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Immunohistochemical demonstration of neuron specific antigen, HPC-1 in the enteric nervous system of the guinea-pig distal colon
Neuroscience Letters, 160 (1993) 37-40 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights i'e~e~'~l 0 3 0 4 - 3 9 ~ $ ~ . ~ - .
37 :~..~-~.-,--
NSL 09799
Immunohistochemical demonstration of neuron specific antigen, HPC-1 in the enteric nervous system of the guinea-pig distal colon M a s a t o N a g a h a m a a, Yoshihiro Tsutsui a, Atsukazu K u w a h a r a b and Kimio A k a g a w a b alnstitute for Developmental Research, Kasugai, Aichi (Japan} and bNational Institute for Physiological Sciences, Okazaki, Aichi (Japan} (Received 1 April 1993; Revised version received 4 June 1993; Accepted 4 June 1993)
Key words: Enteric nervous system; HPC-1 antigen; Antiserum; Immunohistochemistry; Guinea-pig distal colon The HPC-1 antigen is a newly identified neuron specific membrane protein in the central nervous system. The HPC-1 antigen was revealed similarity to epimorphin. The presence of HPC-1 antigen in the enteric nervous system of guinea-pig distal colon was immunohistochemically demonstrated using the antibody against the HPC-1. Immunohistochemical study clearly revealed the topography and structure of the enteric nervous system of the guinea-pig distal colon. HPC-1 was present only in the nervous system and entirely distributed. HPC-1 antigen is present at the surfaces of ganglion cells, but not in the cytoplasm.
The HPC-1 antigen is a newly identified neuron specific membrane protein but the function of this antigen is not understood yet. The HPC-1 antigen is 35-kDa protein [9]. Immunohistological study showed that HPC-1 antigen was widely distributed in rat central nervous system [3, 4, 11]. The sequence analysis was suggested that the HPC-1 antigen was an integrated membrane protein [10]. It was reported that the HPC-1 antigen could bind both to a synaptic vesicle protein and to N-type Ca channel [5, 9, 15]. The HPC-1 antigen reveals strong homology to epimorphin that is mesenchymal factor related to the morphogenesis of primitive epidermal tissues in embryonic stage [7, 9]. In rat retinal cultures, the HPC-1 antigen was identified in neuronal cells not in glial cells [1-3]. The presence of HPC-I mRNA in neuronal cells not in glial cells has also been demonstrated in rat central nervous system by the in situ hybridization study [11]. Although HPC-1 antigen has been noted in the entire central nervous system, its distribution and identification in the enteric nervous system have not been studied. To determine this issue, we have attempted to demonstrate the presence of HPC-1 antigen in the enteric nervous system in the guinea-pig distal colon. The guinea-pig distal colon was prepared for whole mount sampling using a method modified from previous studies [6, 12, 13]. In brief, five male guinea-pigs (200300 g) were used in this study. Under Nembutal anestheCorrespondence: M. Nagahama, Institute for Developmental Research, 713-8 Kamiya-cho, Kasugai, Aichi 4804)3, Japan.
sia, animals were perfused through the aorta with a fixative containing 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) at room temperature. After 20 min of perfusion, portions of the distal colon were dissected and immersed into the same fixative ~ 1 h. Next, tissues were rinsed in 0.3% Triton × 100 in PBS (0.01 M phosphate buffer (pH 7.3) containing 0.88% NaC1) at 4°C for 24 h. Portions of the distal colon were split open along the mesenteric side and then separated into two layers (mucosal + submucosal layers, circular muscle layer and longitudinal layer) with fine forceps under a dissecting microscope. The specimens were immersed in 0.6% H 2 0 2 diluted with methanol at 4°C for l0 min to block endogenous peroxidase and rinsed in PBS. The whole mount samples were incubated with rabbit antibody against HPC-1 (1:2000 dilution) in PBS with 1% normal goat serum at 4°C for 3 days. The HPC-1 antibody was already characterized in previous paper [10]. After washing in PBS, they were stained by the ABC method [8]. These samples were examined under light microscope. For control experiment, we used PBS solution instead of the HPC-1 antibody in same Immunohistochemical procedure. The immunoreactivity of HPC-1 was confined to the nervous system throughout the submucosal and muscle layers of guinea-pig distal colon. The HPC-1 immunoreactive, myenteric neuronal network consisted of the ganglion strands (containing numerous ganglion cells and nerve fibers) with internodal strands (nerve bundles interconnecting the each ganglion strand) (Fig. 1). The sur-
38
Fig. 1. a: Low-magnification view of HPC-1 positive myenteric plexus. Micrograph shows HPC-t immunoreactive ganglionic network consisting of ganglia (G) with internodal strands (arrow). Scale bar, 100/~m. b: High-magnification view of ganglia. Surface of ganglion cells are HPC-1 positive but cytoplasm of ganglion cells are negative. Scale bar, 100/tm. Fig. 2. a: Low-magnification view of HPC-I immunoreactivty in submucosal plexus in guinea-pig colon. Micrograph shows HPC-1 immunoreactive meshwork consisting of ganglia (G) with internordal strands (arrow). Cytoplasm of ganglion cells are HPC-1 negative. Scale bar, 100/lm. b: High-magnification view of ganglia. Surface of ganglion cells are HPC-I positive but cytoplasm of ganglion cells are negative. Scale bar, 100/lm.
39 faces of many ganglion cells were prominently stained, but not the cytoplasm (Fig. lb). The HPC-1 immunoreactive nerve fibers surrounding ganglion cells were numerous in ganglia. The internodal connecting strands contained many HPC-1 immunoreactive nerve fibers. The submucosal plexus in the connective tissue of submucosa was intensely immunoreactive to HPC-1 (Fig. 2). The HPC-1 immunoreactive products displayed the meshwork of submucosal plexus consisting of ganglia with internodal strands (Fig. 2a). The cytoplasm of ganglion cells was not stained like as myenteric ganglion cells (Fig. 2b). The many nerve fibers in internodal connecting strand in the submucosal plexus were stained densely by the HPC-1 antibody (Fig. 2b). Many of the positive fibers were traced to the submucosal plexus or the myenteric plexus. The HPC-1 immunoreactive nerve fibers originated from the myenteric plexus penetrated into the circular muscle layer. In the circular muscle layer, the many HPC-1 immunoreactive nerve fibers were parallel to the muscle bundles forming an elaborate network (Fig. 3a). The varicosities were densely stained by HPC-1 antibody (Fig. 3b). The regions between varicosities were also immunoreactive for HPC-1 antibody (Fig. 3b). The HPC-1 immunoreactive nerve fibers also penetrated into longitudinal muscle layer. The present observations show that immunoreactivity for HPC-1 is observed only in the nervous system throughout the colon. The presence of HPC-I immunoreactivity is demonstrated in the almost, if not all, of components of enteric nervous system. The presence of HPC-1 immunoreactivity is confined to the surface of the ganglion cells. Though, it is hard to tell the detailed localization of HPC- 1 immunoreactivity from our observation, there is no positive staining in cytoplasm. These observations are very similar to the observations of a GTP-binding protein (Go) which is another neuron specific membrane-bound protein and supposed to interact with various neurotransmitter receptors [12, 14]. Thus, the present results may be possible to support that HPC1 antigen is a membrane-bound protein in enteric nervous system similar to that of central nervous system [10]. Enteric nervous system consists of neurons and glial cells like central nervous system. Since enteric ganglia, different from the other peripheral ganglia, possess many properties that are characteristics of the central nervous system and HPC-1 m R N A only express in neuronal cells not in glial cells in the central nervous system [11, 16]. Thus, it is unlikely that HPC-1 antibody detected the membrane protein of enteric glial cells in the present study. The HPC-1 immunoreactivity is very dense at varicosities. This seems consistent with that the HPC-1 antigen is identical to p35A (Syntaxin) that is capable of binding to a synaptic vesicle protein [5]. The HPC-1 im-
Fig. 3. a: Low-magnificationviewof HPC-1 immunoreactivyin circular muscle layer in guinea-pig colon. Micrograph shows HPC-1 immunoreactivenerve bundles running parallel to muscle bundles. Scale bar, 100/lm.b: High-magnificationview.Note that many immunoreactive varicosities(arrow head) were observed in distal end of fibers and that regions between varicosities (between arrow heads) also immunoreactive. Scalebar, 100/lm.
40 m u n o r e a c t i v i t y is entirely distributed t h r o u g h o u t enteric n e r v o u s system. This result strongly suggests a possibility that HPC-1 m a y be involved in n o t only synaptic m e c h a n i s m s b u t also other f u n c t i o n s of the n e u r o n s . In this context, the similarity between HPC-1 a n d epimorphin, which was a n essential factor for epithelial morphogenesis, implied a possible f u n c t i o n o f HPC-1 in neur o n a l differentiation. However, further studies will be necessary to assess these possibilities. I n conclusion, the discovery o f HPC-1 i m m u n o r e a c tivity in the enteric n e u r o n s m a y provide a n i m p o r t a n t key for the analysis o f n e u r o n a l cell f u n c t i o n s o f the enteric n e r v o u s system. This work was s u p p o r t e d by a g r a n t - i n - a i d from the M i n i s t r y o f E d u c a t i o n , Science a n d C u l t u r e of J a p a n (to A. K u w a h a r a ) a n d grants from N i s s a n Science F o u n d a tion a n d N a i t o F o u n d a t i o n (to K. Akagawa).
1 Akagawa, K. and Barnstable, C.J., Identification and characterization of cell types in monolayer cultures of rat retina using monoclonal antibodies, Brain Res., 383 (1986) 110-120. 2 Akagawa, K. and Barnstable, C.J., Identification and characterization of cell types accumulating GABA in rat retinal cultures using cell type specific monoclonal antibodies, Brain Res., 408 (1987) 154-162. 3 Akagawa, K., Takada, M., Hayashi, H. and Uemura, K., Calciumand voltage-dependent potassium channel in the rat retinal amacrine cells identified in vitro using a cell type-specific monoclonal antibody, Brain Res., 518 (1990) 1 5. 4 Barnstable, C.J., Akagawa, K., Hofstein, R. and Horn, J.P., Monoclonal antibodies that label discrete cell types in the mammalian nervous system, Cold Spring Harbor Symp. Quant. Biol., 48 (1983) 863 876.
5 Bennet, M.K., Calakos, N. and Scheller, R.H., Syntaxin: A synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones, Science,257 (1992) 255-259. 6 Costa, M., Buffa, R. and Furness, J.B., Immunohistochemical localization of polypeptide in peripheral autonomic nerves using whole mount preparations, Histochemistry, 65 (1980) 157--165. 7 Hirai, Y., Takebe, K., Takashima, M., Kobayashi, S. and Takeichi, M., Epimorphin: a mesenchymal protein essential for epithelial morphogenesis. Cell, 69 (1992) 4714 8 I. 8 Hsu, S.-M., Raine, L. and Fanger, H., Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577-580. 9 Inoue, A. and Akagawa, K., Neuron-specific antigen HPC-1 from bovine brain reveals strong homology to epimorphin, an essentioal factor involved in epithelial morphogenesis: Identification of a novel protein family, Biochem. Biophys. Res. Commun., 187 (1992) 1144-1150.
10 Inoue, A., Obata, K. and Akagawa, K., Cloning and sequence analysis ofcDNA for a neuronal cell membrane antigen, HPC-1, J. Biol. Chem., 267 (1992) 10613-10619. 11 Inoue, A. and Akagawa, K., Neuron specific expression of a membrane protein, HPC-1: tissue distribution, and cellular and subcellular localization of immunoreacfivity and mRNA, Mol. Brain Res., 19 (1993) 121-128. 12 Nagahama, M., Kato, K., Tsutsui, Y. and Asano, T., Localization of GTP-binding protein Go in the enteric nervous system in rat ileum, Biol. Signals, 1 (1992) 266-274. 13 Nagahama, M., Ozaki, T. and Hama, K., A study of the myenteric plexus of the congenital aganglionosis rat (spotting lethal), Anat. Embryol. 171 (1985) 285-296. 14 Neer, E.J. and Clapham D.E., Roles of G protein subunits in transmembrane signalling, Nature (London), 333 (1988) 129-134. 15 Yoshida, A., Oho, C., Omori, A., Kuwahara, R., Ito, T. and Takahashi, M. HPC-1 is associated with synaptotagmin and w-conotoxin receptor, J. Biol. Chem. 267(35) (1992) 24925-24928. 16 Wood, J.D., Physiology of enteric nervous system, In L.R. Johnson (Ed.), Physiology of the Gastrointestinal Tract, 2nd Ed., Raven, New York, pp. 67 109.
37 :~..~-~.-,--
NSL 09799
Immunohistochemical demonstration of neuron specific antigen, HPC-1 in the enteric nervous system of the guinea-pig distal colon M a s a t o N a g a h a m a a, Yoshihiro Tsutsui a, Atsukazu K u w a h a r a b and Kimio A k a g a w a b alnstitute for Developmental Research, Kasugai, Aichi (Japan} and bNational Institute for Physiological Sciences, Okazaki, Aichi (Japan} (Received 1 April 1993; Revised version received 4 June 1993; Accepted 4 June 1993)
Key words: Enteric nervous system; HPC-1 antigen; Antiserum; Immunohistochemistry; Guinea-pig distal colon The HPC-1 antigen is a newly identified neuron specific membrane protein in the central nervous system. The HPC-1 antigen was revealed similarity to epimorphin. The presence of HPC-1 antigen in the enteric nervous system of guinea-pig distal colon was immunohistochemically demonstrated using the antibody against the HPC-1. Immunohistochemical study clearly revealed the topography and structure of the enteric nervous system of the guinea-pig distal colon. HPC-1 was present only in the nervous system and entirely distributed. HPC-1 antigen is present at the surfaces of ganglion cells, but not in the cytoplasm.
The HPC-1 antigen is a newly identified neuron specific membrane protein but the function of this antigen is not understood yet. The HPC-1 antigen is 35-kDa protein [9]. Immunohistological study showed that HPC-1 antigen was widely distributed in rat central nervous system [3, 4, 11]. The sequence analysis was suggested that the HPC-1 antigen was an integrated membrane protein [10]. It was reported that the HPC-1 antigen could bind both to a synaptic vesicle protein and to N-type Ca channel [5, 9, 15]. The HPC-1 antigen reveals strong homology to epimorphin that is mesenchymal factor related to the morphogenesis of primitive epidermal tissues in embryonic stage [7, 9]. In rat retinal cultures, the HPC-1 antigen was identified in neuronal cells not in glial cells [1-3]. The presence of HPC-I mRNA in neuronal cells not in glial cells has also been demonstrated in rat central nervous system by the in situ hybridization study [11]. Although HPC-1 antigen has been noted in the entire central nervous system, its distribution and identification in the enteric nervous system have not been studied. To determine this issue, we have attempted to demonstrate the presence of HPC-1 antigen in the enteric nervous system in the guinea-pig distal colon. The guinea-pig distal colon was prepared for whole mount sampling using a method modified from previous studies [6, 12, 13]. In brief, five male guinea-pigs (200300 g) were used in this study. Under Nembutal anestheCorrespondence: M. Nagahama, Institute for Developmental Research, 713-8 Kamiya-cho, Kasugai, Aichi 4804)3, Japan.
sia, animals were perfused through the aorta with a fixative containing 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) at room temperature. After 20 min of perfusion, portions of the distal colon were dissected and immersed into the same fixative ~ 1 h. Next, tissues were rinsed in 0.3% Triton × 100 in PBS (0.01 M phosphate buffer (pH 7.3) containing 0.88% NaC1) at 4°C for 24 h. Portions of the distal colon were split open along the mesenteric side and then separated into two layers (mucosal + submucosal layers, circular muscle layer and longitudinal layer) with fine forceps under a dissecting microscope. The specimens were immersed in 0.6% H 2 0 2 diluted with methanol at 4°C for l0 min to block endogenous peroxidase and rinsed in PBS. The whole mount samples were incubated with rabbit antibody against HPC-1 (1:2000 dilution) in PBS with 1% normal goat serum at 4°C for 3 days. The HPC-1 antibody was already characterized in previous paper [10]. After washing in PBS, they were stained by the ABC method [8]. These samples were examined under light microscope. For control experiment, we used PBS solution instead of the HPC-1 antibody in same Immunohistochemical procedure. The immunoreactivity of HPC-1 was confined to the nervous system throughout the submucosal and muscle layers of guinea-pig distal colon. The HPC-1 immunoreactive, myenteric neuronal network consisted of the ganglion strands (containing numerous ganglion cells and nerve fibers) with internodal strands (nerve bundles interconnecting the each ganglion strand) (Fig. 1). The sur-
38
Fig. 1. a: Low-magnification view of HPC-1 positive myenteric plexus. Micrograph shows HPC-t immunoreactive ganglionic network consisting of ganglia (G) with internodal strands (arrow). Scale bar, 100/~m. b: High-magnification view of ganglia. Surface of ganglion cells are HPC-1 positive but cytoplasm of ganglion cells are negative. Scale bar, 100/tm. Fig. 2. a: Low-magnification view of HPC-I immunoreactivty in submucosal plexus in guinea-pig colon. Micrograph shows HPC-1 immunoreactive meshwork consisting of ganglia (G) with internordal strands (arrow). Cytoplasm of ganglion cells are HPC-1 negative. Scale bar, 100/lm. b: High-magnification view of ganglia. Surface of ganglion cells are HPC-I positive but cytoplasm of ganglion cells are negative. Scale bar, 100/lm.
39 faces of many ganglion cells were prominently stained, but not the cytoplasm (Fig. lb). The HPC-1 immunoreactive nerve fibers surrounding ganglion cells were numerous in ganglia. The internodal connecting strands contained many HPC-1 immunoreactive nerve fibers. The submucosal plexus in the connective tissue of submucosa was intensely immunoreactive to HPC-1 (Fig. 2). The HPC-1 immunoreactive products displayed the meshwork of submucosal plexus consisting of ganglia with internodal strands (Fig. 2a). The cytoplasm of ganglion cells was not stained like as myenteric ganglion cells (Fig. 2b). The many nerve fibers in internodal connecting strand in the submucosal plexus were stained densely by the HPC-1 antibody (Fig. 2b). Many of the positive fibers were traced to the submucosal plexus or the myenteric plexus. The HPC-1 immunoreactive nerve fibers originated from the myenteric plexus penetrated into the circular muscle layer. In the circular muscle layer, the many HPC-1 immunoreactive nerve fibers were parallel to the muscle bundles forming an elaborate network (Fig. 3a). The varicosities were densely stained by HPC-1 antibody (Fig. 3b). The regions between varicosities were also immunoreactive for HPC-1 antibody (Fig. 3b). The HPC-1 immunoreactive nerve fibers also penetrated into longitudinal muscle layer. The present observations show that immunoreactivity for HPC-1 is observed only in the nervous system throughout the colon. The presence of HPC-I immunoreactivity is demonstrated in the almost, if not all, of components of enteric nervous system. The presence of HPC-1 immunoreactivity is confined to the surface of the ganglion cells. Though, it is hard to tell the detailed localization of HPC- 1 immunoreactivity from our observation, there is no positive staining in cytoplasm. These observations are very similar to the observations of a GTP-binding protein (Go) which is another neuron specific membrane-bound protein and supposed to interact with various neurotransmitter receptors [12, 14]. Thus, the present results may be possible to support that HPC1 antigen is a membrane-bound protein in enteric nervous system similar to that of central nervous system [10]. Enteric nervous system consists of neurons and glial cells like central nervous system. Since enteric ganglia, different from the other peripheral ganglia, possess many properties that are characteristics of the central nervous system and HPC-1 m R N A only express in neuronal cells not in glial cells in the central nervous system [11, 16]. Thus, it is unlikely that HPC-1 antibody detected the membrane protein of enteric glial cells in the present study. The HPC-1 immunoreactivity is very dense at varicosities. This seems consistent with that the HPC-1 antigen is identical to p35A (Syntaxin) that is capable of binding to a synaptic vesicle protein [5]. The HPC-1 im-
Fig. 3. a: Low-magnificationviewof HPC-1 immunoreactivyin circular muscle layer in guinea-pig colon. Micrograph shows HPC-1 immunoreactivenerve bundles running parallel to muscle bundles. Scale bar, 100/lm.b: High-magnificationview.Note that many immunoreactive varicosities(arrow head) were observed in distal end of fibers and that regions between varicosities (between arrow heads) also immunoreactive. Scalebar, 100/lm.
40 m u n o r e a c t i v i t y is entirely distributed t h r o u g h o u t enteric n e r v o u s system. This result strongly suggests a possibility that HPC-1 m a y be involved in n o t only synaptic m e c h a n i s m s b u t also other f u n c t i o n s of the n e u r o n s . In this context, the similarity between HPC-1 a n d epimorphin, which was a n essential factor for epithelial morphogenesis, implied a possible f u n c t i o n o f HPC-1 in neur o n a l differentiation. However, further studies will be necessary to assess these possibilities. I n conclusion, the discovery o f HPC-1 i m m u n o r e a c tivity in the enteric n e u r o n s m a y provide a n i m p o r t a n t key for the analysis o f n e u r o n a l cell f u n c t i o n s o f the enteric n e r v o u s system. This work was s u p p o r t e d by a g r a n t - i n - a i d from the M i n i s t r y o f E d u c a t i o n , Science a n d C u l t u r e of J a p a n (to A. K u w a h a r a ) a n d grants from N i s s a n Science F o u n d a tion a n d N a i t o F o u n d a t i o n (to K. Akagawa).
1 Akagawa, K. and Barnstable, C.J., Identification and characterization of cell types in monolayer cultures of rat retina using monoclonal antibodies, Brain Res., 383 (1986) 110-120. 2 Akagawa, K. and Barnstable, C.J., Identification and characterization of cell types accumulating GABA in rat retinal cultures using cell type specific monoclonal antibodies, Brain Res., 408 (1987) 154-162. 3 Akagawa, K., Takada, M., Hayashi, H. and Uemura, K., Calciumand voltage-dependent potassium channel in the rat retinal amacrine cells identified in vitro using a cell type-specific monoclonal antibody, Brain Res., 518 (1990) 1 5. 4 Barnstable, C.J., Akagawa, K., Hofstein, R. and Horn, J.P., Monoclonal antibodies that label discrete cell types in the mammalian nervous system, Cold Spring Harbor Symp. Quant. Biol., 48 (1983) 863 876.
5 Bennet, M.K., Calakos, N. and Scheller, R.H., Syntaxin: A synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones, Science,257 (1992) 255-259. 6 Costa, M., Buffa, R. and Furness, J.B., Immunohistochemical localization of polypeptide in peripheral autonomic nerves using whole mount preparations, Histochemistry, 65 (1980) 157--165. 7 Hirai, Y., Takebe, K., Takashima, M., Kobayashi, S. and Takeichi, M., Epimorphin: a mesenchymal protein essential for epithelial morphogenesis. Cell, 69 (1992) 4714 8 I. 8 Hsu, S.-M., Raine, L. and Fanger, H., Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577-580. 9 Inoue, A. and Akagawa, K., Neuron-specific antigen HPC-1 from bovine brain reveals strong homology to epimorphin, an essentioal factor involved in epithelial morphogenesis: Identification of a novel protein family, Biochem. Biophys. Res. Commun., 187 (1992) 1144-1150.
10 Inoue, A., Obata, K. and Akagawa, K., Cloning and sequence analysis ofcDNA for a neuronal cell membrane antigen, HPC-1, J. Biol. Chem., 267 (1992) 10613-10619. 11 Inoue, A. and Akagawa, K., Neuron specific expression of a membrane protein, HPC-1: tissue distribution, and cellular and subcellular localization of immunoreacfivity and mRNA, Mol. Brain Res., 19 (1993) 121-128. 12 Nagahama, M., Kato, K., Tsutsui, Y. and Asano, T., Localization of GTP-binding protein Go in the enteric nervous system in rat ileum, Biol. Signals, 1 (1992) 266-274. 13 Nagahama, M., Ozaki, T. and Hama, K., A study of the myenteric plexus of the congenital aganglionosis rat (spotting lethal), Anat. Embryol. 171 (1985) 285-296. 14 Neer, E.J. and Clapham D.E., Roles of G protein subunits in transmembrane signalling, Nature (London), 333 (1988) 129-134. 15 Yoshida, A., Oho, C., Omori, A., Kuwahara, R., Ito, T. and Takahashi, M. HPC-1 is associated with synaptotagmin and w-conotoxin receptor, J. Biol. Chem. 267(35) (1992) 24925-24928. 16 Wood, J.D., Physiology of enteric nervous system, In L.R. Johnson (Ed.), Physiology of the Gastrointestinal Tract, 2nd Ed., Raven, New York, pp. 67 109.