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Substance P in the intestine of the cod, Gadus morhua
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II~,4UNOCHEMICAL CHARACTERISATION OF LPLRFm'nide AND FMRFm'nide-LIKE SPINAL C~[:N~D,FROG BRAIN AND CHICKEN BRAIN.
PEPTIDES
IN RAT
S. H O L M E S A N D G.3. D O C K R A Y . Physiological Laboratory, University of LiverpooL, Liverpool, U.K. Antibodies raised to the important molluscan neuropeptide, Phe-Met-Arg-Phe-amide (FMR, Famide) also react with material in vertebrate nervous systems. One of these peptides has been characterized from chicken brain as the pentapeptide Leu-Pro-Leu-Arg-Phe-amide (LPLRFamide). W e have now used radioimmunoassays employing antibodies to F M R F a m i d e and L P L R F a m i d e to examine the immunoreactive material in rat spinal cord, frog brain and chicken brain extracts. With the F M R F a m i d e antibody, L P L R F a m i d e shows about 10% ~rossreactivity, and with the L P L R F a m i d e antibody, F M R F a m i d e reacts about 5%. Neither assay reacts more than 0.1% with ~ I M S H or pancreatic polypeptide-related peptides (APP, BPP, NPY). In extracts of rat spinal cord prepared By boiling in water followed by ] % acetic acid there were ] peaks of FMRFamide-like immunoreactivity which were separated by H P L C ; none of these peaks showed more than trace immunoreactivity with the antibody to LPLRFamide. Immunoreactive material in boiling water/acid extracts of frog brain was separated into two peaks of immunoreactivity by both gel filtrationon Sephadex G-50 and HPLC. Both peaks reacted poorly with the L P L R F a m i d e antibody compared with the F M R F a m i d e antibody. Immunoreactive material in acid alcohol extracts of chicken brain was separated into two groups of peptides by gel filtration and subsequent H P L C . One group reacted similarly with antibodies to both L P L R F a m i d e and F M R F a m i d e when L P L R F a m i d e was used as radioimmunoassay standard; the other group, represented by a single major peptide, reacted 50 times better with the antibody to FMRFamide. Conclusions I. Antibodies to the chicken brain peptide, LPLRFamide, reveal a group of peptides in chicken brain extracts. 2. similar peptides cannot be found in frog brain or rat spinal cord, but the latter tissues contain material reacting with antibodies to FMRFamide. 3. There are several different types of peptide with F M R F a m i d e immunoreactivity in vertebrates.
SUBSTANCE P IN THE INTESTINE OF THE COD, GADUSMORHUA. JORGEN JENSEN, SUSANNE HOLMGREN & ANN-CATHRINE JONSSON, Comparative Neuroscience Unit, Department of Zoophysiology, University of G~teborg, Sweden. Substance P, a memberof the group of peptides called tachykinins which have the C-terminus Phe-XGly-Leu-Met-NH2 in common, has previously been found to have an excitatory action on the vascularly perfused intestine of the cod, Gadus morhua. The presence of a substance P-like peptide in the intestine has also been demonstrated with immunohistochemicalmethods (Jensen & Holmgren 1985, Comp. Biochem. Physiol. 82C, 81-89). But, the exact nature of the tachykinin present in the fish gut is not known. The present study was undertaken to further characterize the substance P-like peptide in the intestine of the cod. Radioimmunoassay(RIA), using antiserum GIO raised in a rabbit against SPzl, was employed to measure the amount of substance P:l~ke material in water and acetic acid (0,5 M) extracts of the intestine. Synthetic SPIt and SPIz labelled with 125I Bolton Hunter Reagent (NEN) were used as standard and label respectively. Immunohistochemistry (IHC) was performed on whole mount preparations using the same antiserum (GIO) as in RIA. The effect of substance P on the vascularly perfused intestine compared to the effects of four other tachykinins, eledoisin, physalaemin, neurokinin A and neurokinin B, was also tested. Substance P was found to be the more potent in the lower concentrations, having a threshold value between I0-~I-I0 -9 M, while the other tachykinins usually had t h e i r threshold value between 10-9-10-8 M. Maximal contractions were obtained with a l l the five tachykinins in concentrations between 10-7-3.10 -7 M. IHC confirmed the previous findings of substance P-like immunoreactivity in the intestine. Furthermore, adsorptions of the antiserum with eledoisin, physalaemin, neurokinin A or neurokinin B did not change i t s staining of immunoreactive material. The amount of substance P-like material was 48,7 ± 3,4 and 19,9 ± 1,9 fmol/g tissue in the water and acetic acid extracts, respectively (mean ± SEM; n=6). The antiserum showed no or low crossreactivity with the other tachykinins. In conclusion, substance P was found to be the most potent of the the tested tachykinins in contracting the intestine. Both RIA and IHC demonstrates the presence of a substance P-like peptide in the cod intestine. The low or absent crossreactivity of the antiserum with other tachykinins indicates that the tachykinin present i s , i f not identical, more closely related to substance P than to the other tachykinins tested. There is s t i l l however, the p o s s i b i l i t y that more than one tachykinin is present in the fish gut.
II~,4UNOCHEMICAL CHARACTERISATION OF LPLRFm'nide AND FMRFm'nide-LIKE SPINAL C~[:N~D,FROG BRAIN AND CHICKEN BRAIN.
PEPTIDES
IN RAT
S. H O L M E S A N D G.3. D O C K R A Y . Physiological Laboratory, University of LiverpooL, Liverpool, U.K. Antibodies raised to the important molluscan neuropeptide, Phe-Met-Arg-Phe-amide (FMR, Famide) also react with material in vertebrate nervous systems. One of these peptides has been characterized from chicken brain as the pentapeptide Leu-Pro-Leu-Arg-Phe-amide (LPLRFamide). W e have now used radioimmunoassays employing antibodies to F M R F a m i d e and L P L R F a m i d e to examine the immunoreactive material in rat spinal cord, frog brain and chicken brain extracts. With the F M R F a m i d e antibody, L P L R F a m i d e shows about 10% ~rossreactivity, and with the L P L R F a m i d e antibody, F M R F a m i d e reacts about 5%. Neither assay reacts more than 0.1% with ~ I M S H or pancreatic polypeptide-related peptides (APP, BPP, NPY). In extracts of rat spinal cord prepared By boiling in water followed by ] % acetic acid there were ] peaks of FMRFamide-like immunoreactivity which were separated by H P L C ; none of these peaks showed more than trace immunoreactivity with the antibody to LPLRFamide. Immunoreactive material in boiling water/acid extracts of frog brain was separated into two peaks of immunoreactivity by both gel filtrationon Sephadex G-50 and HPLC. Both peaks reacted poorly with the L P L R F a m i d e antibody compared with the F M R F a m i d e antibody. Immunoreactive material in acid alcohol extracts of chicken brain was separated into two groups of peptides by gel filtration and subsequent H P L C . One group reacted similarly with antibodies to both L P L R F a m i d e and F M R F a m i d e when L P L R F a m i d e was used as radioimmunoassay standard; the other group, represented by a single major peptide, reacted 50 times better with the antibody to FMRFamide. Conclusions I. Antibodies to the chicken brain peptide, LPLRFamide, reveal a group of peptides in chicken brain extracts. 2. similar peptides cannot be found in frog brain or rat spinal cord, but the latter tissues contain material reacting with antibodies to FMRFamide. 3. There are several different types of peptide with F M R F a m i d e immunoreactivity in vertebrates.
SUBSTANCE P IN THE INTESTINE OF THE COD, GADUSMORHUA. JORGEN JENSEN, SUSANNE HOLMGREN & ANN-CATHRINE JONSSON, Comparative Neuroscience Unit, Department of Zoophysiology, University of G~teborg, Sweden. Substance P, a memberof the group of peptides called tachykinins which have the C-terminus Phe-XGly-Leu-Met-NH2 in common, has previously been found to have an excitatory action on the vascularly perfused intestine of the cod, Gadus morhua. The presence of a substance P-like peptide in the intestine has also been demonstrated with immunohistochemicalmethods (Jensen & Holmgren 1985, Comp. Biochem. Physiol. 82C, 81-89). But, the exact nature of the tachykinin present in the fish gut is not known. The present study was undertaken to further characterize the substance P-like peptide in the intestine of the cod. Radioimmunoassay(RIA), using antiserum GIO raised in a rabbit against SPzl, was employed to measure the amount of substance P:l~ke material in water and acetic acid (0,5 M) extracts of the intestine. Synthetic SPIt and SPIz labelled with 125I Bolton Hunter Reagent (NEN) were used as standard and label respectively. Immunohistochemistry (IHC) was performed on whole mount preparations using the same antiserum (GIO) as in RIA. The effect of substance P on the vascularly perfused intestine compared to the effects of four other tachykinins, eledoisin, physalaemin, neurokinin A and neurokinin B, was also tested. Substance P was found to be the more potent in the lower concentrations, having a threshold value between I0-~I-I0 -9 M, while the other tachykinins usually had t h e i r threshold value between 10-9-10-8 M. Maximal contractions were obtained with a l l the five tachykinins in concentrations between 10-7-3.10 -7 M. IHC confirmed the previous findings of substance P-like immunoreactivity in the intestine. Furthermore, adsorptions of the antiserum with eledoisin, physalaemin, neurokinin A or neurokinin B did not change i t s staining of immunoreactive material. The amount of substance P-like material was 48,7 ± 3,4 and 19,9 ± 1,9 fmol/g tissue in the water and acetic acid extracts, respectively (mean ± SEM; n=6). The antiserum showed no or low crossreactivity with the other tachykinins. In conclusion, substance P was found to be the most potent of the the tested tachykinins in contracting the intestine. Both RIA and IHC demonstrates the presence of a substance P-like peptide in the cod intestine. The low or absent crossreactivity of the antiserum with other tachykinins indicates that the tachykinin present i s , i f not identical, more closely related to substance P than to the other tachykinins tested. There is s t i l l however, the p o s s i b i l i t y that more than one tachykinin is present in the fish gut.