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Muscarinic receptors (M2, M3) in rat and guinea pig thoracic dorsal root ganglia
437
Xth International Symposium on Cholinergic Mechanisms
“H
N2
Figure
D state Allosteric transitions on AChR (from J.-P. Changeux) Figure
1.
Allosteric
transkxv
on AChR (from J.-P. Changeux).
with either agonist (carbamylcholine) or antagonists (d-tubocurarine, hexamethonium and a-bungarotoxin). The stoechiometry of photocoupling was measured: 1 mol of [jH]PA is incorporated per mol of cl-bungarotoxin binding site inactivated demonstrating the high efficiency of r3HJPA as photoalkylating agent. The specific incorporation of L3H]PA into the cx and y subunits is saturable for increasing probe concentration. Preparative photolabeling yields about 35 pmol of site-specific alkylated materiel per experiment (- 7% of the total amount of ACh binding site into a-subunit) allowing us to prepare large scale of photolabeled AChR. In a first step, large amounts of [3H]PA alkylated polypeptides will lead us, after proteolytic cleavages, purification of tritiated peptides and microsequencing, to characterize the amino acids belonging to .4Ch binding sites in the D state.
Muscarinic
2. Structure of the photoactivatable
agomst 1‘HIPA.
In a second step, we plan to do time-resolved photolabeling with rapid mixing apparatus combined with laser photoirradiation. These experiments might permit us to label AChR with 13H]PA on its functional activated state A. A comparative study at the molecular level of the dynamic photolabeling experiments and those obtained at equilibrium for the D state may elucidate the topographical changes occurring at the ACh binding sites during agonist activation. References I 2 3
Dennis, M.. Giraudat. I.. Kotzyba-Hibert. F.. Goeldner, M., Hirth, C., Chang, J.-Y., Lazure, C., Chr&ien, M., and Changeux, J.-P (198X) Biochemistry 27, 2337-2345 Galzi, J.-L., Black, D., Goeldner. M., Hinh, C., and Changcux, J.-P (1990) 1. Biol. Chem. 265, 1043&10437 Kotzyba-Hibett, F., Kessler, P., Zerbib, V., Grutter T., Bogen, C., Takeda, K., Hammadi, A.. Knerr, L., and Goeldner, M. (1997) Bmconj. Chem. 8. 472380
receptors (M2, M3) in rat and guinea pig thoracic dorsal root ganglia R. Haberberger,
M. Henrich, W. Kummer
Acetylcholine (ACh) excites mechanoreceptors and produces pain when applied to human skin. In the rat skin. a group of carbachol sensitive C-nociceptors can be excited by muscarine (7). Binding sites for ACb are present in rat dorsal root ganglia (DRG) and accumulate at the proximal site of a sciatic nerve ligature (8). Functional studies of cultured neurons from rat DRG show that stimulation of muscarinic receptors is followed by activation of the nitric oxide (NO)-cGMP signalling system (2), and muscarinic regulation of Ca*+ -currents has also been observed (9). For a better understanding of cholinergic regulation of sensory neuron function, the localization and distribution of muscarinic receptors, in particular its localization to defined subpopulations of sensory neurons, is of interest. Therefore, in the present study, thoracic DRG of rat and guinea pig were examined using histochemical and immunohistochemical methods for the presence of muscarinic M2- and M3-receptors (M2R, M3R) and markers for select neuronal populations. Sensory neurons of DRG can be subdivided in different groups including capsaicin-sensitive, non-peptidergic, small sensory neurons with binding sites for the alpha-D-galactose specific lectin, I-B4 (6), and small-sized primary sensory neurons
containing the peptide, substance P (SP, 5). A group of SP-containing neurons also shows positive NADPH-diaphorase reaction (I) which indicates the presence of nitric oxide synthase (NOS, 3). Thus, I-B4, NOWNADPH-diaphorase reaction, and SP were used as markers for further characterization of M2R- and M3R-immunoreactive (-IR) sensory neurons. These investigations were performed at formaldehyde-fixed tissues processed either as cryostat sections or as free floating sections of polyethylenglycol-entbedded specimens. Both rat and guinea pig DRG contained neurons with immunoreactivity for both subtypes of muscarinic receptors; the labelling densities of individual neurons varied over a wide range. In guinea pig thoracic DRG, M2R- and M3R-IR were present in nerve cell somata, axonal processes and in the endothelium and smooth muscle cells of intraganglionic blood vessels. Intense filamentous M3R-IR was observed in perikarya of medium-sized neurons (25-30 lrn in diameter) which mostly expressed l-B4 binding sites. These cells contained neither NADPH-diaphorase activity nor SP-IR. Intense to slight M3R-IR was present in larger somata (40-X) pm in diameter) which exhibit 18-4 binding sites
ABSTRACTS
438
and, occasionally, SP-IR and/or NADPH-diaphorase activity. M2R-IR was predominantly present in larger neurons, both with and without I-B4 binding sites. A minor population of mediumsized neurons contained also M?R-IR and I-B4 binding sites. None of the M2R-IR neurons expressed SP-IR. In rat thordcic DRG, M2Rand M3R-IR were present in medium-sized and larger neurons, Predominantly, they expressed 1-B-l binding sites. but some perikarya without 18-4 staining were also seen. Medium-sired M2R-IR perikarya often showed SP-IR and positive NADPH-diaphorase reaction whereas medium-sized M2R-IR perikarya and all M3R-IR neurons were virtually devoid of SP-IR and NADPH-diaphorase activity. The present demonstration of M2R-IR on SP-IR neurons IS in accordance with previous pharmacological and electrophysiological experiments describing M2R or M4R dependent inhibition of peptide release from sensory nerve terminals (4, 9). On the other hand, perikarya with intense M3R-IR contained neilher SPIR nor hADPH-diaphorase activity, but expressed l-84 binding sites. Thus. presumably capsaicin-sensitive. non-peptidergic nociceptive neurons bear M3R. Accordingly, the ACh-dependent NO/cGMP signalling pathway can be abolished by capsaicin trcatment (2). This study was supported by SFB 547. project C2
References I)
AImi. Y.. Fujlmura. M., Vincent, S. R.. Kimura. H. Locall/dtmn of NADPH-diaphorase-containing neuron?. in sensory ganglid of the rar. J. Comp Neu~ol.. 306 (1991) 3X2-392.
Neuronal gated ion
nicotinic channels
acetylcholine comprised
receptors (nAChRs) of c( and B subunits.
are ligandPresently six
a (~2.a7) and three p (p2-p4) subunits have been identified and cloned from human brain (Mc Gehee and Role, 1995: Elliott et al, 19Y6; Gotti et al, 1997). Precise subunh compositlon of the various subtypes and their regional distribution in human brain i.\ still unclear. The nAChRs appear to habe a functional role during brain development. since periods of transient high receptor density have been reported in the frontal cortex, hippocampus. cerebellum and brain stem in humans during mid-gestation and neonatal periods (Kinney et al, 1993; Court and Clementi. 1995). The earliest reported binding of [‘HI-nicotine 50 far has been demonstrdtcd in whole brain homogenates from I?-week-old feetuaes (Cairns and Wonnacott, 1988) and choline acetyltransferaceand acetylcholine esterase activity has been detected in human forebrdm from 8-9 gestational weeks (Candy et al, 1985; Perry et al, 1986). Few data are available concerning of nAChRs in hrain during the first trimester. are no reports on their regional distribution and
the ontogcnesis Moreover. there the gene exprea-
sion of the different nAChR subunits. In this study the development of nicolinic acetylcholine receptors (nAChRs) in brains from human fetuses of 4-12 weeks gestational age was investigated. By using RT-PCR technique the expression of a.?. al, a.5, a7, a2, [J3 and 83 mRNA subunits were all detected in the pre-
2)
Beurr, M. B., Murphy, S , Gebhart, G. F. Muscarimc \rlmulation of the nitoc oaide-cyclic GMP signaling cultured rat sensory neurons, Neuro\ci., 62 (19941
chalmzrgic \ystrtn 111 35 l-359.
3)
Da~r<)n, T. D.. Bredt. D. S., Fotuhi. M., Hwang. S. H Nitric oxide synthavs and neur~nill NADPH arc ~denncal in lhrain and peripheral tissues. Proc Sci. LISA KX (IY9I I 77Y7-7XOI.
51.. Snyder diaphoravz N.itl. Ac;ld
JJ
Delaunni\, A.. (;u~tln, P.. Ansay. M. ceptar subtype\ mediating p ulmonary Pulm. Phnrmac4 7 (lY9J) 185~193.
Mulnplc oederna
mu\cJl!nic rc111 tt,e rahhlt.
5)
6)
7J
XI
Warn&y, Ibnergic (11)X1)
J K.. Larhm. rrcept<,r< flow 155-161
0)
Wanhe. E. l31ancl11. 1.. Mante:al/a. M , Guatteo. t... Macmelh, I:, Frrron~, A. Muscarmic regulation of Ca” currem. in rat entry nrurons:
natal spinal cord, cephalon, subcortical
M. A., Kuhsr. M. J. Muscar~mc cho1n the sC,iltlc nerve, Brain Res.. 217
medulla oblongata. lorehrain and
pan\. cerebral
cerebellum, mesencortex during first
trimester development. Relative quantification of mRNA tHellqtriim-LindahI et al., 1997) rhowed that highest levels fol- ~3. a3 and a7 were expressed in spinal cord and w5 was most abundant in cortex. For the a-subunit mRNAs, p2 was high in co1’lex and cerebellum. 83 was highest in cerebellum whereas fi4 seemed to be equally distributed in all regions. A comparison of expression of nAChR subunit tnRNAa in the cortex and ccrehellum of prenatal and aged (54-8 I years) brain showed that mRNA levels for a-l, ~5, a7, p2 and p4 were all significantly higher in prenatal corlex and ccrehellum than in aged brain, whereah the Icvel of a3 transcript ~‘as similar, and a3 significantly higher in aged cortex. Specific binding of [‘HI-epihatidine to branes was detected a.\ early as 4-S weeks brain regions and the number of nAChRs tional age. The highecr specific binding of
prenatal brain memof gestation in many increased with gcsla[‘HI-epibatidlnc and
[‘HI-cytisinc wab detected in spinal cord, pona and medulla oblongara and the IowesI in cortex. Saturation analysis of bindmg data for both prenatal and ogcd brain revefled a single site for [‘HIcytisine and two bindin? siler for [ HI-epibatidine. The Bmax values obtained with j HI-cytisinc and [ HI-epibatidinc were about five and ten time\ to aged frontal cortex.
higher The
for prenatal whole reduced levels of
brain compared nAChR subunit
Xth International Symposium on Cholinergic Mechanisms
“H
N2
Figure
D state Allosteric transitions on AChR (from J.-P. Changeux) Figure
1.
Allosteric
transkxv
on AChR (from J.-P. Changeux).
with either agonist (carbamylcholine) or antagonists (d-tubocurarine, hexamethonium and a-bungarotoxin). The stoechiometry of photocoupling was measured: 1 mol of [jH]PA is incorporated per mol of cl-bungarotoxin binding site inactivated demonstrating the high efficiency of r3HJPA as photoalkylating agent. The specific incorporation of L3H]PA into the cx and y subunits is saturable for increasing probe concentration. Preparative photolabeling yields about 35 pmol of site-specific alkylated materiel per experiment (- 7% of the total amount of ACh binding site into a-subunit) allowing us to prepare large scale of photolabeled AChR. In a first step, large amounts of [3H]PA alkylated polypeptides will lead us, after proteolytic cleavages, purification of tritiated peptides and microsequencing, to characterize the amino acids belonging to .4Ch binding sites in the D state.
Muscarinic
2. Structure of the photoactivatable
agomst 1‘HIPA.
In a second step, we plan to do time-resolved photolabeling with rapid mixing apparatus combined with laser photoirradiation. These experiments might permit us to label AChR with 13H]PA on its functional activated state A. A comparative study at the molecular level of the dynamic photolabeling experiments and those obtained at equilibrium for the D state may elucidate the topographical changes occurring at the ACh binding sites during agonist activation. References I 2 3
Dennis, M.. Giraudat. I.. Kotzyba-Hibert. F.. Goeldner, M., Hirth, C., Chang, J.-Y., Lazure, C., Chr&ien, M., and Changeux, J.-P (198X) Biochemistry 27, 2337-2345 Galzi, J.-L., Black, D., Goeldner. M., Hinh, C., and Changcux, J.-P (1990) 1. Biol. Chem. 265, 1043&10437 Kotzyba-Hibett, F., Kessler, P., Zerbib, V., Grutter T., Bogen, C., Takeda, K., Hammadi, A.. Knerr, L., and Goeldner, M. (1997) Bmconj. Chem. 8. 472380
receptors (M2, M3) in rat and guinea pig thoracic dorsal root ganglia R. Haberberger,
M. Henrich, W. Kummer
Acetylcholine (ACh) excites mechanoreceptors and produces pain when applied to human skin. In the rat skin. a group of carbachol sensitive C-nociceptors can be excited by muscarine (7). Binding sites for ACb are present in rat dorsal root ganglia (DRG) and accumulate at the proximal site of a sciatic nerve ligature (8). Functional studies of cultured neurons from rat DRG show that stimulation of muscarinic receptors is followed by activation of the nitric oxide (NO)-cGMP signalling system (2), and muscarinic regulation of Ca*+ -currents has also been observed (9). For a better understanding of cholinergic regulation of sensory neuron function, the localization and distribution of muscarinic receptors, in particular its localization to defined subpopulations of sensory neurons, is of interest. Therefore, in the present study, thoracic DRG of rat and guinea pig were examined using histochemical and immunohistochemical methods for the presence of muscarinic M2- and M3-receptors (M2R, M3R) and markers for select neuronal populations. Sensory neurons of DRG can be subdivided in different groups including capsaicin-sensitive, non-peptidergic, small sensory neurons with binding sites for the alpha-D-galactose specific lectin, I-B4 (6), and small-sized primary sensory neurons
containing the peptide, substance P (SP, 5). A group of SP-containing neurons also shows positive NADPH-diaphorase reaction (I) which indicates the presence of nitric oxide synthase (NOS, 3). Thus, I-B4, NOWNADPH-diaphorase reaction, and SP were used as markers for further characterization of M2R- and M3R-immunoreactive (-IR) sensory neurons. These investigations were performed at formaldehyde-fixed tissues processed either as cryostat sections or as free floating sections of polyethylenglycol-entbedded specimens. Both rat and guinea pig DRG contained neurons with immunoreactivity for both subtypes of muscarinic receptors; the labelling densities of individual neurons varied over a wide range. In guinea pig thoracic DRG, M2R- and M3R-IR were present in nerve cell somata, axonal processes and in the endothelium and smooth muscle cells of intraganglionic blood vessels. Intense filamentous M3R-IR was observed in perikarya of medium-sized neurons (25-30 lrn in diameter) which mostly expressed l-B4 binding sites. These cells contained neither NADPH-diaphorase activity nor SP-IR. Intense to slight M3R-IR was present in larger somata (40-X) pm in diameter) which exhibit 18-4 binding sites
ABSTRACTS
438
and, occasionally, SP-IR and/or NADPH-diaphorase activity. M2R-IR was predominantly present in larger neurons, both with and without I-B4 binding sites. A minor population of mediumsized neurons contained also M?R-IR and I-B4 binding sites. None of the M2R-IR neurons expressed SP-IR. In rat thordcic DRG, M2Rand M3R-IR were present in medium-sized and larger neurons, Predominantly, they expressed 1-B-l binding sites. but some perikarya without 18-4 staining were also seen. Medium-sired M2R-IR perikarya often showed SP-IR and positive NADPH-diaphorase reaction whereas medium-sized M2R-IR perikarya and all M3R-IR neurons were virtually devoid of SP-IR and NADPH-diaphorase activity. The present demonstration of M2R-IR on SP-IR neurons IS in accordance with previous pharmacological and electrophysiological experiments describing M2R or M4R dependent inhibition of peptide release from sensory nerve terminals (4, 9). On the other hand, perikarya with intense M3R-IR contained neilher SPIR nor hADPH-diaphorase activity, but expressed l-84 binding sites. Thus. presumably capsaicin-sensitive. non-peptidergic nociceptive neurons bear M3R. Accordingly, the ACh-dependent NO/cGMP signalling pathway can be abolished by capsaicin trcatment (2). This study was supported by SFB 547. project C2
References I)
AImi. Y.. Fujlmura. M., Vincent, S. R.. Kimura. H. Locall/dtmn of NADPH-diaphorase-containing neuron?. in sensory ganglid of the rar. J. Comp Neu~ol.. 306 (1991) 3X2-392.
Neuronal gated ion
nicotinic channels
acetylcholine comprised
receptors (nAChRs) of c( and B subunits.
are ligandPresently six
a (~2.a7) and three p (p2-p4) subunits have been identified and cloned from human brain (Mc Gehee and Role, 1995: Elliott et al, 19Y6; Gotti et al, 1997). Precise subunh compositlon of the various subtypes and their regional distribution in human brain i.\ still unclear. The nAChRs appear to habe a functional role during brain development. since periods of transient high receptor density have been reported in the frontal cortex, hippocampus. cerebellum and brain stem in humans during mid-gestation and neonatal periods (Kinney et al, 1993; Court and Clementi. 1995). The earliest reported binding of [‘HI-nicotine 50 far has been demonstrdtcd in whole brain homogenates from I?-week-old feetuaes (Cairns and Wonnacott, 1988) and choline acetyltransferaceand acetylcholine esterase activity has been detected in human forebrdm from 8-9 gestational weeks (Candy et al, 1985; Perry et al, 1986). Few data are available concerning of nAChRs in hrain during the first trimester. are no reports on their regional distribution and
the ontogcnesis Moreover. there the gene exprea-
sion of the different nAChR subunits. In this study the development of nicolinic acetylcholine receptors (nAChRs) in brains from human fetuses of 4-12 weeks gestational age was investigated. By using RT-PCR technique the expression of a.?. al, a.5, a7, a2, [J3 and 83 mRNA subunits were all detected in the pre-
2)
Beurr, M. B., Murphy, S , Gebhart, G. F. Muscarimc \rlmulation of the nitoc oaide-cyclic GMP signaling cultured rat sensory neurons, Neuro\ci., 62 (19941
chalmzrgic \ystrtn 111 35 l-359.
3)
Da~r<)n, T. D.. Bredt. D. S., Fotuhi. M., Hwang. S. H Nitric oxide synthavs and neur~nill NADPH arc ~denncal in lhrain and peripheral tissues. Proc Sci. LISA KX (IY9I I 77Y7-7XOI.
51.. Snyder diaphoravz N.itl. Ac;ld
JJ
Delaunni\, A.. (;u~tln, P.. Ansay. M. ceptar subtype\ mediating p ulmonary Pulm. Phnrmac4 7 (lY9J) 185~193.
Mulnplc oederna
mu\cJl!nic rc111 tt,e rahhlt.
5)
6)
7J
XI
Warn&y, Ibnergic (11)X1)
J K.. Larhm. rrcept<,r< flow 155-161
0)
Wanhe. E. l31ancl11. 1.. Mante:al/a. M , Guatteo. t... Macmelh, I:, Frrron~, A. Muscarmic regulation of Ca” currem. in rat entry nrurons:
natal spinal cord, cephalon, subcortical
M. A., Kuhsr. M. J. Muscar~mc cho1n the sC,iltlc nerve, Brain Res.. 217
medulla oblongata. lorehrain and
pan\. cerebral
cerebellum, mesencortex during first
trimester development. Relative quantification of mRNA tHellqtriim-LindahI et al., 1997) rhowed that highest levels fol- ~3. a3 and a7 were expressed in spinal cord and w5 was most abundant in cortex. For the a-subunit mRNAs, p2 was high in co1’lex and cerebellum. 83 was highest in cerebellum whereas fi4 seemed to be equally distributed in all regions. A comparison of expression of nAChR subunit tnRNAa in the cortex and ccrehellum of prenatal and aged (54-8 I years) brain showed that mRNA levels for a-l, ~5, a7, p2 and p4 were all significantly higher in prenatal corlex and ccrehellum than in aged brain, whereah the Icvel of a3 transcript ~‘as similar, and a3 significantly higher in aged cortex. Specific binding of [‘HI-epihatidine to branes was detected a.\ early as 4-S weeks brain regions and the number of nAChRs tional age. The highecr specific binding of
prenatal brain memof gestation in many increased with gcsla[‘HI-epibatidlnc and
[‘HI-cytisinc wab detected in spinal cord, pona and medulla oblongara and the IowesI in cortex. Saturation analysis of bindmg data for both prenatal and ogcd brain revefled a single site for [‘HIcytisine and two bindin? siler for [ HI-epibatidine. The Bmax values obtained with j HI-cytisinc and [ HI-epibatidinc were about five and ten time\ to aged frontal cortex.
higher The
for prenatal whole reduced levels of
brain compared nAChR subunit