- Email: [email protected]
Expression of purinergic receptors in bipolar cells of the rat retina
Molecular Brain Research 76 Ž2000. 415–418 www.elsevier.comrlocaterbres
Short communication
Expression of purinergic receptors in bipolar cells of the rat retina Thomas H. Wheeler-Schilling a
a,)
, Katja Marquordt a , Konrad Kohler a , Ronald Jabs b, Elke Guenther a
Department of Pathophysiology of Vision and Neuroophthalmology, DiÕision of Experimental Ophthalmology, UniÕersity Eye Hospital Tuebingen Roentgenweg 11, 72076 Tuebingen, Germany b Clinic of Neurosurgery, Department of Experimental Neurobiology, UniÕersity of Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany Accepted 28 December 1999
Abstract P2X receptors are ligand-gated ion channels which are activated by excitatory neurotransmitter ATP. Despite considerable evidence of signaling by extracellular nucleotides in other sensory systems, P2X receptors in the visual system have only rarely been studied, and almost nothing is known about their functional significance in the retina. To determine whether ATP plays a role in the modulation of vertical retinal signal pathways, we examined the expression of P2X receptor mRNA in freshly isolated bipolar cells of the rat retina Ž Brown Norway, P25. using the single-cell RT-PCR technique. Positive amplification signals were found in about 33% of the bipolar cells for P2X 3 , P2X 4 and P2X 5 but not for P2X 7 mRNA. We conclude that at least a subpopulation of bipolar cells in the rat retina expresses ionotropic P2 receptors of the P2X type and that these possibly exert a neuromodulatory influence on information processing in the retina. q 2000 Elsevier Science B.V. All rights reserved. Keywords: P2X receptor; ATP, single-cell RT-PCR; Ligand-gated ion channel; Retina; Bipolar cell
The extracellular purines adenosine and adenosine 5X triphosphate ŽATP. exert their actions via P1 and P2 receptors respectively w5x. P2 receptors in turn are classified into P2X Žionotropic. receptors and P2Y Žmetabotropic, G-protein-coupled. receptors. P2X receptor subunits assemble to form nucleotid-gated ion channels, which mediate fast excitatory neurotransmission and help regulate transmitter release and neuronal excitability w1,13x. Eight P2X receptor subunits have been cloned to date ŽP2X 1 – 7 , P2XM. w2x. Despite considerable evidence that extracellular ATP modulates signals in other sensory systems, i.e. the ear, only some studies have focused on its role in the eye w3,4,11,12,15,16x. We recently demonstrated the presence of mRNA in the retina for the ionotropic P2 receptor subtypes P2X 2 , P2X 3 , P2X 4 , and P2X 7 w3,4x. With the exception of the P2X 2 subunit w7x, little is known about the cell-type specificity of P2X receptor expression and thus the possible physiological functions of these subtypes in the mammalian retina. In order to investigate whether P2X receptors are involved in signal modulation within the vertical pathways
)
Corresponding author. Fax: q49-7071-295-777; [email protected]
e-mail:
of the mammalian retina, we used the single cell RT-PCR technique to analyze P2X receptor expression in acutely isolated bipolar cells. We found that different types of P2X receptors are expressed in a subpopulation of retinal bipolar cells, indicating that ATP can modulate signal transmission in the mammalian retina. This study was carried out in accordance with the European Communities Council Directive Ž86r609rEEC.. All chemicals were obtained from Sigma ŽDeisenhofen, Germany. unless stated otherwise. Brown Norway rats Ž) P25. were sacrificed by short CO 2 incubation. The eyes were dissected by hemisection along the ora serrata, and the retina was carefully removed from the underlying tissue and incubated for 30–40 min at 378C in phosphate buffered solution ŽPBS. containing 250 mgrml Nargase ŽServa, Germany.. After wash in HEPES based, 100 unitsrml DNAse I containing minimum essential medium ŽMEM, Hank’s salt, Live Technologies, Germany. the tissue was dissociated by gentle trituration through a fine Pasteur pipette. The cell suspension was stored within the MEM solution at 48C until use. Individual bipolar cells were selected at 630 = magnification using an Olympus BX50 WI microscope. The cell cytosol was collected with tiny, patch clamp pipette-like glass capillaries with a tip diameter of 2–5 mm. The capillaries contained 90 mM
0169-328Xr00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 0 0 . 0 0 0 2 0 - 6
T. H. Wheeler-Schilling et al.r Molecular Brain Research 76 (2000) 415–418
416
CsAc, 40 mM CsCl 2 , 10 mM EGTA, 1 mM MgCl 2 , 0.2 mM CaCl 2 , and 10 mM HEPES ŽpH 7.2. and were positioned via a micro manipulator system Ž5171, Eppendorf, Germany.. Each pipette was placed on a bipolar cell soma, and the cytosol was harvested by applying negative pressure under visual control. The contents of the glass capillaries were expelled into 0.2 ml tubes containing 1 ml of hexamer random primers Ž2 mgrml, Boehringer Mannheim, Germany., 1 ml of dNTPs Ž10 mM in each case. ŽMBI Fermentas, St. Leon-Rot, Germany., 1 ml MgCl 2 25 mM ŽPerkin-Elmer, Weiterstadt, Germany., 1 ml 10 = Reaction Buffer Ž100 mM Tris–HCl, pH 8.3, 500 mM KCl ŽPerkin-Elmer., 0,5 ml RNasin ribonuclease inhibitor 50Urml ŽPromega, Madison, WI, USA. 0,5 ml MuLV reverse transcriptase 50 Urml ŽPerkin-Elmer.. The tubes were incubated at 258C for 10 min followed by 428C for 60 min for RT reaction. Samples without reverse transcriptase as well as samples without the cytosol of bipolar cells served as negative controls. Two rounds of PCR Ž40 and 35 cycles on a Perkin-Elmer GeneAmp 2400. were used with four sets of nested primers. The sequences of all primers used for PCR-reactions are listed in Table 1. The entire product of the reverse transcription reaction was utilized for the first amplification. 4 ml 10 = Reaction Buffer, 1 ml 25 mM MgCl 2 , 1 ml of each outer primer Ž10 mM, up and low. and 18.75 ml H 2 O was added to the reverse transcription reaction. A hot start PCR was performed by incubating the reaction at 948C for 3 min and adding 0.5 ml AmpliTaq DNA polymerase Ž5Urml; Perkin-Elmer.. Reaction conditions were: denaturation at 948C for 30 s, annealing at 578C for 30 s, extension at 728C for 30 s, followed by a final extension step at 728C for 5 min. Aliquots of each first PCR were
diluted 100-fold and reamplified in a second PCR amplification. All reactions had the following composition: 5 ml 10 = Reaction Buffer, 3 ml 25 mM MgCl 2 , 1 ml dNTP’s Ž10 mM each., 38 ml H 2 O, 0,5 ml AmpliTaq DNA polymerase Ž5Urml. and 0,5 ml of the first PCR product. The amplification conditions were as described, with the exception that the nested primer pairs were used Ž10 mM of each nested up and low primer per reaction; Table 1.. The cycling parameters were: 948C for 3 min followed by 35 cycles of 948C for 30 s, 588C Žfor P2X 3 , P2X 5 and P2X 7 . or 618C Žfor P2X 4 . for 30 s, and 728C for 30 s. A final extension step of 728C for 5 min followed. Amplification products were analyzed by agarose gel electrophoresis. The P2X-specific PCR products were identified by Southern blot analysis using subunit specific oligonucleotides ŽP2X 3 , P2X 4 , P2X 5 and P2X 7 probes; Table 1. as hybridization probes, according to standard procedure. Labeling of the oligonucleotides with digoxigenin-dUTP was performed with the DIG Oligonucleotide Tailing Kit ŽBoehringer Mannheim, Germany.. After hybridization to the target nucleic acids, oligonucleotides were detected by enzyme-linked immunoassay using an anti-digoxigenin alkaline phosphatase conjugate with subsequent enzymecatalyzed color reaction ŽBCIPrNBT, Boehringer Mannheim, Germany.. As an additional control, PCR products of the P2X subtypes were cloned in pCR Script Amp SKŽq. plasmid ŽStratagene, Heidelberg, Germany.. The identity of the PCR products was determined by fluorescent DNA sequencing on an ABI PRISM 310 Genetic Analyzer ŽPerkin-Elmer; data not shown.. Only bipolar cells without osmotic damage in the soma or processes were selected. The morphology of a typical
Table 1 Nucleotide sequences of P2X primer sets used for the SC-RT-PCR amplification of cDNAs and hybridization probes for Southern Blot analysis P2X 3 P2X 3 P2X 3 P2X 3 P2X 3 P2X 4 P2X 4 P2X 4 P2X 4 P2X 4 P2X 5 P2X 5 P2X 5 P2X 5 P2X 5 P2X 7 P2X 7 P2X 7 P2X 7 P2X 7
up low nested up nested low probe up low nested up nested low probe up low nested up nested low probe up low nested up nested low probe
X
X
5 -CAA CTT CAG GTT TGC CAA A-3 X X 5 -TGA ACA GTG AGG GCC TAG AT-3 X X 5 -ATC ATC CCC ACC ATT ATC-3 X X 5 -AAA TAG CAG CCC TTC TTC-3 X X 5 -AGG GTA CTG CGT CAA CCA ACC CAG TGT TCG-3 X X 5 -CCC AAC ATC ACC ACG TC-3 X X 5 -CAG GAG ACC TTC CGT GAA A-3 X X 5 -TCC CTT CTG CCC CAT ATT CC-3 X X 5 -TTC ATC TCC CCC GAA AGA CC-3 X X 5 -GGC TAC AAT TTC AGG TTT GCC AAG TAC TAC-3 X X 5 -AGT CAT CAA CAT TGG TTC TG-3 X X 5 -CAG GAG ACC TTC CGT GAA A-3 X X 5 -CGA CCT GGT ACT TAT CTA CCT C-3 X X 5 -ACG TTC ACA ATG GCA TTC-3 X X 5 -AGG TTG AGG CCA ACG AGA TGG AGC AGG AGC-3 X X 5 -AGG AGC CCC TTA TCA GCT CT-3 X X 5 -CAT TGG TGT ACT TGT CGT CC-3 X X 5 -AAC AGT GCC ATT CTG ACC-3 X X 5 -GCC ACC TCT GTA AAG TTC TC-3 X X 5 -AGC GCC GAA AAC TTC ACC GTA CTC ATC AAG-3
1005–1023 bp 1523–1503 bp 1123–1140 bp 1472–1455 bp 1261–1290 bp 719–735 bp 1342–1323 bp 772–791 bp 1260–1241 bp 971–1000 bp 1192–1211 bp 1733–1715 bp 1246–1267 bp 1515–1498 bp 1341–1370 bp 282–301 bp 974–955 bp 501–518 bp 858–839 bp 671–700 bp
T. H. Wheeler-Schilling et al.r Molecular Brain Research 76 (2000) 415–418
417
Fig. 1. Morphology of a typical, freshly isolated bipolar cell of the rat retina. Scale bar: 10 mm.
rat bipolar cell is shown in Fig. 1. mRNA of the P2X 3 , P2X 4 , P2X 5 , or P2X 7 subunit was found in 33% of the bipolar cells Ž n s 24.. PCR products of all cells were distinctly visible in one band per specific primer with the following base pair Žbp. lengths: 350 bp ŽP2X 3 ., 489 bp ŽP2X 4 ., and 270 bp ŽP2X 5 .. An example of gel electrophoresis is shown in Fig. 2. In contrast, no PCR product of the predicted size of 358 bp was detected for P2X 7 in any bipolar cells examined for the expression of this subunit Ž n s 8.. In the case of P2X 2 , unfortunately, we were not able to perform the RT-PCR for this particular subunit successfully at the single cell level due to unresolved technical problems. It is not trivial to scale RT-PCR experiments down to the single cell level, whilst performing well in whole tissue experiments Žrefer to our previous studies.. ATP is by far the most abundant purine in the retina w10x. Binding studies of a ,b-meATP in rabbit eyecup preparations first indicated the existence of P2X receptors in the vertebrate retina w11x. Using indirect RT-PCR in situ hybridization and immunolabeling, Greenwood et al. w7x
demonstrated expression of the P2X 2 subunit in the somata of photoreceptors, retinal ganglion cells, and neurons of the inner nuclear layer of rat retina. Recently, we demonstrated the presence of the P2X receptor subtypes P2X 2 , P2X 3 , P2X 4 , P2X 5 and P2X 7 mRNAs in the retina. Moreover, P2X 2 and P2X 4 mRNAs are also expressed in the choroid w3,4x. The P2X 7 receptor protein was found in the ganglion cell layer and in some cells of the inner nuclear layer, indicating that P2X receptors might be involved in the modulation of vertical signal transmission in the retina. The results of the present study provide direct molecular evidence for the expression of distinct P2X receptor subunits within acutely isolated bipolar cells. Since it has already been shown that cell culture conditions modify the expression pattern of purinergic receptors w8,9,17x, this study analyzed only freshly isolated bipolar cells using serum-free solutions. Application of RT-PCR at the single-cell level revealed an expression of P2X 3 , P2X 4 , and P2X 5 mRNA in approximately one third of the bipolar cells. In contrast, no
Fig. 2. Detection of P2X 3 , P2X 4 , P2X 5 -mRNA in individual bipolar cells of pigmented rats Ž Brown Norway, P25.. M: 100 bp molecular weight standard; lanes 1, 4, and 6: P2X 3,4,5 in individual bipolar cells; lanes 2, 5 and 8: negative controls; lanes 3, 6, and 9: P2X 3,4,5 positive controls Žspecific clones, .. The 1.5% agarose gel is stained with ethidium bromide. generous gift of Dr. U. Brandle ¨
418
T. H. Wheeler-Schilling et al.r Molecular Brain Research 76 (2000) 415–418
specific RT-PCR product for the P2X 7 subunit was found. Together with the distribution of P2X 7 immunoreactivity in the INL w4x, this strongly indicates that amacrine cells andror Muller cells express P2X 7 receptors but not bipo¨ lar or horizontal cells. Moreover, since the P2X 7 subunit is thought to be the only purinoreceptor involved in processes of apoptosis, the lack of P2X 7 mRNA makes it likely that bipolar cell death is not mediated by the activation of purinoceptors w6,14x. Although purinergic signaling is known to take place in the central nervous system ŽCNS., the exact function of most P2X receptors remains to be determined. Among the recently described functions of P2X receptors in the CNS are fast synaptic transmission, modulation of transmitter release, and modulation of neuron excitability Žsee Ref. w13x for a review.. The expression of P2X receptor in bipolar cells of the mammalian retina is the first evidence that purinoreceptors also may be involved in these processes of signal modulation within the vertical retinal pathways w12x. Further functional analysis with immunohistochemical and electrophysiological techniques will be required for a better understanding of the complex physiology of purinoceptors in the retina.
w5x w6x
w7x
w8x
w9x
w10x
w11x
w12x w13x w14x
References w15x w1x M.P. Abbracchio, G. Burnstock, Purinoceptors: are there families of P2X and P2Y purinoceptors? Pharmacol. Ther. 64 Ž1994. 445–475. w2x A.J. Brake, D. Julius, Signaling by extracellular nucleotides, Annu. Rev. Cell Dev. Biol. 12 Ž1996. 519–541. w3x U. Brandle, E. Guenther, C. Irrle, T.H. Wheeler-Schilling, Gene ¨ expression of the P2X receptors in the rat retina, Mol. Brain Res. 59 Ž1998. 269–272. w4x U. Brandle, K. Kohler, T.H. Wheeler-Schilling, Expression of the ¨
w16x
w17x
P2X 7-receptor subunit in neurons of the rat retina, Mol. Brain Res. 62 Ž1998. 106–109. G. Burnstock, The past, present and future of purine nucleotides as signalling molecules, Neuropharmacology 36 Ž1997. 1127–1139. K.C. Fernando, C.E. Gargett, J.S. Wiley, Activation of the P2ZrP2X7 receptor in human lymphocytes produces a delayed permeability lesion: involvement of phospholipase D, Arch. Biochem. Biophys. 362 Ž1999. 197–202. D. Greenwood, W.P. Yao, G.D. Housley, Expression of the P2X2 receptor subunit of the ATP-gated ion channel in the retina, NeuroReport 8 Ž1997. 1083–1088. R. Jabs, I.A. Paterson, W. Walz, Qualitative analysis of membrane currents in glial cells from normal and gliotic tissue in situ: downregulation of Naq current and lack of P2 purinergic responses, Neuroscience 81 Ž1997. 847–860. H.K. Kimelberg, Z. Cai, P. Rastogi, C.J. Charniga, S. Goderie, V. Dave, T.O. Jalonen, Transmitter-induced calcium responses differ in astrocytes acutely isolated from rat brain and in culture, J. Neurochem. 68 Ž1997. 1088–1098. M.J. Neal, J.R. Cunningham, Z. Dent, Modulation of extracellular GABA levels in the retina by activation of glial P2X-purinoceptors, Br. J. Pharmacol. 124 Ž1998. 317–322. M.J. Neal, J.R. Cunningham, S.J. Paterson, Binding sites for alpha,beta-methylene ATP are present in the vertebrate retina, Br. J. Pharmacol. 114 Ž1995. 101P, Abstract. A. Peral, J. Pintor, Purinergic transmission in the retina, Neurosci. Res. Commun. 23 Ž1998. 129–138. S.J. Robertson, Tissue distribution and functional contribution of P2X receptors in the CNS, Drug Dev. Res. 45 Ž1998. 336–341. L.E. Schulze, C. Hugo, S. Rost, S. Arnold, A. Gruber, B. Brune, R.B. Sterzel, Extracellular ATP causes apoptosis and necrosis of cultured mesangial cells via P2ZrP2X7 receptors, Am. J. Physiol. 275 Ž1998. F962–F971. M. Sugioka, Y. Fukuda, M. Yamashita, Ca2q responses to ATP via purinoceptors in the early embryonic chick retina, J. Physiol. ŽLondon. 493 Ž1996. 855–863. H. Taschenberger, R. Juttner, R. Grantyn, Ca2q-permeable P2X receptor channels in cultured rat retinal ganglion cells, J. Neurosci. 19 Ž1999. 3353–3366. W. Walz, S. Ilschner, C. Ohlemeyer, R. Banati, H. Kettenmann, Extracellular ATP activates a cation conductance and a Kq conductance in cultured microglial cells from mouse brain, J. Neurosci. 13 Ž1993. 4403–4411.
Short communication
Expression of purinergic receptors in bipolar cells of the rat retina Thomas H. Wheeler-Schilling a
a,)
, Katja Marquordt a , Konrad Kohler a , Ronald Jabs b, Elke Guenther a
Department of Pathophysiology of Vision and Neuroophthalmology, DiÕision of Experimental Ophthalmology, UniÕersity Eye Hospital Tuebingen Roentgenweg 11, 72076 Tuebingen, Germany b Clinic of Neurosurgery, Department of Experimental Neurobiology, UniÕersity of Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany Accepted 28 December 1999
Abstract P2X receptors are ligand-gated ion channels which are activated by excitatory neurotransmitter ATP. Despite considerable evidence of signaling by extracellular nucleotides in other sensory systems, P2X receptors in the visual system have only rarely been studied, and almost nothing is known about their functional significance in the retina. To determine whether ATP plays a role in the modulation of vertical retinal signal pathways, we examined the expression of P2X receptor mRNA in freshly isolated bipolar cells of the rat retina Ž Brown Norway, P25. using the single-cell RT-PCR technique. Positive amplification signals were found in about 33% of the bipolar cells for P2X 3 , P2X 4 and P2X 5 but not for P2X 7 mRNA. We conclude that at least a subpopulation of bipolar cells in the rat retina expresses ionotropic P2 receptors of the P2X type and that these possibly exert a neuromodulatory influence on information processing in the retina. q 2000 Elsevier Science B.V. All rights reserved. Keywords: P2X receptor; ATP, single-cell RT-PCR; Ligand-gated ion channel; Retina; Bipolar cell
The extracellular purines adenosine and adenosine 5X triphosphate ŽATP. exert their actions via P1 and P2 receptors respectively w5x. P2 receptors in turn are classified into P2X Žionotropic. receptors and P2Y Žmetabotropic, G-protein-coupled. receptors. P2X receptor subunits assemble to form nucleotid-gated ion channels, which mediate fast excitatory neurotransmission and help regulate transmitter release and neuronal excitability w1,13x. Eight P2X receptor subunits have been cloned to date ŽP2X 1 – 7 , P2XM. w2x. Despite considerable evidence that extracellular ATP modulates signals in other sensory systems, i.e. the ear, only some studies have focused on its role in the eye w3,4,11,12,15,16x. We recently demonstrated the presence of mRNA in the retina for the ionotropic P2 receptor subtypes P2X 2 , P2X 3 , P2X 4 , and P2X 7 w3,4x. With the exception of the P2X 2 subunit w7x, little is known about the cell-type specificity of P2X receptor expression and thus the possible physiological functions of these subtypes in the mammalian retina. In order to investigate whether P2X receptors are involved in signal modulation within the vertical pathways
)
Corresponding author. Fax: q49-7071-295-777; [email protected]
e-mail:
of the mammalian retina, we used the single cell RT-PCR technique to analyze P2X receptor expression in acutely isolated bipolar cells. We found that different types of P2X receptors are expressed in a subpopulation of retinal bipolar cells, indicating that ATP can modulate signal transmission in the mammalian retina. This study was carried out in accordance with the European Communities Council Directive Ž86r609rEEC.. All chemicals were obtained from Sigma ŽDeisenhofen, Germany. unless stated otherwise. Brown Norway rats Ž) P25. were sacrificed by short CO 2 incubation. The eyes were dissected by hemisection along the ora serrata, and the retina was carefully removed from the underlying tissue and incubated for 30–40 min at 378C in phosphate buffered solution ŽPBS. containing 250 mgrml Nargase ŽServa, Germany.. After wash in HEPES based, 100 unitsrml DNAse I containing minimum essential medium ŽMEM, Hank’s salt, Live Technologies, Germany. the tissue was dissociated by gentle trituration through a fine Pasteur pipette. The cell suspension was stored within the MEM solution at 48C until use. Individual bipolar cells were selected at 630 = magnification using an Olympus BX50 WI microscope. The cell cytosol was collected with tiny, patch clamp pipette-like glass capillaries with a tip diameter of 2–5 mm. The capillaries contained 90 mM
0169-328Xr00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 0 0 . 0 0 0 2 0 - 6
T. H. Wheeler-Schilling et al.r Molecular Brain Research 76 (2000) 415–418
416
CsAc, 40 mM CsCl 2 , 10 mM EGTA, 1 mM MgCl 2 , 0.2 mM CaCl 2 , and 10 mM HEPES ŽpH 7.2. and were positioned via a micro manipulator system Ž5171, Eppendorf, Germany.. Each pipette was placed on a bipolar cell soma, and the cytosol was harvested by applying negative pressure under visual control. The contents of the glass capillaries were expelled into 0.2 ml tubes containing 1 ml of hexamer random primers Ž2 mgrml, Boehringer Mannheim, Germany., 1 ml of dNTPs Ž10 mM in each case. ŽMBI Fermentas, St. Leon-Rot, Germany., 1 ml MgCl 2 25 mM ŽPerkin-Elmer, Weiterstadt, Germany., 1 ml 10 = Reaction Buffer Ž100 mM Tris–HCl, pH 8.3, 500 mM KCl ŽPerkin-Elmer., 0,5 ml RNasin ribonuclease inhibitor 50Urml ŽPromega, Madison, WI, USA. 0,5 ml MuLV reverse transcriptase 50 Urml ŽPerkin-Elmer.. The tubes were incubated at 258C for 10 min followed by 428C for 60 min for RT reaction. Samples without reverse transcriptase as well as samples without the cytosol of bipolar cells served as negative controls. Two rounds of PCR Ž40 and 35 cycles on a Perkin-Elmer GeneAmp 2400. were used with four sets of nested primers. The sequences of all primers used for PCR-reactions are listed in Table 1. The entire product of the reverse transcription reaction was utilized for the first amplification. 4 ml 10 = Reaction Buffer, 1 ml 25 mM MgCl 2 , 1 ml of each outer primer Ž10 mM, up and low. and 18.75 ml H 2 O was added to the reverse transcription reaction. A hot start PCR was performed by incubating the reaction at 948C for 3 min and adding 0.5 ml AmpliTaq DNA polymerase Ž5Urml; Perkin-Elmer.. Reaction conditions were: denaturation at 948C for 30 s, annealing at 578C for 30 s, extension at 728C for 30 s, followed by a final extension step at 728C for 5 min. Aliquots of each first PCR were
diluted 100-fold and reamplified in a second PCR amplification. All reactions had the following composition: 5 ml 10 = Reaction Buffer, 3 ml 25 mM MgCl 2 , 1 ml dNTP’s Ž10 mM each., 38 ml H 2 O, 0,5 ml AmpliTaq DNA polymerase Ž5Urml. and 0,5 ml of the first PCR product. The amplification conditions were as described, with the exception that the nested primer pairs were used Ž10 mM of each nested up and low primer per reaction; Table 1.. The cycling parameters were: 948C for 3 min followed by 35 cycles of 948C for 30 s, 588C Žfor P2X 3 , P2X 5 and P2X 7 . or 618C Žfor P2X 4 . for 30 s, and 728C for 30 s. A final extension step of 728C for 5 min followed. Amplification products were analyzed by agarose gel electrophoresis. The P2X-specific PCR products were identified by Southern blot analysis using subunit specific oligonucleotides ŽP2X 3 , P2X 4 , P2X 5 and P2X 7 probes; Table 1. as hybridization probes, according to standard procedure. Labeling of the oligonucleotides with digoxigenin-dUTP was performed with the DIG Oligonucleotide Tailing Kit ŽBoehringer Mannheim, Germany.. After hybridization to the target nucleic acids, oligonucleotides were detected by enzyme-linked immunoassay using an anti-digoxigenin alkaline phosphatase conjugate with subsequent enzymecatalyzed color reaction ŽBCIPrNBT, Boehringer Mannheim, Germany.. As an additional control, PCR products of the P2X subtypes were cloned in pCR Script Amp SKŽq. plasmid ŽStratagene, Heidelberg, Germany.. The identity of the PCR products was determined by fluorescent DNA sequencing on an ABI PRISM 310 Genetic Analyzer ŽPerkin-Elmer; data not shown.. Only bipolar cells without osmotic damage in the soma or processes were selected. The morphology of a typical
Table 1 Nucleotide sequences of P2X primer sets used for the SC-RT-PCR amplification of cDNAs and hybridization probes for Southern Blot analysis P2X 3 P2X 3 P2X 3 P2X 3 P2X 3 P2X 4 P2X 4 P2X 4 P2X 4 P2X 4 P2X 5 P2X 5 P2X 5 P2X 5 P2X 5 P2X 7 P2X 7 P2X 7 P2X 7 P2X 7
up low nested up nested low probe up low nested up nested low probe up low nested up nested low probe up low nested up nested low probe
X
X
5 -CAA CTT CAG GTT TGC CAA A-3 X X 5 -TGA ACA GTG AGG GCC TAG AT-3 X X 5 -ATC ATC CCC ACC ATT ATC-3 X X 5 -AAA TAG CAG CCC TTC TTC-3 X X 5 -AGG GTA CTG CGT CAA CCA ACC CAG TGT TCG-3 X X 5 -CCC AAC ATC ACC ACG TC-3 X X 5 -CAG GAG ACC TTC CGT GAA A-3 X X 5 -TCC CTT CTG CCC CAT ATT CC-3 X X 5 -TTC ATC TCC CCC GAA AGA CC-3 X X 5 -GGC TAC AAT TTC AGG TTT GCC AAG TAC TAC-3 X X 5 -AGT CAT CAA CAT TGG TTC TG-3 X X 5 -CAG GAG ACC TTC CGT GAA A-3 X X 5 -CGA CCT GGT ACT TAT CTA CCT C-3 X X 5 -ACG TTC ACA ATG GCA TTC-3 X X 5 -AGG TTG AGG CCA ACG AGA TGG AGC AGG AGC-3 X X 5 -AGG AGC CCC TTA TCA GCT CT-3 X X 5 -CAT TGG TGT ACT TGT CGT CC-3 X X 5 -AAC AGT GCC ATT CTG ACC-3 X X 5 -GCC ACC TCT GTA AAG TTC TC-3 X X 5 -AGC GCC GAA AAC TTC ACC GTA CTC ATC AAG-3
1005–1023 bp 1523–1503 bp 1123–1140 bp 1472–1455 bp 1261–1290 bp 719–735 bp 1342–1323 bp 772–791 bp 1260–1241 bp 971–1000 bp 1192–1211 bp 1733–1715 bp 1246–1267 bp 1515–1498 bp 1341–1370 bp 282–301 bp 974–955 bp 501–518 bp 858–839 bp 671–700 bp
T. H. Wheeler-Schilling et al.r Molecular Brain Research 76 (2000) 415–418
417
Fig. 1. Morphology of a typical, freshly isolated bipolar cell of the rat retina. Scale bar: 10 mm.
rat bipolar cell is shown in Fig. 1. mRNA of the P2X 3 , P2X 4 , P2X 5 , or P2X 7 subunit was found in 33% of the bipolar cells Ž n s 24.. PCR products of all cells were distinctly visible in one band per specific primer with the following base pair Žbp. lengths: 350 bp ŽP2X 3 ., 489 bp ŽP2X 4 ., and 270 bp ŽP2X 5 .. An example of gel electrophoresis is shown in Fig. 2. In contrast, no PCR product of the predicted size of 358 bp was detected for P2X 7 in any bipolar cells examined for the expression of this subunit Ž n s 8.. In the case of P2X 2 , unfortunately, we were not able to perform the RT-PCR for this particular subunit successfully at the single cell level due to unresolved technical problems. It is not trivial to scale RT-PCR experiments down to the single cell level, whilst performing well in whole tissue experiments Žrefer to our previous studies.. ATP is by far the most abundant purine in the retina w10x. Binding studies of a ,b-meATP in rabbit eyecup preparations first indicated the existence of P2X receptors in the vertebrate retina w11x. Using indirect RT-PCR in situ hybridization and immunolabeling, Greenwood et al. w7x
demonstrated expression of the P2X 2 subunit in the somata of photoreceptors, retinal ganglion cells, and neurons of the inner nuclear layer of rat retina. Recently, we demonstrated the presence of the P2X receptor subtypes P2X 2 , P2X 3 , P2X 4 , P2X 5 and P2X 7 mRNAs in the retina. Moreover, P2X 2 and P2X 4 mRNAs are also expressed in the choroid w3,4x. The P2X 7 receptor protein was found in the ganglion cell layer and in some cells of the inner nuclear layer, indicating that P2X receptors might be involved in the modulation of vertical signal transmission in the retina. The results of the present study provide direct molecular evidence for the expression of distinct P2X receptor subunits within acutely isolated bipolar cells. Since it has already been shown that cell culture conditions modify the expression pattern of purinergic receptors w8,9,17x, this study analyzed only freshly isolated bipolar cells using serum-free solutions. Application of RT-PCR at the single-cell level revealed an expression of P2X 3 , P2X 4 , and P2X 5 mRNA in approximately one third of the bipolar cells. In contrast, no
Fig. 2. Detection of P2X 3 , P2X 4 , P2X 5 -mRNA in individual bipolar cells of pigmented rats Ž Brown Norway, P25.. M: 100 bp molecular weight standard; lanes 1, 4, and 6: P2X 3,4,5 in individual bipolar cells; lanes 2, 5 and 8: negative controls; lanes 3, 6, and 9: P2X 3,4,5 positive controls Žspecific clones, .. The 1.5% agarose gel is stained with ethidium bromide. generous gift of Dr. U. Brandle ¨
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specific RT-PCR product for the P2X 7 subunit was found. Together with the distribution of P2X 7 immunoreactivity in the INL w4x, this strongly indicates that amacrine cells andror Muller cells express P2X 7 receptors but not bipo¨ lar or horizontal cells. Moreover, since the P2X 7 subunit is thought to be the only purinoreceptor involved in processes of apoptosis, the lack of P2X 7 mRNA makes it likely that bipolar cell death is not mediated by the activation of purinoceptors w6,14x. Although purinergic signaling is known to take place in the central nervous system ŽCNS., the exact function of most P2X receptors remains to be determined. Among the recently described functions of P2X receptors in the CNS are fast synaptic transmission, modulation of transmitter release, and modulation of neuron excitability Žsee Ref. w13x for a review.. The expression of P2X receptor in bipolar cells of the mammalian retina is the first evidence that purinoreceptors also may be involved in these processes of signal modulation within the vertical retinal pathways w12x. Further functional analysis with immunohistochemical and electrophysiological techniques will be required for a better understanding of the complex physiology of purinoceptors in the retina.
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