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Gastrin, acting via the cholecystokinin-2 receptor (CCK2R), activates its own promoter in a positive-feed-forward loop that may result in hypergastrinemia. Activity of the gastrin promoter is also stimulated by exogenous Zn 2+ ions. Here, the role of intracellular zinc and calcium signaling in the gastrin positive-feed-forward loop was investigated. Gastrin promoter activity was measured in the human gastric carcinoma cell line AGS-CCK2R and in Jurkat cells transfected with various gastrin promoter-luciferase constructs after treatment with gastrin in the presence and absence of zinc- and calcium-chelating agents. The free intracellular zinc ion concentrations were measured in the same cells with the fluorescent indicator FluoZin-3. Cell proliferation and migration/invasion were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide cell proliferation assay and in Boyden chamber assays, respectively. The zinc chelator N,N,N,N-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) abolished gastrin-stimulated gastrin promoter activity, and the inhibition was completely reversed by exogenous Zn 2+ ions. In contrast, the calcium chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) potentiated gastrin-stimulated gastrin promoter activity. Treatment with gastrin increased the intracellular concentration of free Zn 2+ ions, and the increase was blocked by TPEN, but not by BAPTA-AM. TPEN also inhibited the stimulation of cell proliferation and migration/invasion by gastrin, but BAPTA-AM had no effect. These results, which are the first report of the existence of Zn 2+ signaling downstream of CCK2R activation, suggest that zinc chelation therapies may be effective in counteracting gastrin-dependent tumor growth.

Over-expression of growth factors can contribute to the development and progression of cancer, and gastrins in particular have been implicated in accelerating the development of gastrointestinal cancers. Previously our group showed that hypoxia, cobalt chloride (a hypoxia mimetic) and zinc chloride could activate the expression of the gastrin gene in vitro. To characterise activation of the gastrin promoter by zinc ions further in vivo, TALEN technology was used to engineer a luciferase reporter construct into the endogenous human gastrin gene promoter in SW480 colon cancer cells. Gastrin promoter activity in the resultant Gast(luc) SW480 colon cancer cells was then measured by bioluminescence in cell culture and in tumour xenografts in SCID mice. Activation of intracellular signalling pathways was assessed by Western blotting. Activation of the gastrin promoter by zinc ions was concentration dependent in vitro and in vivo. Zinc ions significantly stimulated phosphorylation of ERK1/2 (MAPK pathway) but not of Akt (PI3K pathway). We conclude that the endogenous gastrin promoter is responsive to zinc ions, likely via activation of the MAPK pathway.

Gastrin and its precursors have been shown to promote mitogenesis and angiogenesis in gastrointestinal tumors. Hypoxia stimulates tumor growth, but its effect on gastrin gene regulation has not been examined in detail. Here we have investigated the effect of hypoxia on the transcription of the gastrin gene in human gastric cancer (AGS) cells. Gastrin mRNA was measured by real-time PCR, gastrin peptides were measured by RIA, and gastrin promoter activity was measured by dual-luciferase reporter assay. Exposure to a low oxygen concentration (1%) increased gastrin mRNA concentrations in wild-type AGS cells (AGS) and in AGS cells overexpressing the gastrin receptor (AGS-cholecystokinin receptor 2) by 2.1 ± 0.4- and 4.1 ± 0.3-fold (P < 0.05), respectively. The hypoxia mimetic, cobalt chloride (300 μM), increased gastrin promoter activity in AGS cells by 2.4 ± 0.3-fold (P < 0.05), and in AGS-cholecystokinin receptor 2 cells by 4.0 ± 0.3-fold (P < 0.05), respectively. The observations that either deletion from the gastrin promoter of the putative binding sites for the transcription factor hypoxia-inducible factor 1 (HIF-1) or knockdown of either the HIF-1α or HIF-1β subunit did not affect gastrin promoter inducibility under hypoxia indicated that the hypoxic activation of the gastrin gene is likely HIF independent. Mutational analysis of previously identified Sp1 regulatory elements in the gastrin promoter also failed to abrogate the induction of promoter activity by hypoxia. The observations that hypoxia up-regulates the gastrin gene in AGS cells by HIF-independent mechanisms, and that this effect is enhanced by the presence of gastrin receptors, provide potential targets for gastrointestinal cancer therapy.

Amidated gastrin-releasing peptide (GRP) is the prototypical autocrine growth factor. Nonamidated peptides derived from the C terminus of pro-GRP are also biologically active in colorectal cancer (CRC) cell lines in vitro, via a receptor distinct from the GRP receptor. The aims of this study were to measure the amounts of pro-GRP-derived peptides in human CRC cell lines and tumors, characterize the immunoreactive peptide, and investigate its effect on proliferation in vitro and in vivo. Pro-GRP-derived peptides were quantitated by region-specific ELISA in extracts of five human CRC cell lines and 20 tumors. The immunoreactive material was purified by HPLC and its mass and sequence established by mass spectrometry. The concentration of GRPamide was determined by RIA. Proliferation of DLD-1 cells and murine gastrointestinal mucosa was measured by [(3)H]-thymidine incorporation and mitotic index, respectively. In CRC cell extracts, ELISA for pro-GRP-derived peptides detected 3-152 fmol/10(6) cells. The immunoreactive peptide was purified and identified as pro-GRP42-98. Resected stage III tumors contained significantly less pro-GRP immunoreactivity than stage II tumors, and no amidated GRP was detected in cell lines or tumors. Stable transfection of DLD-1 cells with pro-GRP short hairpin RNA, or treatment with a monoclonal anti-pro-GRP antibody, significantly reduced proliferation. Pro-GRP42-98, pro-GRP47-68, and pro-GRP80-97 significantly stimulated mitosis in colonic, but not small intestinal, mucosa of 10-wk-old mice. We conclude that nonamidated peptides derived from the C terminus of pro-GRP are expressed in significant quantities in CRC cell lines and tumors and stimulate the proliferation of CRC cells and of normal colonic mucosa. Such peptides are attractive targets for novel CRC therapies.

Gastrins are peptide hormones important for gastric acid secretion and growth of the gastrointestinal mucosa. We have previously demonstrated that ferric ions bind to gastrins, that the gastrin-ferric ion complex interacts with the iron transport protein transferrin in vitro, and that circulating gastrin concentrations positively correlate with transferrin saturation in vivo. Here we report the effect of long-term dietary iron modification on gastrin-deficient (Gas(-/-)) and hypergastrinemic cholecystokinin receptor 2-deficient (Cck2r(-/-)) mice, both of which have reduced basal gastric acid secretion. Iron homeostasis in both strains appeared normal unless the animals were challenged by iron deficiency. When fed an iron-deficient diet, Gas(-/-) mice, but not Cck2r(-/-) mice, developed severe anemia. In iron-deficient Gas(-/-) mice, massive splenomegaly was also apparent with an increased number of splenic megakaryocytes accompanied by thrombocytosis. The expression of the mRNA encoding the iron-regulatory peptide hepcidin, Hamp, was down-regulated in both Cck2r(-/-) and Gas(-/-) mice on a low-iron diet, but, interestingly, the reduction was greater in Cck2r(-/-) mice and smaller in Gas(-/-) mice than in the corresponding wild-type strains. These data suggest that gastrins play an important direct role, unrelated to their ability to stimulate acid secretion, in hematopoiesis under conditions of iron deficiency.

There are many precedents for the production from a single precursor of multiple peptides, with independent receptors and different bioactivities. Gastrin-releasing peptide (GRP) is initially synthesized as amino acids 1-27 of a 125-residue precursor, proGRP, and is subsequently cleaved and amidated to form GRP18-27. We investigated the hypothesis that C-terminal proGRP peptides are also biologically active. Human proGRP18-125 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. Recombinant proGRP18-125 stimulated proliferation and migration of the human colorectal carcinoma cell line DLD-1. The observations that an antagonist selective for the GRP receptor did not inhibit activity in either proliferation or migration assays and that the recombinant peptide did not bind to either the GRP receptor or orphan receptor BRS-3 indicated that neither activity was mediated by the known GRP receptors. Recombinant human proGRP31-125 and proGRP42-98 were also prepared and shown to stimulate proliferation of DLD-1 cells and the human prostate carcinoma cell line DU145. The synthetic peptides proGRP47-68 and [Tyr79]proGRP80-97 stimulated inositol phosphate production, MAPK kinase activity, and proliferation and migration of DLD-1 cells. Binding sites for both radioiodinated synthetic peptides were demonstrated on DLD-1 cells. Each peptide was able to compete with the other for binding, and a GRP receptor antagonist did not inhibit binding of either peptide. We conclude that peptides derived from the C terminus of proGRP are biologically active and that their activity is mediated by a receptor distinct from the two known GRP receptors.

Gastrin-releasing peptide (GRP) has a widespread distribution and multiple stimulating effects on endocrine and exocrine secretions and metabolism. The prohormone for GRP (ProGRP, 125 amino acids) is processed to the amidated, biologically active end products GRP(1-27) and GRP(18-27). Amidated forms of GRP are putative autocrine or paracrine growth factors in a number of cancers including colorectal cancer. However, the potential role and biological activity of proGRP has not been investigated. Using a newly developed antisera directed to the N terminus of human proGRP, proGRP immunoreactivity was detected in all of the endometrial, prostate, and colon cancer cell lines tested and in nine of 10 resected colorectal carcinomas. However, no amidated forms were detected, suggesting an attenuation of processing in tumors. Recombinant proGRP was expressed as a His-tag fusion protein and purified by metal affinity chromatography and HPLC. ProGRP stimulated proliferation of a colon cancer cell line and activated MAPK, but unlike GRP(18-27)amide had no effect on inositol phosphate production. ProGRP did not compete with iodinated bombesin in binding assays on Balb-3T3 cells transfected with the known GRP receptors, GRP-R or BRS-3. We conclude that proGRP is present in a number of cancer cell lines and in resected colorectal tumors and is biologically active. Our results suggest that antagonists to GRP precursors rather than the amidated end products should be developed as a treatment for colorectal and other cancers that express proGRP-derived peptides.

Amidated and nonamidated progastrin-derived peptides have distinct biological activities that are mediated by a range of receptor subtypes. The objective was to determine the nature of the stored and secreted progastrin-derived peptides and to investigate whether progastrin release is regulated by gastric acidity. Using an antiserum directed to the C terminus of progastrin for identification and to monitor purification, C-terminal flanking peptides (CTFP) of progastrin (prog(76-83), prog(77-83), and prog(78-83) in approximately equivalent amounts) were isolated and identified from extracts of sheep antrum using ion exchange, HPLC, and mass spectrometry. Only trace amounts of full-length progastrin were present. Progastrin CTFP was the predominant progastrin-derived peptide in the antrum [progastrin CTFP/gastrin amide (Gamide) = 3]. Similarly, progastrin CTFP was the major circulating form in the antral (CTFP, 710 +/- 62 pmol/liter; Gamide, 211 +/- 35 pmol/liter) and jugular (CTFP, 308 +/- 16 pmol/liter; gastrin amide, 32 +/- 3 pmol/liter) veins. Alteration of gastric acidity in sheep by iv infusion of a H/K-adenosine triphosphatase inhibitor or somatostatin or by intragastric infusion of HCl demonstrated that the CTFP concentrations changed, although to a lesser extent than the changes in circulating gastrin amide. We conclude that the CTFP of progastrin is the major stored and circulating species of the gastrin gene, and that it is secreted in a regulated fashion rather than constitutively. Because full-length progastrin is bioactive, but is only a minor antral and secreted form, determination of the biological activity of the C-terminal flanking peptides will be important for a complete understanding of gastrin endocrinology.

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.

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.

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.

It has been proposed that regular restoration of blood flow is beneficial during liver surgery with vascular isolation. The aim of this study was to compare intermittent versus continuous occlusion of blood flow to the resected liver, as measured by survival, liver function tests and histological examination. Male Wistar rats were allocated to have either sham operation, 80 per cent liver resection with 30 min continuous occlusion, or resection with intermittent occlusion (two 15-min periods of ischaemia separated by 5 min reperfusion). There was no significant difference in the survival rate, with 17 of 20 animals surviving in both ischaemia groups. There was a significantly higher serum alanine aminotransferase concentration on day 1 in animals receiving continuous occlusion, and significantly higher concentrations of bilirubin on days 8 and 23 and of serum alkaline phosphatase on day 23 in those having intermittent ischaemia (P < 0.001). There was a significantly greater loss and slower regaining of weight when occlusion was intermittent. Histological changes were significantly more pronounced at day 23 in animals undergoing intermittent ischaemia (P < 0.05), although these were in only one grading. Continuous and intermittent occlusion affected the components of liver function tests differently, with no advantage for one technique. These findings suggest that periodic release of inflow occlusion during liver surgery is not necessary.

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.

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.

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.

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.

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.

Vasoactive Intestinal Peptide (VIP) is a 28-amino-acid putative neurotransmitter that may have a role in the regulation of myometrial blood flow and uterine contractility. The chronically cannulated fetal sheep preparation was used to examine the fetal clearance and placental transfer of VIP. Metabolic Clearance Rate (MCR) and placental transfer of VIP were measured by alternate steady-state infusion of VIP into the mother and fetus. Plasma concentrations of VIP were measured by radioimmunoassay. MCR was similar in the pregnant (45 +/- 10 ml/kg/min) and nonpregnant ewes (35 +/- 5 ml/kg/min). However, compared to both pregnant and nonpregnant ewes, fetal MCR was significantly increased at 77 +/- 15 ml/kg/min, indicating highly developed clearance mechanisms in the fetus. VIP did not cross the placenta in either direction. Both the placenta and fetal liver metabolized VIP and contributed to the elevated fetal clearance of VIP. The results show that VIP in fetal tissue is unlikely to influence maternal uterine activity with any VIP-mediated effects emanating from maternal and/or placental sources.

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.

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.

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.