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Vesicular monoamine transporter expression in enteric neurons and ECL cells
A960
AGA ABSTRACTS
• AVIAN MOTILIN: ISOLATION, SEQUENCE, BIOLOGICAL ACTIVITY AND PHYSIOLOGICAL R O L E . P.De ClercQ, I.Depoortere, " P.Vergara and T.L.Peeters. Gut Hormone Lab, Gasthuisberg ON, Leuven, Belgium and " Unitat de Fisiologia Animal de Veterinaria, Un. Autonoma de Barcelona, Spain. The biological role of motilin has been mostly studied in rabbit, dog and in man. We here report the first study of a nonmammalian motilin. Avian motilin was isolated from acid extracts of the small intestinal mucosa of the chicken by a combination of gel and ion-exchange chromatography followed by reverse phase HPLC. The purification was monitored by radio-receptor assay. Sequencing showed that, compared to porcine motilin, residues 4,7-10 and 12 were replaced b y Phe, Gin, Ser, Asp, lie and Lys respectively. The biological activity of synthetic avian motilin and of the (1-14) fragment of avian motilin was evaluated in the tissue bath and in chickens chronically implanted with electrodes in stomach, duodenum, jejunum and ileum. In the tissue bath, under isotonic conditions, segments of the chicken small intestine showed a tonic contractile response towards avian motilin, which was 88 + 1 % of the response towards ACh in the jejunum, compared to 82 + 1% in the duodenum and 80 + 2% in the ileum. Avian motilin had a higher potency than porcine motilin. The pECso's (negative logarithm of the dose producing 50% of the maximum response) were 7.41 and 6.48 respectively. The potency of the (1-14) fragment was about tenfold reduced (6.61). The response was unaffected by TTX, atropine, L-NNA, guanethidine, prasozine, or yohimbine but was blocked by verapamil. 0 H M - 1 1 5 2 6 a specific motilin antagonist i n the rabbit, was an agonist equipotent to porcine motilin in the chicken (pECso: 6.56). In vitro avian motilin also affected segments from the rabbit small intestine, but was less potent than porcine motilin in this preparation (pECso'S of 7.69 and 8.25 respectively). In vivo the IV injection of avian motilin (1 gg/kg), induced within 30 seconds the appearance of a motor pattern recently described by one of us (PV), the rhythmic oscillatory pattern (ROC), but did not induce an MMC. No effect was seen after tenfold higher doses of mammalian motilins. Our studies suggest that the structure of motilin, the structure of the motitin receptor and the role of motilin have been changed during phylogenesis.
• EXPRESSION OF SSTR2 SOMATOSTATIN RECEPTOR SUBTYPE INHIBTED PANCREATIC TUMOR CELL GR,OWTH N Delesoue I Rauly, L Buscail, J.P. Est~ve, G.I. Bell , A. V. Schally~, N . Vaysse, C. Susini. INSERM U 151, CHU Rangueil, Toulouse, France; * Howard Hugues Medical Institute, University of Chicago, 1L; § Tulane University, Medical School, New Orleans, L. Somatostatin is a negative growth factor for normal and tumoral cells of GI tract that exerts its effect by interacting with specific receptors: We recently demonstrated that SSTR2 somatostatin receptor subtype mediates the antiproliferative effect of somatostatin analogues (Buscail et al Proc. Natl. Acad. S c i USA, 1994). SSTR2 mRNA is expressed in human pancreas whereas it is undetectable in almost human pancreatic cell lines including CAPAN-1 and BxPC-3. In this study, we investigated the effect of SSTR2 express!on in human" pancreatic tumor cells CAPAN 1, on cell proliferation. Human SSTR2 cDNA was stably transfected into CAPAN-1 cells using lipofectin. In cells expressing SSTR2, the somatostatin analogue RC-160 binds to SSTR2 with high affinity (Kd 0.3 nM) and induced an inhibition of serum-stimulated cell proliferation (ECs0 21 pM) reaching a maximum (-30 + 1 % ) at 1 nM RC-160. Furthermore, the proliferation of cells expressing SSTR2 was decreased compared to that of control cells, expressing the vector alone (doubling :time 37 hr vs 31 hr). The observed inhibition of cell growth induced by expression o f SSTR2 is not due to the clonal line since it is also observed in human pancreatic cancer BxPC-3 cells, stably expressing SSTR2 (doubling time 51 hr vs 36 hr). CAPAN-1 cells are capable of forming colonies in soft agar, consistent with their transformation potential. Expression of SSTR2 affected the growth rate of CAPAN-1 cells in agar as measured by the 2-fold decrease in diameter of colonies formed by cells expressing SSTR2 compared to that of control cells. Cells were analyzed for somatostatin mRNA expression using Reverse transcriptase-Polymerase chain reaction. Somatostatin mRNA was detected in SSTR2-transfected CAPAN-1 and BxPC-3 cells suggesting the existence of an autocrine somatostatin-SSTR2 loop in these cells. In conclusion, expression of SSTR2 in human pancreatic cancer cells results in decrease of cell growth probably as a consequence of an autocrine receptor activation. Therefore, SSTR2 may be of major importance as a therapeutic principle.
GASTROENTEROLOGY,Vol. 108, No. 4
• VESICULAR MONOAMINE TRANSPORTER EXPRESSION IN ENTERIC NEURONS AND EeL CELLS. R. De Giomio. D. Su, D. Peter, R.H. Edwards, N.C. Breeha, C. Stemini. DepL Clin. Phannacol. & Therapeutics, Univ. Bologna, Italy;CURE: UCLA/VA Gastroenteric Biology Center, VAMC-Wadsworth, Depts. Med., Anat. & Cell Biol., Neurol. & Biol. Chem., & Immunol.; UCLA School of Medicine, Los Angeles, CA.
Biogenic amines are abundant and play important roles in the digestive system. Recent molecular cloning studies identified two types of vesicular amine transporters (VMAT): the VMAT- 1 expressed in chromaffin cells of the adrenal medulla and the VMAT-2 expressed in monoamine cell groups of the central nervous system. The availability of specific antibodies to these transporters allows for the elucidation of sites of packaging and transmitter release in amine-containing neurons. In this study, we used a newly developed anti-VMAT-2 polyclonal antibody to localize sites of expression of VMAT-2-immunoreaetivity (IR) in the rat: gastmenteropanereatie tract. ~Aldehyde-fixed specimens of the rat gut and pancreas were processed for single and double labeling immunohistocbemistry. VMAT-2-IR was detected in a subset of nerve fibers and cell bodies of myentefic, submucous and pancreatic ganglia. The major target of VMAT-2-1R innervation was the vaseulature in both the gut and pancreas. Interestingly, VMAT-2-1R was identified in endocrine-like cells of the stomach. These were mainly distributed to the fundus and corpus, whereas they were rare in the antrum. Double labeling experiments with calbindin antibodies (a marker for EeL cells) showed that the vast majority, if not all, of the calbindin-containiog cells contained VMAT-2-1R, while a small number displayed immunolabeling only for VMAT-2-IR. VMAT-2-IR cells did not contain gastrin. Chemical (64)HDA) and surgical s~mpathectomy resulted in the disappearance ofVMAT-2 innervatton ofthe vaseulature. In conclusion: 1) VMAT-2 is a valuable marker to study the distribution and localization of amine-handling neural and EeL cells; 2) the identification of VMAT-2-1R in neurons and endocrine cells suggests that biogenic amines use the same vesicular transporter;, and 3) denervation studies indicate that at least a subpopulation of VMAT-2-1R nerve fibers supplying the pancreas (i.e. those innervating the blood vessels) is of extrinsic (sympathetic) origin. Supported by Morphology/Imaging Core DK 41301, GM 08375 and VA Medical Research Funds.
MOTILIN IS A NEUROPEPTIDE: DEMONSTRATION OF MOTILIN RECEPTORS IN THE RABBIT CEREBELLUM. I.Deooortere. G.Van Assche, T.L.Peeters. Gut Hormone Lab, Gasthuisber-g ON, Leuven, Belgium. Motilin has been mostly Studied in the perspective of the hormonal regulation o f interdigestive motility. However, motilin immunoreactivity has been demonstrated in the cerebellum, where it is almost exclusively found in the Purkinje cells, and motilin has an inhibitory effect on Deiters' neurons of the lateral vestibular nucleus in rabbits. We now report the first demonstration of central motilin receptors by binding studies a n d macro-autoradiography. Homogenates from rabbit cerebellum bound 12SI-nle13-porcine motilin in a reversible, time and concentration dependent manner. Analysis of displacement curves showed the presence of two classes of receptors, one with a high affinity (pK0t=9.00) and a low capacity (7.7 fmol/mg protein) for porcine motilin, the other with a low affinity (PKa2=6.45) and a high capacity (1969 fmol/mg protein). The same results were obtained with rabbit motilin (pKd's 9.16 and 6.87) and the motilin antagonist OHM-11526 (PKd'S 9.35 and 6.32). Feline and canine motilin had a markedly lower affinity for the low affinity site (pKd~=5.12 and 4.65 respectively) and avian motilin had a lower affinity for both sites (PKdt=8.28, pKd2=4.18). Structure-activity studies were performed with N-terminal and C-terminal fragments, The low affinity of the [2-22] fragment (PKdt =6.27, pKd~=4.89) illustrates the importance o f the Phe t residue. All other C-terminal fragments had very little affinity. In N,terminal fragments 16 residues were required to reach a reasonable affinity2 For example the PKdl values for the [1-11], [1-13] and [1-16] fragments were:' 5.38, 5.04 and 7.16. Erythromycin and its enolether derivative also bound to cerebellar motilin receptors (PKdi----- 6.49 and 7.69 respectively) Motilin binding was unaffected by serotonin, .GABA, DOPA, Substance P, sandostatin, neurotensin and enkephalin. GTP-~,S (0.1 raM) reduced the number of high affinity sites with 52 %, suggesting that the receptor is G-protein coupled. Autoradiographic studies on 20 #m cerebellar slices clearly showed that the motilin binding sites were located in the molecular layer of the cerebellar cortex (lateral lobe, flocculus and vermis). Displacement could be accomplished with unlabelled motilin and with erythromycin enolether. Together with the representation of motilin in this region and in the major innervation of deep cerebellar and vestibular nuclei, we postulate that motilin is a mediator of the Purldnje cell cerebellar output paths.
AGA ABSTRACTS
• AVIAN MOTILIN: ISOLATION, SEQUENCE, BIOLOGICAL ACTIVITY AND PHYSIOLOGICAL R O L E . P.De ClercQ, I.Depoortere, " P.Vergara and T.L.Peeters. Gut Hormone Lab, Gasthuisberg ON, Leuven, Belgium and " Unitat de Fisiologia Animal de Veterinaria, Un. Autonoma de Barcelona, Spain. The biological role of motilin has been mostly studied in rabbit, dog and in man. We here report the first study of a nonmammalian motilin. Avian motilin was isolated from acid extracts of the small intestinal mucosa of the chicken by a combination of gel and ion-exchange chromatography followed by reverse phase HPLC. The purification was monitored by radio-receptor assay. Sequencing showed that, compared to porcine motilin, residues 4,7-10 and 12 were replaced b y Phe, Gin, Ser, Asp, lie and Lys respectively. The biological activity of synthetic avian motilin and of the (1-14) fragment of avian motilin was evaluated in the tissue bath and in chickens chronically implanted with electrodes in stomach, duodenum, jejunum and ileum. In the tissue bath, under isotonic conditions, segments of the chicken small intestine showed a tonic contractile response towards avian motilin, which was 88 + 1 % of the response towards ACh in the jejunum, compared to 82 + 1% in the duodenum and 80 + 2% in the ileum. Avian motilin had a higher potency than porcine motilin. The pECso's (negative logarithm of the dose producing 50% of the maximum response) were 7.41 and 6.48 respectively. The potency of the (1-14) fragment was about tenfold reduced (6.61). The response was unaffected by TTX, atropine, L-NNA, guanethidine, prasozine, or yohimbine but was blocked by verapamil. 0 H M - 1 1 5 2 6 a specific motilin antagonist i n the rabbit, was an agonist equipotent to porcine motilin in the chicken (pECso: 6.56). In vitro avian motilin also affected segments from the rabbit small intestine, but was less potent than porcine motilin in this preparation (pECso'S of 7.69 and 8.25 respectively). In vivo the IV injection of avian motilin (1 gg/kg), induced within 30 seconds the appearance of a motor pattern recently described by one of us (PV), the rhythmic oscillatory pattern (ROC), but did not induce an MMC. No effect was seen after tenfold higher doses of mammalian motilins. Our studies suggest that the structure of motilin, the structure of the motitin receptor and the role of motilin have been changed during phylogenesis.
• EXPRESSION OF SSTR2 SOMATOSTATIN RECEPTOR SUBTYPE INHIBTED PANCREATIC TUMOR CELL GR,OWTH N Delesoue I Rauly, L Buscail, J.P. Est~ve, G.I. Bell , A. V. Schally~, N . Vaysse, C. Susini. INSERM U 151, CHU Rangueil, Toulouse, France; * Howard Hugues Medical Institute, University of Chicago, 1L; § Tulane University, Medical School, New Orleans, L. Somatostatin is a negative growth factor for normal and tumoral cells of GI tract that exerts its effect by interacting with specific receptors: We recently demonstrated that SSTR2 somatostatin receptor subtype mediates the antiproliferative effect of somatostatin analogues (Buscail et al Proc. Natl. Acad. S c i USA, 1994). SSTR2 mRNA is expressed in human pancreas whereas it is undetectable in almost human pancreatic cell lines including CAPAN-1 and BxPC-3. In this study, we investigated the effect of SSTR2 express!on in human" pancreatic tumor cells CAPAN 1, on cell proliferation. Human SSTR2 cDNA was stably transfected into CAPAN-1 cells using lipofectin. In cells expressing SSTR2, the somatostatin analogue RC-160 binds to SSTR2 with high affinity (Kd 0.3 nM) and induced an inhibition of serum-stimulated cell proliferation (ECs0 21 pM) reaching a maximum (-30 + 1 % ) at 1 nM RC-160. Furthermore, the proliferation of cells expressing SSTR2 was decreased compared to that of control cells, expressing the vector alone (doubling :time 37 hr vs 31 hr). The observed inhibition of cell growth induced by expression o f SSTR2 is not due to the clonal line since it is also observed in human pancreatic cancer BxPC-3 cells, stably expressing SSTR2 (doubling time 51 hr vs 36 hr). CAPAN-1 cells are capable of forming colonies in soft agar, consistent with their transformation potential. Expression of SSTR2 affected the growth rate of CAPAN-1 cells in agar as measured by the 2-fold decrease in diameter of colonies formed by cells expressing SSTR2 compared to that of control cells. Cells were analyzed for somatostatin mRNA expression using Reverse transcriptase-Polymerase chain reaction. Somatostatin mRNA was detected in SSTR2-transfected CAPAN-1 and BxPC-3 cells suggesting the existence of an autocrine somatostatin-SSTR2 loop in these cells. In conclusion, expression of SSTR2 in human pancreatic cancer cells results in decrease of cell growth probably as a consequence of an autocrine receptor activation. Therefore, SSTR2 may be of major importance as a therapeutic principle.
GASTROENTEROLOGY,Vol. 108, No. 4
• VESICULAR MONOAMINE TRANSPORTER EXPRESSION IN ENTERIC NEURONS AND EeL CELLS. R. De Giomio. D. Su, D. Peter, R.H. Edwards, N.C. Breeha, C. Stemini. DepL Clin. Phannacol. & Therapeutics, Univ. Bologna, Italy;CURE: UCLA/VA Gastroenteric Biology Center, VAMC-Wadsworth, Depts. Med., Anat. & Cell Biol., Neurol. & Biol. Chem., & Immunol.; UCLA School of Medicine, Los Angeles, CA.
Biogenic amines are abundant and play important roles in the digestive system. Recent molecular cloning studies identified two types of vesicular amine transporters (VMAT): the VMAT- 1 expressed in chromaffin cells of the adrenal medulla and the VMAT-2 expressed in monoamine cell groups of the central nervous system. The availability of specific antibodies to these transporters allows for the elucidation of sites of packaging and transmitter release in amine-containing neurons. In this study, we used a newly developed anti-VMAT-2 polyclonal antibody to localize sites of expression of VMAT-2-immunoreaetivity (IR) in the rat: gastmenteropanereatie tract. ~Aldehyde-fixed specimens of the rat gut and pancreas were processed for single and double labeling immunohistocbemistry. VMAT-2-IR was detected in a subset of nerve fibers and cell bodies of myentefic, submucous and pancreatic ganglia. The major target of VMAT-2-1R innervation was the vaseulature in both the gut and pancreas. Interestingly, VMAT-2-1R was identified in endocrine-like cells of the stomach. These were mainly distributed to the fundus and corpus, whereas they were rare in the antrum. Double labeling experiments with calbindin antibodies (a marker for EeL cells) showed that the vast majority, if not all, of the calbindin-containiog cells contained VMAT-2-1R, while a small number displayed immunolabeling only for VMAT-2-IR. VMAT-2-IR cells did not contain gastrin. Chemical (64)HDA) and surgical s~mpathectomy resulted in the disappearance ofVMAT-2 innervatton ofthe vaseulature. In conclusion: 1) VMAT-2 is a valuable marker to study the distribution and localization of amine-handling neural and EeL cells; 2) the identification of VMAT-2-1R in neurons and endocrine cells suggests that biogenic amines use the same vesicular transporter;, and 3) denervation studies indicate that at least a subpopulation of VMAT-2-1R nerve fibers supplying the pancreas (i.e. those innervating the blood vessels) is of extrinsic (sympathetic) origin. Supported by Morphology/Imaging Core DK 41301, GM 08375 and VA Medical Research Funds.
MOTILIN IS A NEUROPEPTIDE: DEMONSTRATION OF MOTILIN RECEPTORS IN THE RABBIT CEREBELLUM. I.Deooortere. G.Van Assche, T.L.Peeters. Gut Hormone Lab, Gasthuisber-g ON, Leuven, Belgium. Motilin has been mostly Studied in the perspective of the hormonal regulation o f interdigestive motility. However, motilin immunoreactivity has been demonstrated in the cerebellum, where it is almost exclusively found in the Purkinje cells, and motilin has an inhibitory effect on Deiters' neurons of the lateral vestibular nucleus in rabbits. We now report the first demonstration of central motilin receptors by binding studies a n d macro-autoradiography. Homogenates from rabbit cerebellum bound 12SI-nle13-porcine motilin in a reversible, time and concentration dependent manner. Analysis of displacement curves showed the presence of two classes of receptors, one with a high affinity (pK0t=9.00) and a low capacity (7.7 fmol/mg protein) for porcine motilin, the other with a low affinity (PKa2=6.45) and a high capacity (1969 fmol/mg protein). The same results were obtained with rabbit motilin (pKd's 9.16 and 6.87) and the motilin antagonist OHM-11526 (PKd'S 9.35 and 6.32). Feline and canine motilin had a markedly lower affinity for the low affinity site (pKd~=5.12 and 4.65 respectively) and avian motilin had a lower affinity for both sites (PKdt=8.28, pKd2=4.18). Structure-activity studies were performed with N-terminal and C-terminal fragments, The low affinity of the [2-22] fragment (PKdt =6.27, pKd~=4.89) illustrates the importance o f the Phe t residue. All other C-terminal fragments had very little affinity. In N,terminal fragments 16 residues were required to reach a reasonable affinity2 For example the PKdl values for the [1-11], [1-13] and [1-16] fragments were:' 5.38, 5.04 and 7.16. Erythromycin and its enolether derivative also bound to cerebellar motilin receptors (PKdi----- 6.49 and 7.69 respectively) Motilin binding was unaffected by serotonin, .GABA, DOPA, Substance P, sandostatin, neurotensin and enkephalin. GTP-~,S (0.1 raM) reduced the number of high affinity sites with 52 %, suggesting that the receptor is G-protein coupled. Autoradiographic studies on 20 #m cerebellar slices clearly showed that the motilin binding sites were located in the molecular layer of the cerebellar cortex (lateral lobe, flocculus and vermis). Displacement could be accomplished with unlabelled motilin and with erythromycin enolether. Together with the representation of motilin in this region and in the major innervation of deep cerebellar and vestibular nuclei, we postulate that motilin is a mediator of the Purldnje cell cerebellar output paths.