- Email: [email protected]
Subtype 2 somatostatin receptors expression in human normal and tumoral gastro-intestinal tissues
April 1 9 9 5
• RABBIT ILEAL SUCRASE-ISOMALTASE REPRESENTS A FUNCTIONAL RECEPTOR FOR (2 D I F F I C I L E TOXIN A. C. Pothoulakis, R.J. Gilbert, C. Cladaras, I. Castagliuolo, S. Nikulasson, J.T., J.S. Montcrief, G. Semenza, T.W. Wilkius, J.T. LaMont. Sections of Gastroenterology and Pathology and Molecular Genetics, Boston University School of Medicine, Boston MA, Section of Gastroenterology, St. Elizabeth's Hospital, Boston MA, Department of Biochemistry, Swiss Federal Institute of Technology, Zurich, Switzerland and Virginia Polytechnic Institute, Blacksburg, VA. C. di['/ictle toxin A (TxA) causes secretion and intestinal inflammation following binding to a specific brush receptor (BB). We have reported that-~H-TxA binds avidly to sucrase-isomaltase (S-I) and that S-I lgG blocks binding of TxA to rabbit ileal BB. The aim of this stud3,'was to examine whether S-I represents a functional receptor for TxA using two assay systems: 1) in vivo cnterotoxic response and 2) increase in intracetlular calcium in CHO cells. Methods: Rabbit ileal loops were ir[iected with purified TxA (20 ug, n= 12) or buffer (n= 15). In test animals ileal loops were first injected with either guinea pig anti-S-I polyclonal lgG ( 1 mg/loop, n=12) or control lgG (n=6). We measured fluid secretion at 5 hours as loop ~eight/length (mg/cm) and intestinal pcnncabdity • • by dpm of 3H-manmtol • m luminal fluid (dpm/cm loop). The cDNA of rabbit S-1 was cloned into the expression vector pcDNAI-amp. CHO cells were transfected with the expression vector for S-I and RSV-hygroplasmid and cells resistant to hygromyein B were selected. Receptormediated signal transduction in response to recombinant TxA C-terminal peptidc, a 95 kDa peptide associated with TxA receptor binding, or native TxA (3-6 ug/ml) was assayed as a change in intracellular calcium in fura-2-1oaded normal or transfected CHO cells using digital fluorescence microscopy. Results: Anti-S-I lgG, but not control IgG, inhibited TxA-induced secretion (by 8 1.3%, p<0.01 ), and permeability (by 82.6%, p<0.05). Anti S-I lgG also reduced epithelial cell damage associated with this'toxin. CHO cells transfected with rabbit S-I cDNA showed presence of S-1 mRNA and protein by Northern and Western blot analysis and S-I enzymatic activi~'. TxA recombinant binding peptide resulted in a calcium increase in 8% of control CHO cells (n=25). whereas this peptide resulted in a calcium increase in 72.2% ofS-I transfected cells (n=18, p<0.01). Similar differences in calcium response between control and S-I transfected CHO cells were observed following exposure to TxA. Conclusions: Rabbit ileal S-I and transfected S-I function as a receptor for TxA as evidenced by 1) marked inhibition by anti-S-1 lgG of TxA-induced enterotoxicit 3, in w v o and 2) increased calcium in S-I transfected cells exposed to TxA rcceptor binding peptide or holotoxin. We speculate that, while S-I is not an absolute requirement for TxA binding (e.g. colon). S-I facilitates binding of TxA to membranes and that this in turn enhances toxicity.
Hormones and Receptors
A999
S U B T Y P E 2 S O M A T O S T A T I N R E C E P T O R S E X P R E S S I O N IN HUMAN
NORMAL
AND TUMORAL
GASTRO-INTESTINAL
TISSUES. G. Pr6vost, R. Benamouzi< J. Amouroux, T. Coste, J. Rautureau. Depts of Gastroenterology, Pathology and IOCMH. Hopital Avicenne. Bobigny. France. *supported by Fondation Midy. Previous studies have reported inhibition of cell proliferation and tumoral growth by somatostatin (SST) analogues. The cloning of 5 SST receptor genes was recently reported. SSTR1, SSTR2 and SSTR3 were mainly characterized in brain and in various tumoral cell lines. SSTR4 and SSTR5 were only characterized in brain and pituitary gland. The digestive tract SSTR subtypes remain unknown. The aim of our study was to characterize these receptors in normal and tumoral stomach and large intestine mucosa. Colic (n=16) and gastric (n=5) samples were obtained from the tumor area and from normal mucosa at the time of surgery. Each sample was treated as follow: one part for reutine histopathological evaluation and the other stored in liquid nitrogen until cross-linking assay using both labelled SST-14 and SST analogues (BIM 23014, BIM 23060 and BIM 23068 selective for SSTR2; BIM 23052 and BIM 23056 which do not bind to SSTR2). RT-PCR studies with primers allowing the detection of RNA expression of SST receptors were also performed. A complex of 57 kD was visualized in all the normal and tumoral samples. A complex of 90-100 kD was also present in 3 colic samples. This 57 kD complex was pharmacologically characterized as the subtype 2. C o n c l u s i o n : The presence of subtype 2 sematostatin receptors was asserted by 2 different approaches in normal and tumoral human stomach and large intestine. The biological activity of these receptors remain to be determined.
• PANCREATIC POLYPEPTIDE RECEPTORS IN THE RAT BRAIN LOCALIZE TO A U T O N O M I C CONTROL AREAS OF THE HYPOTHALAMUS AND BRAINSTEM, LIMBIC NUCLEI AND THE OLFACTORY SYSTEM. AM Puccio, GE Hoffman and DC Whitcomb. Depts of Medicine, Neurobiology, Cell Biology and Physiology, Univ. Pittsburgh, Pittsburgh, PA 15261 and VAMC Pittsburgh 15240. Pancreatic polypeptide (PP) is a regulatory peptide that modulates gastrointestinal function. We have previously demonstrated PP receptors in the brainstem and interpeduncular nucleus. Microinjection of PP into the brainstem affects gastric motility and pancreatic exocrine secretion. Furthermore, we have demonstrated that small amounts of PP mRNA is present in specific regions of the rat brain which would be necessary for the existence of central peptidergic system utilizing PP. AIM: the aim of this study is to map regions of the rat brain that have PP receptors in order to determine the systems that are potentially modulated by PP. METHODS: Rats were killed, brains removed, frozen and sectioned on a cryostat in three planes (horizontal, sagittal and coronal). PP receptor autoradiography was performed by the method of Whitcomb (AJP 259: G687, 1990). Brain regions with PP binding sites were confirmed when identified in all three planes. RESULTS: Saturable PP binding was identified in the hypothalamus (archuate n, medial preoptic area and caudal paraventricular n), forebrain (anterior olfactory nucleus and islands of Calleja), the dorsal endopiriform n, piriform cortex, the bed n. of the stria terminalis, the thalamus (anteromedial thai. n, reuniens thai. n. and paraventricular thai n.), the red nucleus, substantia nigra, medial amygdaloid n. pd, the interpeduncular n, parabrachial n, locus coeruleus, mesencephalic trigeminal n, dorsal motor n of the vagus, the n. solitary tract, and the area postrema. CONCLUSIONS: The distribution of PP binding sites corresponds to brain regions regulating digestion and autonomic function. In addition, the pattern of binding in the olfactory and limbic system suggest that PP may also be involved in behavioral aspects of feeding. (Supported by NIH R29 DK45781 to Dr Whitcomb)
ARACHIDONYC ACID IITHIBITS CARBONIC ANHYDRASE I(CAI) WHILE INDOMETHACIN(I~C)ANTAGONIZES THIS E ~ E C T ° !.Pu~cas,~arcela Colt~u,GGabriela Domu~a,Center for D~edical Research an~ Assistanee,Simleu Silvaniei, ROUMANIA. Arachidenic acid,Drecursor of prostaglandin~PGs) is formed from phospholipids by action of phospho!ipase A2°0ur studies have shown that vasodilating PGs~PGE~PGI~.)inhibit CA I and CA II IMC,an inhibi %or of cyclooxygenase,activates CA i-If and an%age nizes the inhibitory effect of PGE~,PGIot~nd of acetazolamide on CA l-il(i°~u~ca~ e~ al.X ~WCOG,Los Amge!es,!994).Ai~[:we followed the effect of araehi denic acid and of !~C on CA l,isozyme also located in the capillaries of gastric m u c o s a ° ~ H O D : w e fel lowed in vitro the effects of arachidonic acid,l~C and of their association on purified CA I at cormen ~ra[:ions between !O-8-!O-4M,according to dose-response relationship. CA activity was determined by a steD~ed-flow method(Khalifah RG~I971).RESULTS: C'on~(!.~).Arachide'nic acid i]~.~C Arachoacid+IMC ---~0-9 '-19~ ~ 16% -5% i0 -~ -5~$ 29% -22% 10 -4 -64~$ 48% -32% ~ N : t h e results show that arachidenic acid inhibits CA l,enzyme involved in the regulation of vascular %onus;CA I inhibition is accompanied by vasodilation, l~{C,knowm as an inhibitor of eye!sexy genase,amtagenizes the inhibitory effect of arachi donic acid on CA l°Considering the vasodilating ef fect of a~achidonic acid and. i~s inhibitory effect on CA i,our results suggest the involvement of this isozyme in araehidonic acid-induced vasedilation. These results suggest that l~C,known as an antagonist of PG-induced vasodi!ation,alse is an antagonist of araohidonic acid-induced vasodilation°The intimate mechanism by which CA I could be involved in vasodilation may rely on the pH modifieatioms initiated by CA I inhibition a~d by arachidonic acid inhibition followed by stimulation of guanylate cyclase and increased productiom of cGN~. ~
-
°
• RABBIT ILEAL SUCRASE-ISOMALTASE REPRESENTS A FUNCTIONAL RECEPTOR FOR (2 D I F F I C I L E TOXIN A. C. Pothoulakis, R.J. Gilbert, C. Cladaras, I. Castagliuolo, S. Nikulasson, J.T., J.S. Montcrief, G. Semenza, T.W. Wilkius, J.T. LaMont. Sections of Gastroenterology and Pathology and Molecular Genetics, Boston University School of Medicine, Boston MA, Section of Gastroenterology, St. Elizabeth's Hospital, Boston MA, Department of Biochemistry, Swiss Federal Institute of Technology, Zurich, Switzerland and Virginia Polytechnic Institute, Blacksburg, VA. C. di['/ictle toxin A (TxA) causes secretion and intestinal inflammation following binding to a specific brush receptor (BB). We have reported that-~H-TxA binds avidly to sucrase-isomaltase (S-I) and that S-I lgG blocks binding of TxA to rabbit ileal BB. The aim of this stud3,'was to examine whether S-I represents a functional receptor for TxA using two assay systems: 1) in vivo cnterotoxic response and 2) increase in intracetlular calcium in CHO cells. Methods: Rabbit ileal loops were ir[iected with purified TxA (20 ug, n= 12) or buffer (n= 15). In test animals ileal loops were first injected with either guinea pig anti-S-I polyclonal lgG ( 1 mg/loop, n=12) or control lgG (n=6). We measured fluid secretion at 5 hours as loop ~eight/length (mg/cm) and intestinal pcnncabdity • • by dpm of 3H-manmtol • m luminal fluid (dpm/cm loop). The cDNA of rabbit S-1 was cloned into the expression vector pcDNAI-amp. CHO cells were transfected with the expression vector for S-I and RSV-hygroplasmid and cells resistant to hygromyein B were selected. Receptormediated signal transduction in response to recombinant TxA C-terminal peptidc, a 95 kDa peptide associated with TxA receptor binding, or native TxA (3-6 ug/ml) was assayed as a change in intracellular calcium in fura-2-1oaded normal or transfected CHO cells using digital fluorescence microscopy. Results: Anti-S-I lgG, but not control IgG, inhibited TxA-induced secretion (by 8 1.3%, p<0.01 ), and permeability (by 82.6%, p<0.05). Anti S-I lgG also reduced epithelial cell damage associated with this'toxin. CHO cells transfected with rabbit S-I cDNA showed presence of S-1 mRNA and protein by Northern and Western blot analysis and S-I enzymatic activi~'. TxA recombinant binding peptide resulted in a calcium increase in 8% of control CHO cells (n=25). whereas this peptide resulted in a calcium increase in 72.2% ofS-I transfected cells (n=18, p<0.01). Similar differences in calcium response between control and S-I transfected CHO cells were observed following exposure to TxA. Conclusions: Rabbit ileal S-I and transfected S-I function as a receptor for TxA as evidenced by 1) marked inhibition by anti-S-1 lgG of TxA-induced enterotoxicit 3, in w v o and 2) increased calcium in S-I transfected cells exposed to TxA rcceptor binding peptide or holotoxin. We speculate that, while S-I is not an absolute requirement for TxA binding (e.g. colon). S-I facilitates binding of TxA to membranes and that this in turn enhances toxicity.
Hormones and Receptors
A999
S U B T Y P E 2 S O M A T O S T A T I N R E C E P T O R S E X P R E S S I O N IN HUMAN
NORMAL
AND TUMORAL
GASTRO-INTESTINAL
TISSUES. G. Pr6vost, R. Benamouzi< J. Amouroux, T. Coste, J. Rautureau. Depts of Gastroenterology, Pathology and IOCMH. Hopital Avicenne. Bobigny. France. *supported by Fondation Midy. Previous studies have reported inhibition of cell proliferation and tumoral growth by somatostatin (SST) analogues. The cloning of 5 SST receptor genes was recently reported. SSTR1, SSTR2 and SSTR3 were mainly characterized in brain and in various tumoral cell lines. SSTR4 and SSTR5 were only characterized in brain and pituitary gland. The digestive tract SSTR subtypes remain unknown. The aim of our study was to characterize these receptors in normal and tumoral stomach and large intestine mucosa. Colic (n=16) and gastric (n=5) samples were obtained from the tumor area and from normal mucosa at the time of surgery. Each sample was treated as follow: one part for reutine histopathological evaluation and the other stored in liquid nitrogen until cross-linking assay using both labelled SST-14 and SST analogues (BIM 23014, BIM 23060 and BIM 23068 selective for SSTR2; BIM 23052 and BIM 23056 which do not bind to SSTR2). RT-PCR studies with primers allowing the detection of RNA expression of SST receptors were also performed. A complex of 57 kD was visualized in all the normal and tumoral samples. A complex of 90-100 kD was also present in 3 colic samples. This 57 kD complex was pharmacologically characterized as the subtype 2. C o n c l u s i o n : The presence of subtype 2 sematostatin receptors was asserted by 2 different approaches in normal and tumoral human stomach and large intestine. The biological activity of these receptors remain to be determined.
• PANCREATIC POLYPEPTIDE RECEPTORS IN THE RAT BRAIN LOCALIZE TO A U T O N O M I C CONTROL AREAS OF THE HYPOTHALAMUS AND BRAINSTEM, LIMBIC NUCLEI AND THE OLFACTORY SYSTEM. AM Puccio, GE Hoffman and DC Whitcomb. Depts of Medicine, Neurobiology, Cell Biology and Physiology, Univ. Pittsburgh, Pittsburgh, PA 15261 and VAMC Pittsburgh 15240. Pancreatic polypeptide (PP) is a regulatory peptide that modulates gastrointestinal function. We have previously demonstrated PP receptors in the brainstem and interpeduncular nucleus. Microinjection of PP into the brainstem affects gastric motility and pancreatic exocrine secretion. Furthermore, we have demonstrated that small amounts of PP mRNA is present in specific regions of the rat brain which would be necessary for the existence of central peptidergic system utilizing PP. AIM: the aim of this study is to map regions of the rat brain that have PP receptors in order to determine the systems that are potentially modulated by PP. METHODS: Rats were killed, brains removed, frozen and sectioned on a cryostat in three planes (horizontal, sagittal and coronal). PP receptor autoradiography was performed by the method of Whitcomb (AJP 259: G687, 1990). Brain regions with PP binding sites were confirmed when identified in all three planes. RESULTS: Saturable PP binding was identified in the hypothalamus (archuate n, medial preoptic area and caudal paraventricular n), forebrain (anterior olfactory nucleus and islands of Calleja), the dorsal endopiriform n, piriform cortex, the bed n. of the stria terminalis, the thalamus (anteromedial thai. n, reuniens thai. n. and paraventricular thai n.), the red nucleus, substantia nigra, medial amygdaloid n. pd, the interpeduncular n, parabrachial n, locus coeruleus, mesencephalic trigeminal n, dorsal motor n of the vagus, the n. solitary tract, and the area postrema. CONCLUSIONS: The distribution of PP binding sites corresponds to brain regions regulating digestion and autonomic function. In addition, the pattern of binding in the olfactory and limbic system suggest that PP may also be involved in behavioral aspects of feeding. (Supported by NIH R29 DK45781 to Dr Whitcomb)
ARACHIDONYC ACID IITHIBITS CARBONIC ANHYDRASE I(CAI) WHILE INDOMETHACIN(I~C)ANTAGONIZES THIS E ~ E C T ° !.Pu~cas,~arcela Colt~u,GGabriela Domu~a,Center for D~edical Research an~ Assistanee,Simleu Silvaniei, ROUMANIA. Arachidenic acid,Drecursor of prostaglandin~PGs) is formed from phospholipids by action of phospho!ipase A2°0ur studies have shown that vasodilating PGs~PGE~PGI~.)inhibit CA I and CA II IMC,an inhibi %or of cyclooxygenase,activates CA i-If and an%age nizes the inhibitory effect of PGE~,PGIot~nd of acetazolamide on CA l-il(i°~u~ca~ e~ al.X ~WCOG,Los Amge!es,!994).Ai~[:we followed the effect of araehi denic acid and of !~C on CA l,isozyme also located in the capillaries of gastric m u c o s a ° ~ H O D : w e fel lowed in vitro the effects of arachidonic acid,l~C and of their association on purified CA I at cormen ~ra[:ions between !O-8-!O-4M,according to dose-response relationship. CA activity was determined by a steD~ed-flow method(Khalifah RG~I971).RESULTS: C'on~(!.~).Arachide'nic acid i]~.~C Arachoacid+IMC ---~0-9 '-19~ ~ 16% -5% i0 -~ -5~$ 29% -22% 10 -4 -64~$ 48% -32% ~ N : t h e results show that arachidenic acid inhibits CA l,enzyme involved in the regulation of vascular %onus;CA I inhibition is accompanied by vasodilation, l~{C,knowm as an inhibitor of eye!sexy genase,amtagenizes the inhibitory effect of arachi donic acid on CA l°Considering the vasodilating ef fect of a~achidonic acid and. i~s inhibitory effect on CA i,our results suggest the involvement of this isozyme in araehidonic acid-induced vasedilation. These results suggest that l~C,known as an antagonist of PG-induced vasodi!ation,alse is an antagonist of araohidonic acid-induced vasodilation°The intimate mechanism by which CA I could be involved in vasodilation may rely on the pH modifieatioms initiated by CA I inhibition a~d by arachidonic acid inhibition followed by stimulation of guanylate cyclase and increased productiom of cGN~. ~
-
°