and Cyclophilin in the present study Subtype
Sequence
Position
Size
ml
5’-GAAGAAGAGGAAGAGGACGAA-3’ 5’-CAGGAGAGGGGACTATCAGCA-3’
(826-847) (1378-1399)
573 bp
5’-GGGTCCTCTCTTTCATCCTCT-3’ 5’-TCCTGGGTTATTTCATCATCT-3’
(443-464) (891-912)
469 bp
5’-GTCTGGCTTGGGTCATCTCCT-3’ 5’-ACTGCTGCTGTGGTCTTGGTC-3’
(669-689) (1102-1082)
434 bp
5’-TGGGTACTGTCC’ITCGTGCTC-3’ 5’-CACACTCA-ITGCCTGTCTGCTTCG-3’
(569-589) (1160-1 134)
592 bp
5’-CTCATCAGTGGAATCTTCTCCA-3’ 5’-GGTCC-ITGGTTCGCTTCTCTGT-3’
(477-498) (927-906)
451 bp
5’-GACAAGGTCCCAAAGACAGC-3’ 5’-GTCCAGCATTTGCCATGGAC-3’
(121-140) (335-355)
235 bp
m2 m3 m4 m5 Cyclophilin
The corresponding upstream and downstream primers are shown in each sequence pair. Numbers in parenthesis indicate the positions of the termini of each primer, corresponding to the numbering based on the coding sequence obtained from the Gene Bank data base.
E. Ht[lstriim-Linc[uh[,A. Nordbprg/Journul oj”Neurvimnrunology68 (1996) 139-144
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Fig. 1. Agarose gel electrophoresis with ethidium bromide staining of PCR products amplified from human blood mononuclear cells, monocytes, B cells and leukemic cell lines Peer and HL-60 with primers specific for the three muscarinic receptor genes m3, m4 and m5. Total RNA (1 Kg) was used as template for first strand synthesis and cDNA was then amplified by PCR using the appropriate primers (Table 1). Typical results are shown for assays repeated at least 2–3 times. PCR performed on negarive controls when reverse transcriptase step was omitted resulted in no amplified products (not shown). A DNA standard lane (100 bp DNA ladder) is shown at the left of the gel, with bands labelled in bp. 1, DNA size marker; 2, control human genomic DNA; 3, blood mononuclear cells; 4, monocytes; 5, EB virus transformed B cells; 6, Peer, a leukemic T cell line; 7, HL-60, a promyelocytic leukemic cell line.
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Fig, 2. Agarose gel electrophoresis with ethidium bromide staining of PCR products amplified from human peripheral T cells with primers specific for the three musmrinic receptor genes m3 (lanes 2–3), m4 (lanes 4-5) and m5 (lanes 6-7). Totrrl RNA (1 pg) was used as template for first strand synthesis and cDNA was then amplified by PCR using the (Table 1). Subpopulations of CD3 and CD4 positive appropriate p T lymphocytes were purified from blood mononuclear cells using magnetic cell sorting witb antihuman CD3 and CD4 antibodies. Typical results are shown for assays repeated at Ieasr rwo rimes. PCR performed on negarive controls when reverse transcriptase step was omitted resulted in no amplified products (not shown). A DNA standard lane ( 100 bp DNA ladder) is shown at the left of the gel with bands Iabelled in hp. lane 1, DNA size marker; lanes 2, 4, 6, CD3 positive T cells; lanes 3, 5, 7, CD4 positive T cells.
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1 W t m b A p s e p ( 1 a d c e r f c n m p l e w a l a p x a f A p h b r ( 1 R a 1 R a 1 - F p l r M1 h h b o ( a 1 E a 1 T p t t a l O f i t n t udm shi unw hpbg ar srti ey tca espm 5 ee a n s tw An 1 d r h bo u m l omt n oc an ahu oTecnc l eadlewa nr l l a d el e s cesd xrl ,pe o rats lems s e sm3d i hon –n t be m f P a H c a c t r i n v e f s t u ei gsr a p tst ee m dhc i e aa lr l y i p t a p p w r b m c s ( a 1 T u p q s e ( a 1 i u w t d d s d i m l q R R p s m d et r a m t l a t i T e m s t l T x d s t m r l c f n s a p d from l P a H c w f d m b c B a l e s p w A d oe s w e T c i f e T d u R p a c a P c b t s w o t t t s t r h a R ( p a n o d as s d P c a r s u t s h d t Tc p l TI c l N b a m sl u –bm t ms y pt 5 eo ts f h b m c e t m f t m a m s w n s m w e t c a 1 p t t e t s c d B c p J c d f P c s t m c a e s c d i P c S an b c A h s t t m s e c h s d c l a g t T w w s g f t S t c c l a e m t o M R C S G T m s ( a 1 A u L a H O F C G p m t t t m o F A W F S T l a t t t c a t R C a G F t D b a n s e d H a S f t k g t c i p J c T h s l R m c c N a l m h r b o s s e m e R m ( a 1 S a B a 1 l . Adem. A., ,Nordberg, A., Bucht, G. and Winblad. B. ( 1986) ExtraneuC a 1 W T p b a s H ronal cholinergic markers in Alzheimer’s and Parkinson”s disease. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 10.247-257. w a d t s T l e Ashkenazi. A., Peralta, E.G., Winslow, J.W.. Ramachandran, J. and p c l h p v u Capon, D.J. (1988) Functional role of muscarinic acetylcholine receps i m d s t c i tor subtype diversity. Cold Spring Harbor Symp. Quant. Biol. 53. i u d e m i 263-’272, g r a v b a r (1993) Retinoic acid-inBaumgartner. M.K., Wei, J. and Aronstam, R.S. duced differentiation of human neurobkcstoma cell line alters musa ( a K 1 T H carinic receptor expression. Dev. Bmin Res. 72, 305–308. u t p s w n i d Bering, B.. Moises, H.W. and Muller, W.E. ( 1987) Muscminic cholinert m p w m e gic receptors on intact human lymphocytes-properties and subclass t e m s H c characterization. Biol. Psychiatry 22, 1451–1458. d f m p b w Bonner, T.I.. Buckley, N.J., Young, A.C. and Bmmr, M.R. ( 1987) Identification of a family of muscarinic acetylcholine receptor genes. t m s w p Science 237, 527–53 1. t p t u w ac Bonner, T.I.. Yong, A.C., Brann, M.R. and Buckley. N.J. ( 1988) Cloning d b a l m e and expression of the human and rat m5 muscarinic acetylcholine p t l m s am f o y receptor genes. Neuron 1, 403–410. a w d d s Burnet, P.W.J.. Eastwood, S.L. and Harrison, P.J. ( 1994) Detection and quantitation of 5-HT [A and 5.HT 2A receptor mRNAs in human d h b d ( a l t
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hippocampus using reverse transcriptase–polymerase chain reaction
(RT-PCR)techniqueand their correlationwith bindingsite densities and age. Neurosci. Left. 178, 85–89. Chomczynski, P. and Sacchi. N. ( 1987) Single step method for RNA isolation by acid guanidinium thiocyiurate-phenol-chloroform extraction. Anal. Biochem. 162, 156– 159. Costa, L.C., Kaylor, G. and Murphy, S.D. ( 1988) Muscarinic cholinergic binding sites on rat lymphocytes. Immunopharmacology 16, 139–149. Costa, P., Traver, D.J. Auger, C.B. and Costa, L.C. (1994) Expression of cholinergic muscarinic receptor subtypes mRNA in rat blood mononuclear cells. Immunopharmacology 28, 113– 123. Costa, P., Auger, C.B.. Traver. D.J. and Costa. L.G. (1995) Idemitication of m3, m4 imd m5 subtypes of muscarinic receptor mRNA in human blood mononuclear cells. J. Neuroimmunol. 60, 45-51. Drescher, D., Upirdhyay, S., Wilcox, E. and Fcx, J. (1992) Analysis of muscarinic receptor subtypes in the mouse cochlea by means of the polymemse chain reaction. J. Neurochem. 59, 765-767. Dulis, B.H., Gordon, M.A. and Wilson, I.B. (1979) Identification of muscarinic binding sites in human neutrophils by direct binding. Mol. Pharmacol. 15, 28-34. Eva, C., Ferrero, P.. Rocca, P., Funaro, A., Bergamasco, B., Ravizza, L. and Genazzani, E. (1989)”[3H]N-methylscopolamine binding to mus‘carinic receptors in human peripheral blood lymphocytes: Characterization, localization on T-lymphocyte subsets and age-dependent changes. Neuropharmacology 28, 7 19–726. Ferrero, P., Rocca, P., Eva, C., Benna, P., Rebaudengo, N., Ravizza, L., Ge,nazzani.E. and Bergamasco, B. ( 1991) An analysis of lymphocyte [’H]-N-methyl-scopolamine binding in neurological patients, Brain 114, 1759-1770. Genaro, A.M., Cremaschi, G.C. and Borda, E.S. (1993) Muscarinic cholinergic rcccptrrrsmr murine lymphocyte subpoptdations. Selective interactions with second messenger response system upon pharmacological stimulation. Immunopharmacology 26, 21-29. Karteda, T., Kitamura, Y. and Nomura, Y. (1993) Presence of m3 subtype muscarinic acetylcholine receptors and receptor-mediated increases in tbe cytoplasmic concentraticfnof Ca2+ in Jurkat, a human leukemic helper T lymphocyte line. Mol. Pbarmacol. 43, 356-364. .:
Neuroimmunoiog>68 (1996) 139-144 Lubbert, M. and Koeftler, H.P. (1988) Leukemic cell lines as a paradigm for myeloid differentiation. Cancer Res. 100, 33-62. Maslinski, W., Krdlberg, M., Nordstrom, O. and Bartfai, T. (1988) Muscarinic receptors and receptor mediated actions on rat thymocytes. J. Neuroimmunol. 17, 265–274. Maslinski, W, (1990) Cholinergic receptors of lymphocytes. Brain B&hav. Immun. 3, 1–14. Nitsch, R.M., Slack, B.E., Wrsrtman, R.J. and Growdon, J.H. (1992) Release of Alzheimer amyloid precursor derivatives stimulated by activation of muscarinic acetylcholine receptors. Science 258, 304– 307. Nordberg, A., Adem, A., Bucht, G., Viitanen, M, and Winblad, B. (1990) Alteratimr~in lymphrrcytereceptor densities in dementia of Alzheime.r type: A possible diagnostic marker. In: ed. C.J. Fowler. L.A. Carlsson, C-G. G(rttfries turd B. Winbhd Biological Markers in Dementia of Alzheimer Type. Smith-Gordon Co. Ltd, London, pp. 149-159. Nordberg, A. ( 1992) Neurorcceptor changes in Alzheimer disease. Cerebrovasc. Bruin Metab. Rev. 4, 303-328. Rabey, J.M., Shenkman. L. and Gilad, G.M, (1986) Cholinergic muscarinic binding by human lymphocytes: Chtmges with aging, antagonist treatment and senile dementia of the Alzheimer type. Ann. Neurol, 20, 628–631. Ravizza, L., Ferrero, P., Eva, C., Rocca, P., Tarenzi, L. and Benna, P. (1988) Alzheimer’s disease. In: ed. E. Giacobini and R. Becker, Current Research in Alzheimer Therapy - Cholinesterase Inhibitors. Taylor and Francis, New York, pp. 355-363. Steel, M, and Buckley, N, (1993) Differential regulation of muscarinic receptor mRNA levels in neuroblastoma cells by chronic agonist exposure: A comparative polymerase chain reaction study. Mol. Pharmacol. 43, 694-701. Strom, T.B., Lane, M.A. and George. K. (1981) The parallel, time-deperrdent, bimodal change in lymphocyte cholinergic binding activity and cholinergic influence upon lymphocyte-mediated cytotoxicity after lymphocyte activation. J. Immunol. 127, 705-710. Wei, J., Walton, E.A,, Milici, A. and Buccafusco, J.J. (1994) ml–m5 Muscarinic receptor distribution in rat CNS by RT-PCR and HLPC. J. Neurochem. 63. 815–821.
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S-14186 H
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I
l
k
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U
a
Received6 October 1995; revised 30 April 1996: accepted I MrIy 1996
A this study we have analysed the expression of mRNA encoding the ml –m5 mAChR subtypes in humrm blood mononuclear cells and subpopulations of lymphocytes u reverse transcriptase–poly merase chain reaction (RT-PCR) technique. Total RNA was extracted from human blood mononuclear cells, T c mmuxytes, EB virus transformed B cells and from two leukemic cell lines and analysed by RT-PCR. Our results indicate that mRNAs for the m3, m4 and m5 muscarinic subtypes are expressed in mononuclear cells and purified T cells while m I and m2 mRNAs were not detected in these cells. No m I-m5 subtype mRNA was detected in B cells and mmrocytes. indicating absence of muscarinic receptors in these cells. The expression of muscarinic subtypes in the leukemic T cell line, Peer, and the promyelocytic leukemic cell line HL-60 was different from peripheral mononuclear cells. Both m3 and m5 subtypes were expressed in Peer cells but not the m4 subtype, whereas the m4 and m5 subtypes were detected in HL-60 cells. K#yMvrd.s: Reverse transcriptase-polymerase reaction; mRNA: Muscarinic receptors: Peripheral blood mononuclear cells: Lymphrrcytcs: Leukemic cells
I functions of the immune system s
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‘ Corresponding author. Tel.: +46-8-7465208; fax: +46-8-6899210. 0165-5728/96/$15.00
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Copyright 0 1996 Elsevier Science B.V. All rights reserved.
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Table 1 Primers used for PCR amplification of the ml–m5 subtypes and Cyclophilin in the present study Subtype
Sequence
Position
Size
ml
5’-GAAGAAGAGGAAGAGGACGAA-3’ 5’-CAGGAGAGGGGACTATCAGCA-3’
(826-847) (1378-1399)
573 bp
5’-GGGTCCTCTCTTTCATCCTCT-3’ 5’-TCCTGGGTTATTTCATCATCT-3’
(443-464) (891-912)
469 bp
5’-GTCTGGCTTGGGTCATCTCCT-3’ 5’-ACTGCTGCTGTGGTCTTGGTC-3’
(669-689) (1102-1082)
434 bp
5’-TGGGTACTGTCC’ITCGTGCTC-3’ 5’-CACACTCA-ITGCCTGTCTGCTTCG-3’
(569-589) (1160-1 134)
592 bp
5’-CTCATCAGTGGAATCTTCTCCA-3’ 5’-GGTCC-ITGGTTCGCTTCTCTGT-3’
(477-498) (927-906)
451 bp
5’-GACAAGGTCCCAAAGACAGC-3’ 5’-GTCCAGCATTTGCCATGGAC-3’
(121-140) (335-355)
235 bp
m2 m3 m4 m5 Cyclophilin
The corresponding upstream and downstream primers are shown in each sequence pair. Numbers in parenthesis indicate the positions of the termini of each primer, corresponding to the numbering based on the coding sequence obtained from the Gene Bank data base.
E. Ht[lstriim-Linc[uh[,A. Nordbprg/Journul oj”Neurvimnrunology68 (1996) 139-144
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Fig. 1. Agarose gel electrophoresis with ethidium bromide staining of PCR products amplified from human blood mononuclear cells, monocytes, B cells and leukemic cell lines Peer and HL-60 with primers specific for the three muscarinic receptor genes m3, m4 and m5. Total RNA (1 Kg) was used as template for first strand synthesis and cDNA was then amplified by PCR using the appropriate primers (Table 1). Typical results are shown for assays repeated at least 2–3 times. PCR performed on negarive controls when reverse transcriptase step was omitted resulted in no amplified products (not shown). A DNA standard lane (100 bp DNA ladder) is shown at the left of the gel, with bands labelled in bp. 1, DNA size marker; 2, control human genomic DNA; 3, blood mononuclear cells; 4, monocytes; 5, EB virus transformed B cells; 6, Peer, a leukemic T cell line; 7, HL-60, a promyelocytic leukemic cell line.
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Fig, 2. Agarose gel electrophoresis with ethidium bromide staining of PCR products amplified from human peripheral T cells with primers specific for the three musmrinic receptor genes m3 (lanes 2–3), m4 (lanes 4-5) and m5 (lanes 6-7). Totrrl RNA (1 pg) was used as template for first strand synthesis and cDNA was then amplified by PCR using the (Table 1). Subpopulations of CD3 and CD4 positive appropriate p T lymphocytes were purified from blood mononuclear cells using magnetic cell sorting witb antihuman CD3 and CD4 antibodies. Typical results are shown for assays repeated at Ieasr rwo rimes. PCR performed on negarive controls when reverse transcriptase step was omitted resulted in no amplified products (not shown). A DNA standard lane ( 100 bp DNA ladder) is shown at the left of the gel with bands Iabelled in hp. lane 1, DNA size marker; lanes 2, 4, 6, CD3 positive T cells; lanes 3, 5, 7, CD4 positive T cells.
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1 W t m b A p s e p ( 1 a d c e r f c n m p l e w a l a p x a f A p h b r ( 1 R a 1 R a 1 - F p l r M1 h h b o ( a 1 E a 1 T p t t a l O f i t n t udm shi unw hpbg ar srti ey tca espm 5 ee a n s tw An 1 d r h bo u m l omt n oc an ahu oTecnc l eadlewa nr l l a d el e s cesd xrl ,pe o rats lems s e sm3d i hon –n t be m f P a H c a c t r i n v e f s t u ei gsr a p tst ee m dhc i e aa lr l y i p t a p p w r b m c s ( a 1 T u p q s e ( a 1 i u w t d d s d i m l q R R p s m d et r a m t l a t i T e m s t l T x d s t m r l c f n s a p d from l P a H c w f d m b c B a l e s p w A d oe s w e T c i f e T d u R p a c a P c b t s w o t t t s t r h a R ( p a n o d as s d P c a r s u t s h d t Tc p l TI c l N b a m sl u –bm t ms y pt 5 eo ts f h b m c e t m f t m a m s w n s m w e t c a 1 p t t e t s c d B c p J c d f P c s t m c a e s c d i P c S an b c A h s t t m s e c h s d c l a g t T w w s g f t S t c c l a e m t o M R C S G T m s ( a 1 A u L a H O F C G p m t t t m o F A W F S T l a t t t c a t R C a G F t D b a n s e d H a S f t k g t c i p J c T h s l R m c c N a l m h r b o s s e m e R m ( a 1 S a B a 1 l . Adem. A., ,Nordberg, A., Bucht, G. and Winblad. B. ( 1986) ExtraneuC a 1 W T p b a s H ronal cholinergic markers in Alzheimer’s and Parkinson”s disease. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 10.247-257. w a d t s T l e Ashkenazi. A., Peralta, E.G., Winslow, J.W.. Ramachandran, J. and p c l h p v u Capon, D.J. (1988) Functional role of muscarinic acetylcholine receps i m d s t c i tor subtype diversity. Cold Spring Harbor Symp. Quant. Biol. 53. i u d e m i 263-’272, g r a v b a r (1993) Retinoic acid-inBaumgartner. M.K., Wei, J. and Aronstam, R.S. duced differentiation of human neurobkcstoma cell line alters musa ( a K 1 T H carinic receptor expression. Dev. Bmin Res. 72, 305–308. u t p s w n i d Bering, B.. Moises, H.W. and Muller, W.E. ( 1987) Muscminic cholinert m p w m e gic receptors on intact human lymphocytes-properties and subclass t e m s H c characterization. Biol. Psychiatry 22, 1451–1458. d f m p b w Bonner, T.I.. Buckley, N.J., Young, A.C. and Bmmr, M.R. ( 1987) Identification of a family of muscarinic acetylcholine receptor genes. t m s w p Science 237, 527–53 1. t p t u w ac Bonner, T.I.. Yong, A.C., Brann, M.R. and Buckley. N.J. ( 1988) Cloning d b a l m e and expression of the human and rat m5 muscarinic acetylcholine p t l m s am f o y receptor genes. Neuron 1, 403–410. a w d d s Burnet, P.W.J.. Eastwood, S.L. and Harrison, P.J. ( 1994) Detection and quantitation of 5-HT [A and 5.HT 2A receptor mRNAs in human d h b d ( a l t
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