Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS): a new autosomal dominant syndrome.

OBJECTIVE: The purpose of this study was the clinical and pathological characterisation of a new autosomal dominant gastric polyposis syndrome, gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS). METHODS: Case series were examined, documenting GAPPS in three families from Australia, the USA and Canada. The affected families were identified through referral to centralised clinical genetics centres. RESULTS: The report identifies the clinical and pathological features of this syndrome, including the predominant dysplastic fundic gland polyp histology, the exclusive involvement of the gastric body and fundus, the apparent inverse association with current Helicobacter pylori infection and the autosomal dominant mode of inheritance. CONCLUSIONS: GAPPS is a unique gastric polyposis syndrome with a significant risk of gastric adenocarcinoma. It is characterised by the autosomal dominant transmission of fundic gland polyposis, including areas of dysplasia or intestinal-type gastric adenocarcinoma, restricted to the proximal stomach, and with no evidence of colorectal or duodenal polyposis or other heritable gastrointestinal cancer syndromes.

Brain neuropeptide Y and CCK and peripheral adipokine receptors: temporal response in obesity induced by palatable diet.

OBJECTIVE: Palatable food disrupts normal appetite regulation, which may contribute to the etiology of obesity. Neuropeptide Y (NPY) and cholecystokinin play critical roles in the regulation of food intake and energy homeostasis, while adiponectin and carnitine palmitoyltransferase (CPT) are important for insulin sensitivity and fatty acid oxidation. This study examined the impact of short- and long-term consumption of palatable high-fat diet (HFD) on these critical metabolic regulators. METHODS: Male C57BL/6 mice were exposed to laboratory chow (12% fat), or cafeteria-style palatable HFD (32% fat) for 2 or 10 weeks. Body weight and food intake were monitored throughout. Plasma leptin, hypothalamic NPY and cholecystokinin, and mRNA expression of leptin, adiponectin, their receptors and CPT-1, in fat and muscles were measured. RESULTS: Caloric intake of the palatable HFD group was 2-3 times greater than control, resulting in a 37% higher body weight. Fat mass was already increased at 2 weeks; plasma leptin concentrations were 2.4 and 9 times higher than control at 2 and 10 weeks, respectively. Plasma adiponectin was increased at 10 weeks. Muscle adiponectin receptor 1 was increased at 2 weeks, while CPT-1 mRNA was markedly upregulated by HFD at both time points. Hypothalamic NPY and cholecystokinin content were significantly decreased at 10 weeks. CONCLUSION: Palatable HFD induced hyperphagia, fat accumulation, increased adiponectin, leptin and muscle fatty acid oxidation, and reduced hypothalamic NPY and cholecystokinin. Our data suggest that the adaptive changes in hypothalamic NPY and muscle fatty acid oxidation are insufficient to reverse the progress of obesity and metabolic consequences induced by a palatable HFD.

The pregnant ovine endometrium constitutively expresses and secretes a highly stable bombesin-like peptide, which shares C-terminal sequence but differs structurally from gastrin-releasing peptide.

Previous studies have shown that a peptide closely related to gastrin releasing peptide (GRP) is expressed by the pregnant ovine endometrium throughout gestation, however its molecular form and the mode of its secretion have not been defined. We have partially purified the endometrial GRP-like peptide and characterised it chromatographically. In contrast to other tissues, the main molecular form of endometrial GRP is larger (6-8 kDa versus 1-3 kDa), and based on the increased hydrophobicity of its circulating form after reduction, contains at least one disulfide bond. Reduction and treatment with chaotropic agents showed that the protein is not a cleavage product of pro-GRP bound to a binding protein. Tryptic cleavage demonstrated that the C-terminus of the peptide is closely related to GRP18-27 suggesting that bioactivity is likely. The partially purified peptide remained intact after incubation in ovine plasma for 16 h indicating that it is extremely stable and consistent with an hormonal role during pregnancy. Quantification of peptide from monolayer cultures of ovine endometrial cells showed that the GRP-like peptide was secreted constitutively. These data show that a stable, GRP-like peptide, distinct from the known processing products of pre-pro-GRP is constitutively expressed by the gravid ovine endometrium. Since endometrial GRP has an intact bioactive C-terminus and is mitogenic for numerous tissues including the uterus, then it is likely to play an important regulatory role in ovine pregnancy.

Concerning the independence of the basis of hypertension due to ACTH or renovascular constriction.

Administration of ACTH to four sheep with chronic renovascular hypertension resulted in an increase in blood pressure which was at least as high as that described in normotensive sheep and could be completely accounted for by an increase in cardiac output.

Overexpression of sense or antisense human gastrin mRNA does not affect proliferation of normal rat kidney fibroblasts.

Progastrin-derived peptides have been reported to stimulate mitogenesis in Swiss 3T3 fibroblasts [P. Singh, A. Owlia, R. Espeijo, B. Dai, Novel gastrin receptors mediate mitogenic effects of gastrin and processing intermediates of gastrin on Swiss 3T3 fibroblasts: Absence of detectable cholecystokinin (CCK)-A and CCK-B receptors. J. Biol. Chem. 270 (1995) 8429-8438]. The aim of the present study was to determine the generality of these findings, by investigating the effect of endogenous and exogenous progastrin-derived peptides on the proliferation of the normal rat kidney fibroblast cell line NRK. Levels of endogenous progastrin-derived peptides were modified by stable transfection of NRK cells with tetracycline-repressible plasmids containing sequences encoding human gastrin in either the sense or antisense orientation. Expression of sense and antisense gastrin mRNA was demonstrated by reverse transcriptase PCR and by radioimmunoassay, and cell proliferation rates were determined by the colorimetric MTT assay. Sense clones produced full length human progastrin, but significant quantities of glycine-extended or amidated gastrin17 were not detected. Concentrations of endogenous rat progastrin in antisense clones were significantly lower than concentrations in clones transfected with vector only. However no difference in proliferation rate was observed between sense, antisense and vector-transfected clones. No stimulation of proliferation was observed in synchronised untransfected NRK cells after supplementation of media with gastrin17 or gastrin17gly in the concentration range 0.3 to 100 nM. Our results do not provide evidence in support of the hypothesis that endogenous or exogenous progastrin-derived peptides act as growth factors in NRK fibroblasts.

Identification of a 70-kDa gastrin-binding protein on DLD-1 human colorectal carcinoma cells.

Gastrin17gly acts as a growth factor for the colonic mucosa. Studies of the receptor involved have generally been restricted to its binding properties, and no investigation of the structure of gastrin17gly receptors on human colorectal carcinoma cell lines has yet been reported. The aim of this study was to optimise the conditions for binding of gastrin17gly to the human colorectal carcinoma cell line DLD-1, and to investigate the structure of the receptor responsible. Binding of 125I[Met15]gastrin17gly to DLD-1 cells was measured in competition experiments with increasing concentrations of either gastrin17gly or gastrin17, or with single concentrations of gastrin receptor antagonists. The molecular weights of the gastrin17gly binding proteins were determined by gel electrophoresis and autoradiography after covalent cross-linking of 125I[Nle15]gastrin2,17gly to cells or membranes with disuccinimidyl suberate. The IC50 value for binding of gastrin17gly to DLD-1 cells was 2.1+/-0.4 microM. Binding was inhibited by the non-selective gastrin/cholecystokinin receptor antagonists proglumide and benzotript, but not by the cholecystokinin-A receptor antagonist L364,718, or the gastrin/cholecystokinin-B receptor antagonist L365,260. The molecular weight of the major gastrin binding protein on DLD-1 cells or membranes was 70,000. We conclude that the major gastrin17gly binding site on the human colorectal carcinoma cell line DLD-1 is clearly distinct from the cholecystokinin-A and gastrin/cholecystokinin-B receptors, but is similar in some respects to the gastrin/cholecystokinin-C receptor.

Regulation of somatostatin secretion by gastrin- and acid-dependent mechanisms.

The regulation of gastric somatostatin is linked to changes in gastric acidity, with a number of studies showing a good correlation between somatostatin secretion and gastrin-stimulated luminal acidity. However, gastrin may also have direct effects on somatostatin secretion independent of the concurrent acid status. We have examined the relative contribution of gastrin itself vs. gastric acid on the increase in somatostatin secretion observed after gastrin administration. Pentagastrin administered to conscious sheep for 2 h caused a 10- to 12-fold increase in both portal venous and peripheral jugular venous plasma somatostatin levels. This was associated with a decrease in gastric pH from 3.5 to 1.7. When the sheep were pretreated with the proton pump inhibitor omeprazole to prevent any change in gastric acidity, pentagastrin caused a similar increase in plasma somatostatin. The increase in somatostatin could also be produced by gastrin-17 infusions. Thus, these studies demonstrate that in the conscious animal gastrin can stimulate somatostatin independent of changes in gastric acidity. It is proposed that there is a negative feedback between somatostatin and gastrin, which may modulate the acid secretory response to gastrin.

A peptide related to gastrin releasing peptide is synthesised and secreted by the ovine endometrium in early pregnancy.

We have previously shown that the peptide immunoreactivity related closely to the mitogen GRP is expressed by the pregnant ovine endometrium during the final third of pregnancy. In this study we have established that GRP is also expressed early in ovine pregnancy and have quantified the temporal changes in synthesis, storage and secretion of GRP in the peri- and post-attachment period. Secreted GRP peptide levels rise 10 fold just prior to implantation, while endometrial peptide and mRNA concentrations increase 4 and 13 fold respectively between day 17 and 20, immediately following attachment and corresponding to the onset of placentome development. The main molecular form of endometrial GRP has similar binding characteristics on RP-HPLC to GRP 18-27, but is larger. We conclude that a GRP-like peptide is expressed by the pregnant ovine endometrium from early in pregnancy until term, and that it is likely to play an important role in fetal or uterine maturation.

Ontogeny of gastrin, somatostatin, and the H+/K(+)-ATPase in the ovine fetus.

In the term human and ovine fetus, plasma gastrin is elevated, but gastric acid secretion is below adult levels, suggesting a developmentally related immaturity in gastrin and gastric acid regulation. This study investigated a number of elements of the gastric acid regulatory system: gastrin and its glycine-extended precursor, somatostatin, and the H+/K(+)-ATPase. Measurements were made in blood, antrum, and fundus of the ovine fetus during the last half of gestation, of 15-day-old lambs, and of adult sheep at the level of mRNA synthesis, tissue storage, and secretion. Plasma amidated gastrin (gastrin-amide) was elevated at or above adult values from 125 days (term is 145 days) and steadily increased with development, peaking in the lamb. Similar changes occurred with plasma glycine-extended gastrin (gastrin-gly). The peak concentration of antral gastrin-amide was present in the lamb, while the maximum antral gastrin-gly level occurred 1 week before birth. Gastrin mRNA paralleled the changes in antral gastrin-gly. The proportion of higher mol wt species of gastrin decreased during gestation in both plasma and antrum. Low amounts of mRNA for the H+/K(+)-ATPase was present from at least 120 days of gestation and antedated gastric acid secretion. However, there was a 3-fold increase in H+/K(+)-ATPase mRNA from the 140-day-old fetus to the lamb, the period when the greatest reduction in gastric pH occurred (pH 5 to 2). Antral and fundic somatostatin increased rapidly in the fetus at 120 days gestation and were above adult values at term and in the lamb. Somatostatin mRNA changed in parallel to somatostatin peptide. Somatostatin-14 was the major species in antrum and fundus throughout development. The increase in circulating and antral gastrin-amide after birth may be the result of increased amidation of gastrin-gly as well as increased expression of gastrin mRNA. Amidation of gastrin may be a regulatory step in the production of biologically active gastrin during development. The major increase in gastrin and the H+/K(+)-ATPase that occurs in the week before and after gestation correlated with the onset of increased gastric acidity.

Hepatic metabolism of neurotensin.

Neurotensin is released from the intestinal mucosa into the portal circulation and, to exert a systemic effect, it must traverse the liver intact. We examined the potential role of the liver in neurotensin clearance using the isolated perfused rat liver model. With N-terminal and C-terminal directed RIAs and HPLC, we demonstrated rapid metabolism of intact neurotensin to inactive N-terminal fragments in the isolated rat liver system. The disappearance half-lives of C-terminal and N-terminal immunoreactivity were 20.4 +/- 6.0 min and 82.7 +/- 7.7 min, respectively, (P less than 0.002). To assess whether this neurotensin disappearance might be due to metabolism within the perfusate itself by a peptidase released from liver, we further incubated neurotensin in perfusate previously circulated through liver. A rapid and progressive breakdown of intact neurotensin to N-terminal fragments was again shown. These data demonstrate that a substantial proportion of the hepatic clearance of neurotensin is attributable to release of a peptidase by the liver into the circulation.

Developmental regulation of gastric somatostatin secretion in the sheep.

Gastric somatostatin (SRIF) regulates gastric acidity by inhibiting gastric acid and gastrin secretion. SRIF secretion is increased by gastric acidity and also directly by regulators of gastric acid secretion such as gastrin. This direct effect has not been described in the developing animal, nor have the roles of intermediaries such as histamine and gastric acidity been defined. The present study aimed to establish the regulatory role of gastrin and histamine during development on SRIF secretion and also to determine whether the effects of gastrin and histamine are independent of gastric pH. Pentagastrin and histamine were infused on separate occasions into fetal sheep, newborn lambs, and 28-day-old lambs. To determine the roles of endogenous histamine and gastric pH, ranitidine (a histamine-2 receptor antagonist) and omeprazole (a H+/K+ ATPase inhibitor) were coinfused with the agonists. Plasma SRIF and gastrin concentrations were measured by RIA. Pentagastrin stimulated SRIF secretion in the fetus after 131 days of gestation (term is 147 days), whereas stimulation by histamine was effective only after birth. The SRIF stimulatory effect of pentagastrin in 28-day-old lambs was abolished by ranitidine, which also reduced this effect in the adult sheep. This inhibitory effect of ranitidine was shown to be a result of blockade of stimulatory H2 receptors, because in the adult blockade of acid secretion with omeprazole failed to attenuate the response of histamine. These results indicate that in the fetus, gastrin receptors, but not histamine receptors, are functionally involved in the stimulation of SRIF secretion. After birth, both gastrin and histamine stimulate SRIF, but the effect of gastrin is mediated at least in part by the release of endogenous histamine. These responses occur independently of changes in gastric acidity, supporting the concept of a direct negative feedback between SRIF and gastrin.

The role of ACTH and adrenal glucocorticoids in the salt appetite of wild rabbits (Oryctolagus cuniculus (L)).

The selective appetites of wild rabbits for 500 mEq/1 solutions of NaC1, KC1, MgC1(2), and CaC1(2) were studied in intact and adrenalectomized rabbits during daily treatment with either 4 IU long acting ACTH, 1.0 or 2.5 mg cortisol acetate, or 2.5 mg corticosterone. The animals were individually caged and external sodium balances performed. In intact rabbits, cortisol or corticosterone produced a significant stimulation of NaC1 appetite. The response to concurrent dosage of cortisol and corticosterone was less than half of that obtained with ACTH which produced a comparable alteration of blood glucocorticoid levels but a 10-fold increase in NaC1 intake. CaC1(2) intake was increased in intact rabbits by cortisol treatment but not by corticosterone or ACTH. Adrenalectomized rabbits maintained on daily steroid replacement therapy of 0.1 mg deoxycorticosterone acetate and 0.75 mg cortisone acetate showed a normal pattern of electolyte, food, and water intake. Under these conditions ACTH produced a 4-fold increase in NaC1 intake. Further addition of cortisol and corticosterone to steroid replacement therapy produced an increase in NaC1 intake comparable to their effect on normal rabbits. Thereupon supplementation with ACTH resulted in an increase to a level at least as great as that found in ACTH treated, normal rabbits. The effects of ACTH and glucocorticoids on NaC1 appetite were synergistic. Sodium balance showed that increases in NaC1 intake were not the result of the treatment initially producing a body sodium deficit, which was then corrected by increased intake. The results provide further evidence for the hypothesis that NaC1 appetite may be hormonally regulated, and demonstrate that ACTH is capable of stimulating NaC1 intake by a previously unsuspected non-adrenal pathway.

Expression of gastrin-releasing peptide (GRP) and GRP receptors in the pregnant human uterus at term.

Synthesis of both gastrin-releasing peptide (GRP) and messenger ribonucleic acid (mRNA) has been demonstrated in pregnant sheep, but studies in women have not been reported. Therefore, we examined the uterus and placenta of pregnant women at term for synthesis of GRP and expression of GRP receptor genes BRS-3 and GRP-R. A transcript of 0.95 kilobases, corresponding to GRP mRNA, was detected in endometrium and myometrium, but not in amnion, chorion, placenta, or nonpregnant endometrium. GRP immunoreactivity (GRP-ir) was detected in half (three of six) of the endometrial (1.23 +/- 0.04 pmol/g) and myometrial (0.73 +/- 0.04 pmol/g) samples and in some, but not all, samples of amnion (one of four subjects; 0.6 pmol/g), chorion (four of five subjects; 0.8 +/- 0.2 pmol/g), placenta (two of six subjects; 0.5 +/- 0.2 pmol/g), and amniotic fluid (four of six subjects; 59 +/- 19 fmol/mL). GRP-ir was present in the maternal circulation (44 +/- 12 fmol/mL) and was higher in plasma obtained from the umbilical artery (152 +/- 14 fmol/mL) and vein (143 +/- 24 fmol/mL). The major peak of GRP-ir in pregnant endometrial tissue was larger than GRP-(1-27), as determined by gel filtration chromatography. Minor peaks were also observed: two larger than the main form and one corresponding to GRP-(18-27). mRNA for GRP receptors GRP-R and BRS-3 was detected by semiquantitative reverse transcription-PCR. For both receptors, mRNA was higher in the pregnant endometrium than in the nonpregnant endometrium but was detected in all of the uteroplacental tissues examined. GRP-R mRNA predominated in the pregnant endometrium, whereas BRS-3 mRNA predominated in the membranes and placenta. In these tissues, PCR for BRS-3 mRNA gave rise to an additional product (approximately 50 bp larger). These studies demonstrated that a peptide larger than, but related to, GRP is synthesized in the pregnant human uterus and is secreted into the maternal and fetal circulations. The detection of mRNA for GRP-R, BRS-3, and possibly a transcript variant of BRS-3 as well as the detection of a peptide larger than, but related to, GRP suggest a novel regulatory unit in the human reproductive tract.

Characterization of a pancreatic tumor containing vasoactive intestinal peptide, neurotensin, and pancreatic polypeptide.

The biochemical characteristics of a pancreatic tumor from a patient with watery diarrhea, hypokalemia, hypochlorhydria, and steatorrhea is described Plasma concentrations of vasoactive intestinal peptide (VIP) were elevated 6-fold, those of neurotensin (NT) were elevated 10-fold, and those of pancreatic polypeptide (PP) were elevated 200-fold above the normal range. The pancreatic tumor removed was found to contain high concentrations of these peptides in a similar ratio to plasma. The tumor content of NT was 6 times higher than any previously reported, but no specific symptoms could be ascribed to NT. After removal of the tumor, plasma levels of VIP, NT, and PP returned to normal, and the diarrhea disappeared. Gel chromatography of plasma and tumor extracts indicated that all of the immunoreactive PP co-eluted with standard human PP. When the tumor extract was subjected to gel chromatography and high pressure liquid chromatography, two forms of VIP were detected, the major one resembling porcine VIP and a smaller more hydrophobic form detected by C- but not N-terminally directed VIP antisera. This smaller form was not present in normal ileum. Immunoreactive NT in plasma was predominantly an N-terminal fragment. High pressure liquid chromatography of the tumor extract revealed that approximately 75% of the NT immunoreactivity consisted of N-terminal fragments. In contrast, normal ileum contained only authentic NT-(1-13). Since N-terminal fragments of NT are thought to be biologically inactive, the nature of the immunoreactive NT should be determined before attempting to assign specific clinical symptoms to NT-secreting tumors.

Expression of neurotensin in endocrine tumors.

Endocrine tumors are useful sources for determining the synthesis and metabolism of secreted regulatory peptides. The present study was performed to compare the synthesis and metabolism of neurotensin (NT) in normal subjects and four patients with NT-producing tumors. NT mRNA was measured and characterized using oligonucleotide probes and Northern blots, while NT-like peptides were quantitated by RIA with region-specific antisera and high pressure liquid chromatography. Northern blot analysis of mRNA isolated from normal human ileum revealed two species of mRNA hybridizing to a heterologous canine oligonucleotide probe; the apparent sizes of the mRNA were 1.4 and 1.0 kilobases. An identical pattern was found in a pancreatic endocrine tumor, a prostatic adenocarcinoma, and a fibrolamellar hepatoma. The ratio of mRNA to peptide varied between the different tissues. For instance, the hepatoma was the richest source of NT mRNA, but the prostatic tumor contained the highest peptide concentration. Measurements with region-specific antisera showed that N-terminal immunoreactive fragments were more abundant than C-terminal fragments in pancreatic, prostatic, and carcinoid tumors (N/C-teminal ratios, 4.0, 1.6, and 5.0) and in equal concentrations in normal ileum. Reverse phase high pressure liquid chromatography revealed the presence of intact NT in addition to a variable number of smaller N-terminal peptides, presumed to be metabolites. In contrast the hepatoma contained a 5-fold excess of C-terminal immunoreactivity. The excess C-terminal immunoreactivity was also present in the circulation of this patient. The chromatographic properties, immunoreactivity, and unusual stability of the C-terminal fragment found in the hepatoma patient suggest that it is a substance distinct from NT itself and is released specifically by the fibrolamellar hepatoma.

Gastrin processing and secretion in patients with end-stage renal failure.

Gastrin circulates at higher than normal concentrations in patients with end-stage renal failure (ESRF). However, it remains unclear which forms of gastrin are elevated and whether there is also an alteration in the secretory profile after stimulation. In the present study all processed and partially processed forms of circulating gastrin were measured in plasma before and after meal stimulation in ESRF patients and control subjects. Since Helicobacter pylori (HP) infection affects gastrin secretion, HP status was determined. Fasting gastrin-amide (36 +/- 8 pmol/L), gastrin-Gly (55 +/- 16 pmol/L), and total gastrin (218 +/- 32 pmol/L) measured in ESRF/HP-patients were all significantly greater than those in the control group (10 +/- 1, 15 +/- 3, and 17 +/- 2 pmol/L, respectively; P < 0.01). Plasma gastrin-amide (126 +/- 67 pmol/L) and total gastrin (397 +/- 164 pmol/L) were highest in the ESRF/HP+ patients. The proportion of nonamidated gastrin products was 4-fold higher in ESRF patients than in control subjects, suggesting structure-specific changes in gastrin secretion and metabolism, and this was confirmed by chromatography. The meal-stimulated increments in control/HP- and ESRF/HP-groups were similar. However, the ESRF/HP+ group had a markedly potentiated gastrin response. Fasting plasma somatostatin, an inhibitor of gastrin secretion, was also measured and was significantly lower in the ESRF patients than that in the control group. These studies show that the hypergastrinemia associated with renal failure has been underestimated. This is because only amidated products were measured. The potentiated gastrin meal response in ESRF attributed previously to changes in gastrin metabolism are in part explained by the effect of HP infection. The observed diminished somatostatin response suggests that the increase in circulating gastrin in ESRF is the result of loss of inhibition of secretion as well as decreased metabolism. As both amidated and nonamidated gastrin are now considered to have trophic and secretory effects, these findings may explain the gastrointestinal tract hypertrophy often associated with ESRF.

Metabolism of neurotensin and pancreatic polypeptide in man: role of the kidney and plasma factors.

Neurotensin (NT) is a 13 amino acid peptide found predominantly in the ileum and it is released into the circulation by a meal. Much of the circulating NT consists of N terminal fragments which have no known biological activity. However, the sites and rates of NT metabolism are not known. In the present study the MCR and half-disappearance time of NT were estimated by infusing NT(1-13) into 10 normal subjects. The role of the kidney was assessed by studies in patients with chronic renal failure (CRF). The nature of the metabolites was characterized using region specific antisera and high pressure liquid chromatography (HPLC). The plasma pancreatic polypeptide response to the NT infusion was also measured. The NT MCR in normal subjects was 88 +/- 25 (SEM) ml/min X kg measured with the C terminal antiserum but only 9.9 +/- 0.8 ml/min X kg measured with the N terminal antiserum, a result consistent with the presence of long lasting N terminal fragments. HPLC of the plasma at equilibrium established that only 20% of the immunoreactivity was present as NT(1-13), with the majority as NT(1-8). No C terminal fragment were detected. Similarly, incubation of NT(1-13), 1-8, and 8-13 in plasma in vitro showed that N terminal fragments were stable in plasma, whereas C terminal fragments were completely metabolized. In patients with CRF, basal plasma NT (measured with the C terminal antisera) was significantly elevated and C terminal MCR was reduced by 82% and N terminal MCR by 32%. Thus the major effect of CRF was on the initial degradation of NT(1-13) to N terminal fragments. HPLC showed that over 60% of the NT was present as NT(1-13). In vitro degradation of NT was also slowed in CRF plasma. The increased proportion of intact biologically active NT in the circulation of the CRF patients could also explain the greater increase in pancreatic polypeptide levels during the NT(1-13) infusion. These studies have established that the metabolism of NT is influenced by the kidney and that the presence of predominantly N terminal fragments of NT in the peripheral circulation of normal subjects can be explained by a combination of renal and extrarenal factors.

Molecular cloning, genomic organization and selective expression of bombesin receptor subtype 3 in the sheep hypothalamus and pituitary.

The bombesin receptor subtype 3 (BRS-3) is considered an orphan receptor as it has a low affinity for bombesin-like peptides and no identified natural ligand. We have reported a novel form of gastrin-releasing peptide (GRP) present in high abundance in the pregnant uterus of women and sheep. As BRS-3 was originally cloned from guinea pig uterus, we postulated that the uterine GRP-like peptide may be its natural ligand. We have therefore cloned the gene for the sheep homologue of BRS-3 and determined its distribution. The sheep BRS-3 gene spans 4 kbp and comprises three exons with intron-exon borders at positions similar to those observed for the human and mouse BRS-3 genes. The predicted amino acid sequence of ovine BRS-3 has approximately 85% identity with the human, mouse and guinea pig receptors. Highly conserved amino acids important in mediating receptor G-protein coupling to second messengers and important in ligand binding were found to be conserved in ovine BRS-3. One potentially important deviation was noted: ovine BRS-3 possesses an arginine residue at position 294 instead of a histidine residue as found in all other BRS-3. His(294) was previously identified as important in ligand-receptor interactions while Arg(294) was implicated in high ligand affinity. Thus ovine BRS-3 may have binding characteristics different from those of the human, mouse and guinea pig BRS-3 receptors. In the ewe, BRS-3 mRNA expression was detected in pituitary and hypothalamus but not in tissues of the pregnant uterus (endometrium, myometrium, chorioallantois or amnion). Nor was BRS-3 expression detected in the non-pregnant uterus or in testis. This pattern of BRS-3 expression is similar to that observed in the mouse but different from that observed in the human, rat and guinea pig. We conclude that there is no local interaction between uterine GRP-like peptide and BRS-3. However, the high expression of BRS-3 in the pituitary coupled with elevated circulating levels of this GRP-like peptide during pregnancy suggests an alternate pathway. Cloning of the ovine BRS-3 gene will permit a detailed functional analysis of this receptor in the sheep and its role in the mediation of action of uterine GRP.

Regional brain concentrations of cholecystokinin in the rat: the effects of kindled and non-kindled seizures.

In an attempt to understand the neurochemical basis of kindling, this study investigated the effects on brain cholecystokinin (CCK) of amygdaloid kindled and non-kindled seizures. Thirteen brain regions were examined in rats sacrificed either 24 hr or 3 weeks after the last kindled seizure, or 24 hr after a suprathreshold stimulation-induced (non-kindled) seizure; and in sham kindled rats. There were no significant differences in CCK immunoreactivity between any of these groups. These results do not confirm a previous report of an increase in CCK in the hippocampus following amygdaloid kindling in the rat.

Regional brain concentrations of calcitonin gene-related peptide in spontaneously hypertensive and normotensive Wistar-Kyoto rats.

The regional brain and spinal cord concentrations of calcitonin gene-related peptide (CGRP) were measured in age-matched (22-23-week-old) spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. The highest concentration of CGRP in the WKY rats was in the spinal cord (172 +/- 9 pmol/g), followed by the medulla oblongata/pons (88 +/- 5 pmol/g). The relative order of distribution in the remaining regions was: hypothalamus (12.6 +/- 0.8 pmol/g) = striatum greater than thalamus greater than midbrain = hippocampus greater than cortex (2.1 +/- 0.3 pmol/g). The concentration of CGRP in the cerebellum was at the level of the assay's sensitivity (0.5 pmol/g). The relative order of distribution in the SHR strain was essentially the same. However, in comparison with the WKY rats, the SHR had significantly lower levels of CGRP in the hippocampus (-47%), striatum (-49%) and medulla oblongata/pons (-24%), and in the spinal cord (-24%). In younger age-matched (16-17-week-old) rats, the spinal cord and medulla oblongata/pons concentrations of CGRP were also lower in SHR than in WKY rats. CGRP is a putative neurotransmitter which, when administered centrally or peripherally, has potent cardiovascular effects. The reduced levels of this peptide may be an important factor in the cardiovascular and/or behavioural abnormalities of the SHR strain.