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Heterogeneity of progastrin in human antrum and gastrinoma: Isolation of a second peptide related to the gastrin precursor
171 INCREASED NLWvtBERS OF C(~RP-IMMUNOREACTIVE ENDOCRINE C E L L S O F THE RAT RESPIRATORY TRACT IN HYPOXIA.
G. COLLINA, D.R. SPRINGALL, G. BARERI, D. BEEI, 3.M. POLAK. Department of Histochemistry, Royal Postgraduate Medical School, London, W12 OHS, UK; IDepartment of Experimental Medicine, University of Sheffield Medical School, Sheffield, Sl0 23F, U K The respiratory tract of mammals is known to contain many endocrine ceils in the airway epithelium and lung parenchyma which occur singly or in clusters; the latter are often innervated forming the so-called 'neuroepithelia] bodies' (NEBs). In the rat respiratory tract, endocrine cells have been shown to contain calcitonin gene-related peptide (CGRP), a regulatory peptide encoded by the ca]citonin gene. For many years it has been hypothesised that NEBs are involved in sensing the composition of inhaled gas and therefore may be expected to respond to hypoxia. However, few studies have been carried out on the effect of chronic hypoxia on the neuroendocrine ceils in the lung, and their conclusions are disparate. We have therefore made a systematic immunocytochemica] study using antiserum to CGRP to see if numbers of immunoreactive cells changed in hypoxia. Wistar rats were exposed to normobaric hypoxia 00% 0 2) for 21 days. Hypoxic rats (n = 5) and normoxic litter-mate controls (n =5) were then killed and tissues were dissected and fixed in Bouin's fluid and processed to paraffin wax. Sections (5 um thick) were immunosta]ned using antiserum to CGRP by the PAP method. Anti-CGRP serum was used at a dilution ten-fold that normally used in order to increase the sensitivity of distinction between high and low levels of peptide. The numbers of CGRP-immunoreactive endocrine cells in 25 non-serial sections from each animal were counted for all animals. Single ceils and groups were counted separately, as were cells in airways and parenchyma of lung. The area of sections was measured using an IBAS image ana]yser, and results expressed as cell/era 2. Immunocytochemistry showed that the number of CGRP-immunoreactive ceils was significantly greater in the lungs of hypoxic then normoxic animals (76.9 + 10.l and 19.7 + 2.4 celts/cm 2, respectively; p 0.0l). This increase was not restricted to any particular area of the tissue, andthere was no obvious change in distribution of the endocrine ceils. These results suggest that there is a larger amount of stored CGRP in endocrine cells in hypoxia, and that the use of high dilutions of primary antiserum may help to reveal changes in concentrations of peptides by immunocytochemistry.
HETER~TY OF PROGASTRIN IN HUMAN ANTRUM AND GASTRINOMA: ISOLATION OF A SECOND PEP'rIDE RELATED TO THE GASTRIN PRECURSOR. H.P. DESMOND, 5. PAUWELS, * AND G.J. DOCKRAY. Department of Physiology, University of Liverpool, Liverpool, U.K. University of Louva]n in Brussels, Belgium.
*Department of Nuclear Medicine,
The gene sequence encoding the human antra] hormone gastrin indicates that the initial product of translation is a precursor peptide of 101 residues. We have recently described the isolation and chemical characterization of intact progastrin (21-101 human progastrin) from a gastrinoma. Purification was monitored by a radioimmunoassay specific for the predicted C-terminal hexapeptide of progastrin. In the present study we have examined the distribution of progastrin-like peptides in 5 antra] and 5 gastrinoma extracts. Tissues were obtained from gastrinoma patients or duodenal ulcer patients having antrectomy. They were extracted in boiling water and fractionated by gel filtration (Sephadex GS0) and the high moleeular eomponent of progastrinlike immunoreactivity was then separated by reversed phase HPLC (1~ Bondapak Cl8 in a Waters Z-module). All of the extracts were found to contain material with chromatographic properties similar to intact progastrin. However, an additional peak of immunoreactivity was observed in 2 out 5 gastrinomas and in 3 out of 5 antra] extracts. It constituted up to 50% of total progastrin-like immunoreactivity, and had a lower retention time than progastrin on HPLC. This early eluting material was purified to homogeneity from a gastrinoma extract by gel filtration, followed by ion exchange chromatography (Whatman DE52 eluted with a gradient of 0 -0.50 M ammonium acetate pH 6.5) and reversed phase HPLC (Waters Radial Pak C18, followed by Vydac C18 51~ and Techsil C18 511 using a gradient from 0.1% aqueous TFA to acetonitrile). Like progastrin the peptide shows low 00%) cross r e a c t i v i t y with some N-terminal G34 antisera. Structural studies are now in progress.
Conclusions, i. Gel filtration and H P L C revealed intact progastrin in 5 antra] and 5 gastrinoma extracts. 2. An additional component with similar gel filtration and immunochemica] properties to progastrin was found in some extracts and has been isolated.
G. COLLINA, D.R. SPRINGALL, G. BARERI, D. BEEI, 3.M. POLAK. Department of Histochemistry, Royal Postgraduate Medical School, London, W12 OHS, UK; IDepartment of Experimental Medicine, University of Sheffield Medical School, Sheffield, Sl0 23F, U K The respiratory tract of mammals is known to contain many endocrine ceils in the airway epithelium and lung parenchyma which occur singly or in clusters; the latter are often innervated forming the so-called 'neuroepithelia] bodies' (NEBs). In the rat respiratory tract, endocrine cells have been shown to contain calcitonin gene-related peptide (CGRP), a regulatory peptide encoded by the ca]citonin gene. For many years it has been hypothesised that NEBs are involved in sensing the composition of inhaled gas and therefore may be expected to respond to hypoxia. However, few studies have been carried out on the effect of chronic hypoxia on the neuroendocrine ceils in the lung, and their conclusions are disparate. We have therefore made a systematic immunocytochemica] study using antiserum to CGRP to see if numbers of immunoreactive cells changed in hypoxia. Wistar rats were exposed to normobaric hypoxia 00% 0 2) for 21 days. Hypoxic rats (n = 5) and normoxic litter-mate controls (n =5) were then killed and tissues were dissected and fixed in Bouin's fluid and processed to paraffin wax. Sections (5 um thick) were immunosta]ned using antiserum to CGRP by the PAP method. Anti-CGRP serum was used at a dilution ten-fold that normally used in order to increase the sensitivity of distinction between high and low levels of peptide. The numbers of CGRP-immunoreactive endocrine cells in 25 non-serial sections from each animal were counted for all animals. Single ceils and groups were counted separately, as were cells in airways and parenchyma of lung. The area of sections was measured using an IBAS image ana]yser, and results expressed as cell/era 2. Immunocytochemistry showed that the number of CGRP-immunoreactive ceils was significantly greater in the lungs of hypoxic then normoxic animals (76.9 + 10.l and 19.7 + 2.4 celts/cm 2, respectively; p 0.0l). This increase was not restricted to any particular area of the tissue, andthere was no obvious change in distribution of the endocrine ceils. These results suggest that there is a larger amount of stored CGRP in endocrine cells in hypoxia, and that the use of high dilutions of primary antiserum may help to reveal changes in concentrations of peptides by immunocytochemistry.
HETER~TY OF PROGASTRIN IN HUMAN ANTRUM AND GASTRINOMA: ISOLATION OF A SECOND PEP'rIDE RELATED TO THE GASTRIN PRECURSOR. H.P. DESMOND, 5. PAUWELS, * AND G.J. DOCKRAY. Department of Physiology, University of Liverpool, Liverpool, U.K. University of Louva]n in Brussels, Belgium.
*Department of Nuclear Medicine,
The gene sequence encoding the human antra] hormone gastrin indicates that the initial product of translation is a precursor peptide of 101 residues. We have recently described the isolation and chemical characterization of intact progastrin (21-101 human progastrin) from a gastrinoma. Purification was monitored by a radioimmunoassay specific for the predicted C-terminal hexapeptide of progastrin. In the present study we have examined the distribution of progastrin-like peptides in 5 antra] and 5 gastrinoma extracts. Tissues were obtained from gastrinoma patients or duodenal ulcer patients having antrectomy. They were extracted in boiling water and fractionated by gel filtration (Sephadex GS0) and the high moleeular eomponent of progastrinlike immunoreactivity was then separated by reversed phase HPLC (1~ Bondapak Cl8 in a Waters Z-module). All of the extracts were found to contain material with chromatographic properties similar to intact progastrin. However, an additional peak of immunoreactivity was observed in 2 out 5 gastrinomas and in 3 out of 5 antra] extracts. It constituted up to 50% of total progastrin-like immunoreactivity, and had a lower retention time than progastrin on HPLC. This early eluting material was purified to homogeneity from a gastrinoma extract by gel filtration, followed by ion exchange chromatography (Whatman DE52 eluted with a gradient of 0 -0.50 M ammonium acetate pH 6.5) and reversed phase HPLC (Waters Radial Pak C18, followed by Vydac C18 51~ and Techsil C18 511 using a gradient from 0.1% aqueous TFA to acetonitrile). Like progastrin the peptide shows low 00%) cross r e a c t i v i t y with some N-terminal G34 antisera. Structural studies are now in progress.
Conclusions, i. Gel filtration and H P L C revealed intact progastrin in 5 antra] and 5 gastrinoma extracts. 2. An additional component with similar gel filtration and immunochemica] properties to progastrin was found in some extracts and has been isolated.