0013.7227/92/1314-2033$03.00/0 Endocrinology CopyrightO1992byThe Endocrine Society
GASTRIN
RF&EASING
Vol. 131, No. 4 Printed
PBPTIDE
IMMUNOREACTIVITy
IS PRESENT
IN
OVINE
AMNIOTIC
FLUID
AND FEW&
in V.S.A
AND MATERNAL
CIRCULATIONS
of
Ob/Gyn’,
University
Physiology" of Western
Medicine3, and Ontario, London,
ABSTRACT: Using antisera directed towards the C-terminal region of gastrin releasing peptide (GRP), significant quantities of GRP-like immunoreactivity (GRPLI) were detected in ovine amniotic fluid and in the fetal and maternal circulations. The highest GRPLI levels were found in amniotic fluid (2135 ? 829 fmol/ml, n=12; mean f SEW), followed by those in ovine fetal (604 f 267 fmol/ml, n=13) and maternal plasma (229 f 89 fmol/ml, n=13). On gel filtration chromatography, the predominant GRPLI form in each fluid eluted in an identical position consistent with the entity being of apparently larger molecular size than porcine GRPlw2,. Certain fetal plasma samples contained a second GRPLI peak eluting at the void volume. Hence, during ovine pregnancy a GRPLI entity circulates in fetal and maternal plasma; the entity is of apparently larger molecular size than GRP,-27 but contains a structure immunologically indistinguishable from the bioactive Cterminal region of GRPI.,7. Given the recoanized bioactivities of GRP, this entity may be an important hormone during ovine fetal life. dilutions of plasma and AF samples and the mean INTRODUCTION concentration in each sample calculated from the Initial studies on the bombesin-related resulting dose-inhibition curves. gastrin releasing peptide (GRP) emphasized its ability to affect memmalian endocrine and exocrine secretory functions (1) but it is now recognized that GRP also may be an important autocrine regulator of cell growth (2,3). Recent studies have demonstrated that GRP and bombesin have mitogenic properties in human and mouse fetal lung (4) and in Because the ovine fetal chondrocytes (5). mechanisms underlying the coordinated growth of the mammalian embryo sne of fundamental imnortance, and given the evidence that GRP is a potential growth factor in fetal life, we initiated studies to evaluate the actions of GRP in an ovine pregnancy model. This study reDorts the nnesence of GRP-like immunoreactivity (GRPLI) in rel&ively high concentrations in ovine fetal and maternal circulations and also in amniotic fluid (AF). I-L,
Materials
NATERIALS
AND
WETRODS
and Procedures Using pregnant ewes of mixed breed and known gestational age (term 145-150 days) and surgical procedures and post-operative management as described (6), polyvinyl catheters were implanted under general anaesthesia into 13 fetal sheep (99128 days gestation), either into the fetal carotid artery and jugular vein O+ into the fetal femoral artery and vein. An open-end catheter was placed were implanted into in the amniotic sac. Catheters the maternal femoral artery and vein. In twin nresnancies onlv one fetus was catheterized. Foliowing at least 5 days recovery from surgery, fetal (3 ml) and maternal (5 ml) blood and RF (5 ml)‘ssmpies were obtained daily between 0800-1200 h. Less than 10% of the estimated fetal blood volume was withdrawn at each sampling. The samples were drawn into heparinized syringes, transferred immediately into chilled polypropylene tubes and centrifuged at 1500 x g for 10 min at 4'C. Plasma and AF samples were stored at -7OOC until further analysis. All animal protocols were approved by University of Western Ontario Animal Care Committees in accordance with guidelines established by the Canadian Council on Animal Care.
Figure
1. RIA dose-response OVinS fetal at gestational ages: FPl-132 FP3-114 days, FP4-114 days,
repreSentatiVe
in
Iowa
City
office
June
16,
of GRPLI in five samples obtained days, FPZ-134 days, FP5-142 days.
n
10000
Immunochemical Analvses Plasma and AF were analyzed for GRPLI by a previously described radioimmunoassay procedure (7). Briefly, the LR-16 antiserum used in the RIA is directed towards the C-terminal GRP21-25 sequence, reacts eguipotently with bombesin and GRP but has less than 0.01% cross-reaction with substance P, NRA and neuromedin 8, and does not crnss-react with and -11 or any structurally acidic FGF, IGF-I unrelated gastroenteropancreatic peptide tested. The sensitivity of the assay is less than 0.625 fmol/tube with half-displacement less than 7.5 fmol/tube. RIA was performed on serial 1:l Received
curves
plaSma
AF
Animal
Figure 2. The mean f SEM of fetal and maternal plasma and amniotic fluid concentrations of GRPLI obtained from 13 and 12 animals respectively, between 106-146 days gestation. Closed bars, amniotic fluid; open bars, fetal plasma; hatched bars, maternal plasma.
92
2033
Downloaded from https://academic.oup.com/endo/article-abstract/131/4/2033/3034799 by University of Otago Library user on 20 January 2019
W. Fra~er’*~, A.M. Carter',', J.R.G. Challis',2 and T.J. McDonald3s4. MRC Group in Fetal and Neonatal Health and Development. Departments Biochemistry4, The Lawson Research Institute and University Hospital, Ontario, Canada N6A 5A5.
2034
RAPID
Endo. Vol131.No4
COMMUNICATIONS
Analvsis AF samples were not available from one animal. The overall mean GRPLI concentrations in fetal and maternal plasma samples and in amniotic fluids for individual animals were calculated from values obtained in successive samples, taken through gestation. Differences in mean GRPLI concentrations between AF and plasma samples within individual sheep were sought using one way ANOVA, Fisher PLSD and Scheffe F-test. All values are presented as mean f SEM.
Representative gel filtration profiles of the GRPLI in fetal and maternal olasma and in Av ilr~ --shown in Fig. 3. The Predornlnant-GRpLI-peak in each fluid eluted in an identical position which occurred earlier than that of the porcine GRP1-27 standard, suggesting an apparently larger molecular size for the GRPLI entity. Co-chromatography of fetal plasma GRPLI with added porcine GRP)-27, did not alter the elution positions of the two entities (data not shown). In certain fetal plasma samples, an additional peak of immunoreactivity occurred in the void volume (Fig.3A).
0
20
40
60
80
100
120
140
Fmctmn volume (ml)
(b)
Data
RESULTS Throughout gestation, all three fluids contained GRPLI which produced dose-inhibition curves parallel to that of the standard curve (data for fetal plasma GRPLI shown in Fig. 1). Boiling AF, to inactivate proteolytic enzymes, did not significantly alter the content nor the parallelism of the dose-inhibition curve (data not shown). The GRPLI levels in plasma and AF of 13 fetal sheep and their mothers (106-146 days gestation) demonstrated a considerable range of values (Fig. 2). The mean average concentration of GRPLI in the AF (2135 k 829 fmolfml; n=12) was 3.5 fold higher than that in fetal plasma (604 f 267 fmol/ml, n=13) and 9 fold higher than that in maternal plasma (229 f 89 fmol/ml, n=13); the mean AF concentration of GRPLI was significantly greater than that of fetal or maternal plasma (Fig. 2; pqO.01). There was no correlation of GRPLI levels in any fluid with gestational age, circulating fetal ACTH levels, blood pO2, blood pH, maternal feeding or time of sampling.
0
20
40
60
80
100
400
140
120
(C)
300
200
100
0 0
20
40
60
60
100
120
140
Fracnon volumr (ml) Figure 3. Gel filtration chromatography profiles of GRPLI in ovine fetal (a) and maternal (b) plasma and amniotic fluid (c) on Sephadex G-SO (SF) columns (1 x 116 cm) eluted with GRP RIA buffer at 4'C. GRPLI was detected by a C-terminally directed antiserum raised against synthetic GRP (see text for details). The elution positions of synthetic porcine GRP)-27 and blue (Vo) and 12'1 are '$'C'; of GRP18.27~ dextran
Downloaded from https://academic.oup.com/endo/article-abstract/131/4/2033/3034799 by University of Otago Library user on 20 January 2019
Aliquots of fetal and maternal plasma and AF were partially purified by loading onto pre-washed Sep-pak C-18 cartridges (Waters Associates, Toronto, washed with 0.1% (v/v) trifluoroacetic acid Ont.), (TFA; Pierce Chemical Company, Rockford, Ill.) in water and subsequently eluted with an acetonitrile (Fisher Chemical Company, Toronto, Ont.): TFA: water The GRPLI recovered in mixture (80:0.1:19.9;v/v). the eluate fractions was 67.4 f 14.7% (n=4; mean f SEM); the eluates were concentrated by evaporation of solvents under a stream of nitrogen. An affinity chromatography column was constructed by coupling the monoclonal bombesin antiserum, designated 2All (a kind gift of Dr. F. Cuttita, National Cancer Institute, Bethesda, Md.) to Affi-gel 10 using procedures suggested by the manufacturer (Bio-Rad, The characteristics of the 2All Mississauga, Ont.). antiserum have been previously described in detail the 2All antiserum is directed (8). Briefly, against the C-terminal region of GRP but does not cross-react with C-terminally extended forms (8). An aliquot of fetal plasma GRPLI, after partial purification on Sep-pak C-18 cartridges, was loaded the column washed with the onto the affinity column, and the GRPLI eluted from the equilibration buffer, The recovery of column with 0.4 M citric acid. fetal GRPLI loaded onto the affinity column was 88%. Gel filtration chromatography was performed on Sephadex G-50 superfine (Pharmacia, Montreal, P.Q.) and eluted at a columns, (1 x 116 cm), equilibrated constant flow of 6 ml/h with GRP RIA buffer (0.06M Na2HP04, 0.01 M EDTA, pH 8, containing 0.05% sodium aside (w/v) and 0.3% bovine serum albumin (w/v) at 4Oc. The void volumes (Vo) and salt peak elution volumes (V,) were defined by determining the elution Montreal, P-Q.) ;;~~;,K+ (D~e~syam~'yor~n~o~~n~*')~ The elution positions of porcine GRP)-27 and GRP1S.27 (Bachem, Torrance, Ca.) were determined by dissolving the peptides in GRP-free plasma (obtained by treating either adult canine plasma or ovine fetal plasma with excess activated charcoal) and monitoring the chromatographic elution patterns with RIA. Aliquots of either fetal or maternal plasma or AF with sufficient GRPLI (>500 fmol/ml) were loaded in 1 ml quantities onto the columnsg One ml fractions were collected and stored at -70 C until performance of RIA.
RAPID
COMMUNICATIONS
I
Medical Research Council of Canada to JRGC and The authors gratefully acknowledge the excellent technical assistance of Mrs. Gail Ward and the excellent secretarial assistance of Mrs. Gloria Kellett.
TJM.
REFERENCES
1
2
3
4
5
6
7
8
9 10
11
12
13 14
,
15
16
17
McDonald TJ 1988 The Gastrin Releasing Polypeptide. In: Mutt V (ed) Adv. Wetabol. Disorders. San Diego, New York, Berkeley, Boston, London, Sydney, Tokyo, Toronto, vol 11:199-250 Zachary I, Wall PJ, Rozengurt E 1987 A role for neuropeptides in the control of cell proliferation. Developmental Biology 124:295308 Cuttita F, Carney DN, Mulshine JL 1985 Bombesin-like peptides can function as autocrine factors in human small cell lung Nature 316:823-826 cancer. Sunday ME, Hua J, Dai HB, Nusrat A, Torday JS 1990 Bombesin increases fetal lung growth and maturation in utero and in organ culture. Am J Respir Cell Mol Biol. 3:199-205 Hill DJ, McDonald TJ 1992 Mitogenic action of gastrin-releasing polypeptide on isolated epiphyseal growth plate chondrocytes from the ovine fetus. Endocrinology 130:2811-2819 Akagi K, Challis JRG 1990 Threshold of hormonal and biophysical responses to acute hypoxemia in fetal sheep at different gestational ages, Can. J. Physiol. Pharmacol. 68:549-555 McDonald TJ, Christofi FL, Brooks BD, Barnett W, Cook MA 1988 Characterization of content and chromatographic forms of neuropeptides in purified nerve varicosities prepared from guinea pig myenteric plexus. Reg Pep. 21:69-83 Lebacq-Verheyden A-M, Kasprzyk PG, Raum MG, Van Wyke Coelingh K, Lebacq JA, Battey JF 1988 Posttranslational processing of endogenous and of baculovirus-expressed human gastrinreleasing peptide precursor. Mol. Cell Biol. 8:3129-3135 Pert CB, Schumacher UK 1982 Plasma bombesin concentrations in patients with extensive small cell carcinoma of the lung. Lancet 1:509 Sorenson GD, Bloom SR, Ghatei MA, Del Prete SA, Cate CC, Pettengill OS 1982 Bombesin production by human small cell carcinoma of the lung. Reg. Pept. 4:59-66 AV, Ghatei MA 1982 Bombesin: Bloom SR, Edwards an important component in neural control of insulin release. Diabetalogia 23:464-465 (abstract) Takeyama M, Kondo K, Takayama F, Kondo R. Murata H, Miyakawa I 1991 High concentration of a gastrin releasing peptide-like immunoreactive substance in pregnant human Biochem. & Biophys. Res. Comm. 176:931milk. 937 Jahnke GD, Lazarus LH 1984 A bombesin immunoreactive peptide in milk. Proc. Natl. Acad. Sci. US 81:578-582 Weber CJ, O'Dorisio TM, McDonald TJ, Howe B, Koschitzky T, Merriam L 1989 GRP-, CGRP- and calcitonin-like immunoreactivity in human breast cyst fluid; and GRP-like immunoreactivity in human breast carcinoma Surgery 106:1134-1140 cell lines. Ulisse S, Gnessi L, Fabbri A, Jannini EA, V, Di Luca Sidozzi A, Moretti C, Bonifacio Isidori A 1988 Bombesin-like Fraioli F, immunoreactivity in human seminal fluid. Ann. NY Acad. Sci. 547:452-454 Spindel ER, Zilberberg MD, Chin WW 1987 Analysis of the gene and multiple messenger ribonucleic acids (mRNAs) encoding human gastrin-releasing peptide: Alternate splicing occurs in neural and endocrine tissue. Mol Endocrinol. 1:224-232 Cuttita F, Fedorko J, Gu J, Lebacq-Verheyden A-M, Linnoila RI, Battey JF 1988 Gastrinreleasing peptide gene-associated peptides are expressed in normal human fetal lung and small cell lung cancer: a novel peptide family found Endocrinol Metab 67:576 in man. J Clin
Downloaded from https://academic.oup.com/endo/article-abstract/131/4/2033/3034799 by University of Otago Library user on 20 January 2019
DISCUSSION With the exception of the occasional patient with small cell carcinoma of the lung (9,lO) and in calf plasma following stimulation of the peripheral ends of splanchnic nerves (11). GRP has not been previously reported as present in significant concentrations in the mammalian peripheral circulation under physiological conditions (1). This studv. for the first time, reoorts the presence of significant concentrations of GRPLI in the circulation during ovine pregnancy. Fetal plasma levels were consistently higher than maternal levels but both were significantly lower than the GRPLI Previous levels present in the amniotic fluid. reports have documented the presence of GRPLI in human (12) and bovine (13) milk, in human breast cyst fluid (14) and human seminal fluid (15). However, this report of high GRPLI levels in plasma is, to the best of our knowledge, unique in the mammal and suggests the potential for an endocrine mode of action during ovine pregnancy. In all three fluids studied, the major GRPLI form eluted before the GRP1-27 standard, a finding consistent with the GRPLI being identical in the three fluids and apparently being of larger molecular size than GRP1-27. Recognition of this ovine GRPLI entity by the two C-terminally directed antisera suggests that the ovine molecule contains a region with a close or identical structure to the important bioactive C-terminal heptapeptide region of GRP. There are a number of possibilities. One obvious explanation for the apparent larger molecular size is that the ovine GRPLI entity could be a C-terminally extended GRP-like peptide analogous to the forms described in normal and neoplastic human tissue (16,17). However, the recognition of this ovine entity by the 2All antisera, which does not recognize C-terminally extended GRP forms, renders this explanation improbable. An alternative possibility is that mature ovine GRP is of larger size than other mammalian GRP molecules: the structure of ovine GRP is at present unknown. 'A further possibility is that the entity is a N-terminally extended form of Although the N-terminus of the mature human GRP. GRP molecule is contiguous with the signal sequence (15), the structure of the ovine GRP precursor molecule is unknown. Finally, it is possible but less probable, that the ovine GRPLI entity is an as yet unknown but structurally related peptide. Obviously, isolation and characterization of this entity is required to address the above speculations. Of interest, human breast milk GRPLI is also apparently larger than GRPl-27 (12). The nature of the fetal plasma GRPLI which occurs in the void volume peak is also unknown. Current studies in this laboratory are addressing the hypothesis that this entity may represent a GRP-like molecule linked to a specific binding protein. Of note, the GRPLI does not occur in appreciable amounts in the void volume peak of either maternal plasma or amniotic fluid. A number of questions arise from these results. Foremost is the question of the precise chemical structure of this circulating ovine GRPLI entity. Of equal importance is the elucidation of the sourcefs1 of the ovine GRPLI secreted into the circulation. The reasons for the presence and differential concentrations of GRPLI in each of the three fluids studied and the reasons for the large variation in fetal plasma GRPLI levels between animals need to be elucidated. Despite the unanswered nature of these and other fundamental questions, the presence of significant concentrations of GRPLI in ovine fetal Dlasma is consistent with a potentially unique roie for GRP in fetal life acting as an endocrine agent. Previous studies have emphasized GRP as playing a paracrinef autocrine/neurotransmitter role. Given GRP's ability to produce mitogenesis in certain fetal tissues and its demonstrated trophic effects on certain adult normal and neoplastic cells, it is possible that this GRPLI entity is a circulating qrowth factor of imoortance durino ovine fetal ljfe. ACKNOWLEDGMENTS These studies were supported by grants from the
GASTRIN
RF&EASING
Vol. 131, No. 4 Printed
PBPTIDE
IMMUNOREACTIVITy
IS PRESENT
IN
OVINE
AMNIOTIC
FLUID
AND FEW&
in V.S.A
AND MATERNAL
CIRCULATIONS
of
Ob/Gyn’,
University
Physiology" of Western
Medicine3, and Ontario, London,
ABSTRACT: Using antisera directed towards the C-terminal region of gastrin releasing peptide (GRP), significant quantities of GRP-like immunoreactivity (GRPLI) were detected in ovine amniotic fluid and in the fetal and maternal circulations. The highest GRPLI levels were found in amniotic fluid (2135 ? 829 fmol/ml, n=12; mean f SEW), followed by those in ovine fetal (604 f 267 fmol/ml, n=13) and maternal plasma (229 f 89 fmol/ml, n=13). On gel filtration chromatography, the predominant GRPLI form in each fluid eluted in an identical position consistent with the entity being of apparently larger molecular size than porcine GRPlw2,. Certain fetal plasma samples contained a second GRPLI peak eluting at the void volume. Hence, during ovine pregnancy a GRPLI entity circulates in fetal and maternal plasma; the entity is of apparently larger molecular size than GRP,-27 but contains a structure immunologically indistinguishable from the bioactive Cterminal region of GRPI.,7. Given the recoanized bioactivities of GRP, this entity may be an important hormone during ovine fetal life. dilutions of plasma and AF samples and the mean INTRODUCTION concentration in each sample calculated from the Initial studies on the bombesin-related resulting dose-inhibition curves. gastrin releasing peptide (GRP) emphasized its ability to affect memmalian endocrine and exocrine secretory functions (1) but it is now recognized that GRP also may be an important autocrine regulator of cell growth (2,3). Recent studies have demonstrated that GRP and bombesin have mitogenic properties in human and mouse fetal lung (4) and in Because the ovine fetal chondrocytes (5). mechanisms underlying the coordinated growth of the mammalian embryo sne of fundamental imnortance, and given the evidence that GRP is a potential growth factor in fetal life, we initiated studies to evaluate the actions of GRP in an ovine pregnancy model. This study reDorts the nnesence of GRP-like immunoreactivity (GRPLI) in rel&ively high concentrations in ovine fetal and maternal circulations and also in amniotic fluid (AF). I-L,
Materials
NATERIALS
AND
WETRODS
and Procedures Using pregnant ewes of mixed breed and known gestational age (term 145-150 days) and surgical procedures and post-operative management as described (6), polyvinyl catheters were implanted under general anaesthesia into 13 fetal sheep (99128 days gestation), either into the fetal carotid artery and jugular vein O+ into the fetal femoral artery and vein. An open-end catheter was placed were implanted into in the amniotic sac. Catheters the maternal femoral artery and vein. In twin nresnancies onlv one fetus was catheterized. Foliowing at least 5 days recovery from surgery, fetal (3 ml) and maternal (5 ml) blood and RF (5 ml)‘ssmpies were obtained daily between 0800-1200 h. Less than 10% of the estimated fetal blood volume was withdrawn at each sampling. The samples were drawn into heparinized syringes, transferred immediately into chilled polypropylene tubes and centrifuged at 1500 x g for 10 min at 4'C. Plasma and AF samples were stored at -7OOC until further analysis. All animal protocols were approved by University of Western Ontario Animal Care Committees in accordance with guidelines established by the Canadian Council on Animal Care.
Figure
1. RIA dose-response OVinS fetal at gestational ages: FPl-132 FP3-114 days, FP4-114 days,
repreSentatiVe
in
Iowa
City
office
June
16,
of GRPLI in five samples obtained days, FPZ-134 days, FP5-142 days.
n
10000
Immunochemical Analvses Plasma and AF were analyzed for GRPLI by a previously described radioimmunoassay procedure (7). Briefly, the LR-16 antiserum used in the RIA is directed towards the C-terminal GRP21-25 sequence, reacts eguipotently with bombesin and GRP but has less than 0.01% cross-reaction with substance P, NRA and neuromedin 8, and does not crnss-react with and -11 or any structurally acidic FGF, IGF-I unrelated gastroenteropancreatic peptide tested. The sensitivity of the assay is less than 0.625 fmol/tube with half-displacement less than 7.5 fmol/tube. RIA was performed on serial 1:l Received
curves
plaSma
AF
Animal
Figure 2. The mean f SEM of fetal and maternal plasma and amniotic fluid concentrations of GRPLI obtained from 13 and 12 animals respectively, between 106-146 days gestation. Closed bars, amniotic fluid; open bars, fetal plasma; hatched bars, maternal plasma.
92
2033
Downloaded from https://academic.oup.com/endo/article-abstract/131/4/2033/3034799 by University of Otago Library user on 20 January 2019
W. Fra~er’*~, A.M. Carter',', J.R.G. Challis',2 and T.J. McDonald3s4. MRC Group in Fetal and Neonatal Health and Development. Departments Biochemistry4, The Lawson Research Institute and University Hospital, Ontario, Canada N6A 5A5.
2034
RAPID
Endo. Vol131.No4
COMMUNICATIONS
Analvsis AF samples were not available from one animal. The overall mean GRPLI concentrations in fetal and maternal plasma samples and in amniotic fluids for individual animals were calculated from values obtained in successive samples, taken through gestation. Differences in mean GRPLI concentrations between AF and plasma samples within individual sheep were sought using one way ANOVA, Fisher PLSD and Scheffe F-test. All values are presented as mean f SEM.
Representative gel filtration profiles of the GRPLI in fetal and maternal olasma and in Av ilr~ --shown in Fig. 3. The Predornlnant-GRpLI-peak in each fluid eluted in an identical position which occurred earlier than that of the porcine GRP1-27 standard, suggesting an apparently larger molecular size for the GRPLI entity. Co-chromatography of fetal plasma GRPLI with added porcine GRP)-27, did not alter the elution positions of the two entities (data not shown). In certain fetal plasma samples, an additional peak of immunoreactivity occurred in the void volume (Fig.3A).
0
20
40
60
80
100
120
140
Fmctmn volume (ml)
(b)
Data
RESULTS Throughout gestation, all three fluids contained GRPLI which produced dose-inhibition curves parallel to that of the standard curve (data for fetal plasma GRPLI shown in Fig. 1). Boiling AF, to inactivate proteolytic enzymes, did not significantly alter the content nor the parallelism of the dose-inhibition curve (data not shown). The GRPLI levels in plasma and AF of 13 fetal sheep and their mothers (106-146 days gestation) demonstrated a considerable range of values (Fig. 2). The mean average concentration of GRPLI in the AF (2135 k 829 fmolfml; n=12) was 3.5 fold higher than that in fetal plasma (604 f 267 fmol/ml, n=13) and 9 fold higher than that in maternal plasma (229 f 89 fmol/ml, n=13); the mean AF concentration of GRPLI was significantly greater than that of fetal or maternal plasma (Fig. 2; pqO.01). There was no correlation of GRPLI levels in any fluid with gestational age, circulating fetal ACTH levels, blood pO2, blood pH, maternal feeding or time of sampling.
0
20
40
60
80
100
400
140
120
(C)
300
200
100
0 0
20
40
60
60
100
120
140
Fracnon volumr (ml) Figure 3. Gel filtration chromatography profiles of GRPLI in ovine fetal (a) and maternal (b) plasma and amniotic fluid (c) on Sephadex G-SO (SF) columns (1 x 116 cm) eluted with GRP RIA buffer at 4'C. GRPLI was detected by a C-terminally directed antiserum raised against synthetic GRP (see text for details). The elution positions of synthetic porcine GRP)-27 and blue (Vo) and 12'1 are '$'C'; of GRP18.27~ dextran
Downloaded from https://academic.oup.com/endo/article-abstract/131/4/2033/3034799 by University of Otago Library user on 20 January 2019
Aliquots of fetal and maternal plasma and AF were partially purified by loading onto pre-washed Sep-pak C-18 cartridges (Waters Associates, Toronto, washed with 0.1% (v/v) trifluoroacetic acid Ont.), (TFA; Pierce Chemical Company, Rockford, Ill.) in water and subsequently eluted with an acetonitrile (Fisher Chemical Company, Toronto, Ont.): TFA: water The GRPLI recovered in mixture (80:0.1:19.9;v/v). the eluate fractions was 67.4 f 14.7% (n=4; mean f SEM); the eluates were concentrated by evaporation of solvents under a stream of nitrogen. An affinity chromatography column was constructed by coupling the monoclonal bombesin antiserum, designated 2All (a kind gift of Dr. F. Cuttita, National Cancer Institute, Bethesda, Md.) to Affi-gel 10 using procedures suggested by the manufacturer (Bio-Rad, The characteristics of the 2All Mississauga, Ont.). antiserum have been previously described in detail the 2All antiserum is directed (8). Briefly, against the C-terminal region of GRP but does not cross-react with C-terminally extended forms (8). An aliquot of fetal plasma GRPLI, after partial purification on Sep-pak C-18 cartridges, was loaded the column washed with the onto the affinity column, and the GRPLI eluted from the equilibration buffer, The recovery of column with 0.4 M citric acid. fetal GRPLI loaded onto the affinity column was 88%. Gel filtration chromatography was performed on Sephadex G-50 superfine (Pharmacia, Montreal, P.Q.) and eluted at a columns, (1 x 116 cm), equilibrated constant flow of 6 ml/h with GRP RIA buffer (0.06M Na2HP04, 0.01 M EDTA, pH 8, containing 0.05% sodium aside (w/v) and 0.3% bovine serum albumin (w/v) at 4Oc. The void volumes (Vo) and salt peak elution volumes (V,) were defined by determining the elution Montreal, P-Q.) ;;~~;,K+ (D~e~syam~'yor~n~o~~n~*')~ The elution positions of porcine GRP)-27 and GRP1S.27 (Bachem, Torrance, Ca.) were determined by dissolving the peptides in GRP-free plasma (obtained by treating either adult canine plasma or ovine fetal plasma with excess activated charcoal) and monitoring the chromatographic elution patterns with RIA. Aliquots of either fetal or maternal plasma or AF with sufficient GRPLI (>500 fmol/ml) were loaded in 1 ml quantities onto the columnsg One ml fractions were collected and stored at -70 C until performance of RIA.
RAPID
COMMUNICATIONS
I
Medical Research Council of Canada to JRGC and The authors gratefully acknowledge the excellent technical assistance of Mrs. Gail Ward and the excellent secretarial assistance of Mrs. Gloria Kellett.
TJM.
REFERENCES
1
2
3
4
5
6
7
8
9 10
11
12
13 14
,
15
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17
McDonald TJ 1988 The Gastrin Releasing Polypeptide. In: Mutt V (ed) Adv. Wetabol. Disorders. San Diego, New York, Berkeley, Boston, London, Sydney, Tokyo, Toronto, vol 11:199-250 Zachary I, Wall PJ, Rozengurt E 1987 A role for neuropeptides in the control of cell proliferation. Developmental Biology 124:295308 Cuttita F, Carney DN, Mulshine JL 1985 Bombesin-like peptides can function as autocrine factors in human small cell lung Nature 316:823-826 cancer. Sunday ME, Hua J, Dai HB, Nusrat A, Torday JS 1990 Bombesin increases fetal lung growth and maturation in utero and in organ culture. Am J Respir Cell Mol Biol. 3:199-205 Hill DJ, McDonald TJ 1992 Mitogenic action of gastrin-releasing polypeptide on isolated epiphyseal growth plate chondrocytes from the ovine fetus. Endocrinology 130:2811-2819 Akagi K, Challis JRG 1990 Threshold of hormonal and biophysical responses to acute hypoxemia in fetal sheep at different gestational ages, Can. J. Physiol. Pharmacol. 68:549-555 McDonald TJ, Christofi FL, Brooks BD, Barnett W, Cook MA 1988 Characterization of content and chromatographic forms of neuropeptides in purified nerve varicosities prepared from guinea pig myenteric plexus. Reg Pep. 21:69-83 Lebacq-Verheyden A-M, Kasprzyk PG, Raum MG, Van Wyke Coelingh K, Lebacq JA, Battey JF 1988 Posttranslational processing of endogenous and of baculovirus-expressed human gastrinreleasing peptide precursor. Mol. Cell Biol. 8:3129-3135 Pert CB, Schumacher UK 1982 Plasma bombesin concentrations in patients with extensive small cell carcinoma of the lung. Lancet 1:509 Sorenson GD, Bloom SR, Ghatei MA, Del Prete SA, Cate CC, Pettengill OS 1982 Bombesin production by human small cell carcinoma of the lung. Reg. Pept. 4:59-66 AV, Ghatei MA 1982 Bombesin: Bloom SR, Edwards an important component in neural control of insulin release. Diabetalogia 23:464-465 (abstract) Takeyama M, Kondo K, Takayama F, Kondo R. Murata H, Miyakawa I 1991 High concentration of a gastrin releasing peptide-like immunoreactive substance in pregnant human Biochem. & Biophys. Res. Comm. 176:931milk. 937 Jahnke GD, Lazarus LH 1984 A bombesin immunoreactive peptide in milk. Proc. Natl. Acad. Sci. US 81:578-582 Weber CJ, O'Dorisio TM, McDonald TJ, Howe B, Koschitzky T, Merriam L 1989 GRP-, CGRP- and calcitonin-like immunoreactivity in human breast cyst fluid; and GRP-like immunoreactivity in human breast carcinoma Surgery 106:1134-1140 cell lines. Ulisse S, Gnessi L, Fabbri A, Jannini EA, V, Di Luca Sidozzi A, Moretti C, Bonifacio Isidori A 1988 Bombesin-like Fraioli F, immunoreactivity in human seminal fluid. Ann. NY Acad. Sci. 547:452-454 Spindel ER, Zilberberg MD, Chin WW 1987 Analysis of the gene and multiple messenger ribonucleic acids (mRNAs) encoding human gastrin-releasing peptide: Alternate splicing occurs in neural and endocrine tissue. Mol Endocrinol. 1:224-232 Cuttita F, Fedorko J, Gu J, Lebacq-Verheyden A-M, Linnoila RI, Battey JF 1988 Gastrinreleasing peptide gene-associated peptides are expressed in normal human fetal lung and small cell lung cancer: a novel peptide family found Endocrinol Metab 67:576 in man. J Clin
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DISCUSSION With the exception of the occasional patient with small cell carcinoma of the lung (9,lO) and in calf plasma following stimulation of the peripheral ends of splanchnic nerves (11). GRP has not been previously reported as present in significant concentrations in the mammalian peripheral circulation under physiological conditions (1). This studv. for the first time, reoorts the presence of significant concentrations of GRPLI in the circulation during ovine pregnancy. Fetal plasma levels were consistently higher than maternal levels but both were significantly lower than the GRPLI Previous levels present in the amniotic fluid. reports have documented the presence of GRPLI in human (12) and bovine (13) milk, in human breast cyst fluid (14) and human seminal fluid (15). However, this report of high GRPLI levels in plasma is, to the best of our knowledge, unique in the mammal and suggests the potential for an endocrine mode of action during ovine pregnancy. In all three fluids studied, the major GRPLI form eluted before the GRP1-27 standard, a finding consistent with the GRPLI being identical in the three fluids and apparently being of larger molecular size than GRP1-27. Recognition of this ovine GRPLI entity by the two C-terminally directed antisera suggests that the ovine molecule contains a region with a close or identical structure to the important bioactive C-terminal heptapeptide region of GRP. There are a number of possibilities. One obvious explanation for the apparent larger molecular size is that the ovine GRPLI entity could be a C-terminally extended GRP-like peptide analogous to the forms described in normal and neoplastic human tissue (16,17). However, the recognition of this ovine entity by the 2All antisera, which does not recognize C-terminally extended GRP forms, renders this explanation improbable. An alternative possibility is that mature ovine GRP is of larger size than other mammalian GRP molecules: the structure of ovine GRP is at present unknown. 'A further possibility is that the entity is a N-terminally extended form of Although the N-terminus of the mature human GRP. GRP molecule is contiguous with the signal sequence (15), the structure of the ovine GRP precursor molecule is unknown. Finally, it is possible but less probable, that the ovine GRPLI entity is an as yet unknown but structurally related peptide. Obviously, isolation and characterization of this entity is required to address the above speculations. Of interest, human breast milk GRPLI is also apparently larger than GRPl-27 (12). The nature of the fetal plasma GRPLI which occurs in the void volume peak is also unknown. Current studies in this laboratory are addressing the hypothesis that this entity may represent a GRP-like molecule linked to a specific binding protein. Of note, the GRPLI does not occur in appreciable amounts in the void volume peak of either maternal plasma or amniotic fluid. A number of questions arise from these results. Foremost is the question of the precise chemical structure of this circulating ovine GRPLI entity. Of equal importance is the elucidation of the sourcefs1 of the ovine GRPLI secreted into the circulation. The reasons for the presence and differential concentrations of GRPLI in each of the three fluids studied and the reasons for the large variation in fetal plasma GRPLI levels between animals need to be elucidated. Despite the unanswered nature of these and other fundamental questions, the presence of significant concentrations of GRPLI in ovine fetal Dlasma is consistent with a potentially unique roie for GRP in fetal life acting as an endocrine agent. Previous studies have emphasized GRP as playing a paracrinef autocrine/neurotransmitter role. Given GRP's ability to produce mitogenesis in certain fetal tissues and its demonstrated trophic effects on certain adult normal and neoplastic cells, it is possible that this GRPLI entity is a circulating qrowth factor of imoortance durino ovine fetal ljfe. ACKNOWLEDGMENTS These studies were supported by grants from the
