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Distinct biological activities of endogenous gastrin and cholecystekinin (CCK) in bullfrog
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Abstracts from the 11th InternationalSymposium on Regulatory Peptides
Developmental Expression of the Pituitary adenylate cyclase activating polypeptide (PACAP) in rat autonomic ganglia and spinal cord. Henri~tte S ]~[iclsen, Jens Hannibal, Jan Fahrenkrug Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen DK- 2400, Copenhagen NV, Denmark. Pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide structurally related to vasoactive intestinal polypeptide (VIP), has been shown to stimulate neuronal growth and differentiation indicating a possible function in the development of the nervous system. However, little is known about the expression of PACAP in foetal life. In the present study we used immunocytochemistry and in situ hybridization histochemistry to examine and compare the expression of PACAP and VIP in autonomic ganglia and spinal cord of rat foetuses aged El2 - E21. PACAP immunoreactivity was detected by the use of a specific monoclonal PACAP antibody, and PACAP mRNA was visualized by use of 35S-labelled cRNA-probes PACAP positive sympathetic fibres were observed in the intermediolateral column of the spinal cord at El3. At El4 PACAP fibres projected to the sympathetic trunk where VIP immunoreaetive cell bodies and fibres were localized. Both peptides were present in the parasympathetic ganglia of the cranial nerves at E 16. At this time, PACAP sympathetic fibres were innervating the medulla of the adrenal gland, and PACAP but no VIP immunoreactivity was demonstrated in cell bodies of sensory ganglia ( the trigeminal ganglion and the dorsal root ganglion). Few VIP cell bodies appeared in the trigeminal ganglion at E20. Both PACAP and V1P immunoreactivity was at E 14-E 16 observed in cells of the myenteric ganglia of the gastrointestinal tract. The distribution of PACAP and VIP immunoreactive cell bodies was confirmed by in situ hybridization histochemistry. Thus, in the early embryonic developmental stages, PACAP occurs in the first neuron in the symphathetic system localized in the spinal cord with fibres projecting to the sympathetic trunk. PACAP appears in the sensory ganglia in the early embryonic stage, and both peptides appear at almost the same time in the parasympathetic ganglia and the enteric nervous system. PACAP and VIP occur with the same distribution pattern as in the adult rat, and consequently there is no evidence for transient expression
Distinct biological activities o f e n d o g e n o u s gastrin a n d c h o l e c y s t e k i n i n ( C C K ) in bullfrog. Kaj G. Nielsen, Peter Bomgren* and Anders H. Johnsen. Dept. Clin. Biochem., Nat. Univ. Hospital, Rigshospitalet, Copenhagen. *Department of Zoophysiology, Univ. of G6teborg. CCK and gastrin share the same amidated carboxyl terminal tetrapeptide, Trp-Met-Asp-Phe.NH2, which is crucial for biological activity of both. In mammals the two peptides are distinguished by the position of a tyrosyl residue. In gastrin Tyr is in position 6 as counted from the C-terminus, and in CCK it is in position 7. CCK peptides are sulfated at the tyrosyl residues, which is essential for biological activity. We have recently identified two different peptides from the bullfrog (Rana catesbeiana): Frog gastrin (FG) from antrum, DY(SO3H)AGWMDF.NH2 and CCK from brain/intestine, DY(SO3H)MGWMDF.NH2 (Johnsen & Rehfeld 1992 and Johnsen 1994). Their structural similarity raised the question whether the two peptides show different stimulatory activity in different target organs as do CCK and gastrin in mammals. The following tissues were studied by isometric muscle contraction experiments: gallbladder, small intestine and stomach. All experiments were performed during summertime. The ECs0-values of different peptides with longitudinal gallbladder strips were CCK-8s: 69.6 nM; FG: 494 nM; CCK-8ns: 17.4 IaM. All potencies were significantly different (p<0.0001). Neither small intestine (longitudinal and circular) nor stomach (pyloric and cardiac both longitudinal and circular) muscle strips showed any contraction, relaxation, increase or decrease in rythmic activity in response to CCK-8s, CCK-8ns and FG. Measurements of acid secretion from isolated mucosa was performed in a pH-stat (pH=5.5) by continous titration of the mucosal bathing solution. ECs0-values were FG: 7.72 nM; CCK-8s: 64.9 nM; CCK-8ns: 150 nM. The differences in potencies of FG and CCK-8ns are as well as the maximal response to FG: 33.8 + 3.41% and CCK-8s: 18.5 + 6.03 % significantly different (p<0.05). Experiments with porcine gallbladder showed no significant difference between FG: EC50=7.18 nM and CCK8s: ECs0=18.7nM (p>0.90). In summary: CCK-8s was 7 times more potent than FG in the gallbladder contraction experiments while FG was 8 times more potent than CCK-8s in the acid secretion experiments. Furthermore, FG showed two times higher efficacy in stimulating gastric acid production than did CCK. Thus, the receptors of the two investigated frog target organs show a high degree of discrimination between the two endogenous peptides. These findings show that not only does the antrum of frog produce a distinct peptide, frog gastrin, but this peptide also shows specific gastrin activity.
Abstracts from the 11th InternationalSymposium on Regulatory Peptides
Developmental Expression of the Pituitary adenylate cyclase activating polypeptide (PACAP) in rat autonomic ganglia and spinal cord. Henri~tte S ]~[iclsen, Jens Hannibal, Jan Fahrenkrug Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen DK- 2400, Copenhagen NV, Denmark. Pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide structurally related to vasoactive intestinal polypeptide (VIP), has been shown to stimulate neuronal growth and differentiation indicating a possible function in the development of the nervous system. However, little is known about the expression of PACAP in foetal life. In the present study we used immunocytochemistry and in situ hybridization histochemistry to examine and compare the expression of PACAP and VIP in autonomic ganglia and spinal cord of rat foetuses aged El2 - E21. PACAP immunoreactivity was detected by the use of a specific monoclonal PACAP antibody, and PACAP mRNA was visualized by use of 35S-labelled cRNA-probes PACAP positive sympathetic fibres were observed in the intermediolateral column of the spinal cord at El3. At El4 PACAP fibres projected to the sympathetic trunk where VIP immunoreaetive cell bodies and fibres were localized. Both peptides were present in the parasympathetic ganglia of the cranial nerves at E 16. At this time, PACAP sympathetic fibres were innervating the medulla of the adrenal gland, and PACAP but no VIP immunoreactivity was demonstrated in cell bodies of sensory ganglia ( the trigeminal ganglion and the dorsal root ganglion). Few VIP cell bodies appeared in the trigeminal ganglion at E20. Both PACAP and V1P immunoreactivity was at E 14-E 16 observed in cells of the myenteric ganglia of the gastrointestinal tract. The distribution of PACAP and VIP immunoreactive cell bodies was confirmed by in situ hybridization histochemistry. Thus, in the early embryonic developmental stages, PACAP occurs in the first neuron in the symphathetic system localized in the spinal cord with fibres projecting to the sympathetic trunk. PACAP appears in the sensory ganglia in the early embryonic stage, and both peptides appear at almost the same time in the parasympathetic ganglia and the enteric nervous system. PACAP and VIP occur with the same distribution pattern as in the adult rat, and consequently there is no evidence for transient expression
Distinct biological activities o f e n d o g e n o u s gastrin a n d c h o l e c y s t e k i n i n ( C C K ) in bullfrog. Kaj G. Nielsen, Peter Bomgren* and Anders H. Johnsen. Dept. Clin. Biochem., Nat. Univ. Hospital, Rigshospitalet, Copenhagen. *Department of Zoophysiology, Univ. of G6teborg. CCK and gastrin share the same amidated carboxyl terminal tetrapeptide, Trp-Met-Asp-Phe.NH2, which is crucial for biological activity of both. In mammals the two peptides are distinguished by the position of a tyrosyl residue. In gastrin Tyr is in position 6 as counted from the C-terminus, and in CCK it is in position 7. CCK peptides are sulfated at the tyrosyl residues, which is essential for biological activity. We have recently identified two different peptides from the bullfrog (Rana catesbeiana): Frog gastrin (FG) from antrum, DY(SO3H)AGWMDF.NH2 and CCK from brain/intestine, DY(SO3H)MGWMDF.NH2 (Johnsen & Rehfeld 1992 and Johnsen 1994). Their structural similarity raised the question whether the two peptides show different stimulatory activity in different target organs as do CCK and gastrin in mammals. The following tissues were studied by isometric muscle contraction experiments: gallbladder, small intestine and stomach. All experiments were performed during summertime. The ECs0-values of different peptides with longitudinal gallbladder strips were CCK-8s: 69.6 nM; FG: 494 nM; CCK-8ns: 17.4 IaM. All potencies were significantly different (p<0.0001). Neither small intestine (longitudinal and circular) nor stomach (pyloric and cardiac both longitudinal and circular) muscle strips showed any contraction, relaxation, increase or decrease in rythmic activity in response to CCK-8s, CCK-8ns and FG. Measurements of acid secretion from isolated mucosa was performed in a pH-stat (pH=5.5) by continous titration of the mucosal bathing solution. ECs0-values were FG: 7.72 nM; CCK-8s: 64.9 nM; CCK-8ns: 150 nM. The differences in potencies of FG and CCK-8ns are as well as the maximal response to FG: 33.8 + 3.41% and CCK-8s: 18.5 + 6.03 % significantly different (p<0.05). Experiments with porcine gallbladder showed no significant difference between FG: EC50=7.18 nM and CCK8s: ECs0=18.7nM (p>0.90). In summary: CCK-8s was 7 times more potent than FG in the gallbladder contraction experiments while FG was 8 times more potent than CCK-8s in the acid secretion experiments. Furthermore, FG showed two times higher efficacy in stimulating gastric acid production than did CCK. Thus, the receptors of the two investigated frog target organs show a high degree of discrimination between the two endogenous peptides. These findings show that not only does the antrum of frog produce a distinct peptide, frog gastrin, but this peptide also shows specific gastrin activity.