Phenobarbital in comparison with carbon tetrachloride and phenobarbital-induced cirrhosis in rat liver regeneration.

BACKGROUND: The simultaneous administration of carbon tetrachloride (CCl4) and phenobarbital in the rat produces one of the most common experimental models of liver cirrhosis. As phenobarbital also has a hepatotrophic effect, its role in liver regeneration following partial hepatectomy (HTX) is not elucidated. PURPOSE: To examine the effect of long-term administration of phenobarbital in liver regeneration after HTX with regard to CCl4-induced cirrhotic rat model. Materials and Methods. The liver regeneration following HTX in phenobarbital-treated rats (PB rats) was compared to that seen in cirrhotic rats (LC rats), induced by oral gavage of CCl4 and phenobarbital, and normal rats. The effect of the withdrawal of phenobarbital was also examined. Liver regeneration was estimated 24 h after the HTX by measuring the liver weight, the DNA content in the liver, and [3H]thymidine incorporation into the DNA. RESULTS: Treatment with CCl4 and phenobarbital caused liver deformity, and the highest percentage of liver weight regeneration was seen in LC rats with this deformity, even though [3H]thymidine incorporation into the DNA was impaired in this group. Phenobarbital had a hepatotrophic effect, but its withdrawal caused a decrease in liver mass and cessation of body weight gain. The change in the DNA content 24 h after HTX was negative in PB rats. CONCLUSIONS: Liver regeneration could not be estimated using liver or body weight in the PB or LC rat model. [3H]Thymidine incorporation into the DNA was reliable indicator of liver regeneration in the different liver states during the early stage after HTX. Although the DNA content with respect to total liver mass was obscured due to liver inflation in PB rats, [3H]thymidine incorporation into the DNA between PB rats and normal rats was similar.

Modulation of basic fibroblast growth factor effect by retinoic acid in cultured retinal pigment epithelium.

PURPOSE: We investigated the effect of retinoic acid (RA) on basic fibroblast growth factor (bFGF)-stimulated proliferation of cultured human retinal pigment epithelial (hRPE) cells and of 125I-bFGF-binding to the bFGF plasma membrane receptors of hRPE. METHODS: Proliferation of hRPE cells in the presence of increasing concentrations of bFGF and bFGF + RA was measured by 3H-thymidine incorporation into hRPE cells. To characterize bFGF receptors, hRPE cells were incubated at 4 degrees C with 125I-bFGF in the presence or absence of heparin. RESULTS: Basic-FGF stimulated 3H-thymidine incorporation into hRPE cells in a dose-dependent manner. RA inhibited bFGF-stimulated 3H-thymidine incorporation in the presence or absence of heparin. Increasing concentrations of unlabeled bFGF decreased the binding of 125I-bFGF to hRPE cells. Scatchared analysis indicated the presence of high and low affinity binding sites for bFGF with an apparent affinity Kd of 50 pM and 330 pM, respectively, and a binding capacity (Bmax) of 1.25 X 10(5) and 3.38 X 10(5) binding sites per cell. Inhibition of 125I-bFGF binding was also possible by the carboxyl-terminal region peptide fragment bFGF-(106-120)-NH2, but not amino-terminal region peptide fragment bFGF-(1-24)-NH2. The addition of heparin to the medium during binding studies did not prevent RA from inhibiting binding of 125I-bFGF to hRPE cells. Scatchard analysis demonstrated that, in the presence of heparin, there is a decrease in the number of high affinity binding sites (from 1.12 +/- 0.11 x 10(5) to 0.7 +/- 0.03 x 10(5) binding sites per cell, a reduction of 36.7 +/- 0.04%, n = 3, p < 0.05). There was no significant change in affinity constants. CONCLUSIONS: These results suggest that RA inhibits bFGF cell proliferation in hRPE cells by decreasing the bFGF receptor number.

Treatment of cirrhotic rats with epidermal growth factor and insulin accelerates liver DNA synthesis after partial hepatectomy.

Prevention of postoperative hepatic failure is important after hepatic resection. In patients with cirrhosis, impaired liver function and regenerative capacity after major hepatic resection are associated with increased morbidity and mortality. In this study, a combination of epidermal growth factor (EGF) and insulin were used as hepatotrophic factors in an attempt to stimulate DNA synthesis after 70% hepatectomy (HTX). Regenerative capacity was evaluated in normal and cirrhotic rat liver by measuring DNA synthesis in vivo. Micronodular liver cirrhosis was established by the simultaneous oral administration of CCl4 and phenobarbital. Epidermal growth factor plus insulin was injected subcutaneously immediately after and 12 h after HTX or sham operation was performed. Rats were killed 24 h after the operation and liver regeneration was estimated by [3H]-thymidine incorporation into DNA as well as an autoradiographic nuclear labelling index. Hepatectomy increased [3H]-thymidine incorporation significantly in both normal and cirrhotic rats. In cirrhotic rats, [3H]-thymidine incorporation after HTX was significantly lower than in normal rats and administration of a combination of EGF and insulin after HTX enhanced [3H]-thymidine incorporation. In conclusion, DNA synthesis 24 h after HTX is decreased in cirrhotic rats compared with normal rats and EGF supplementation with insulin accelerates DNA synthesis in hepatectomized cirrhotic rats. The data suggest that administration of combinations of exogenous hepatotrophic factors may play a useful role in the treatment of cirrhotic patients undergoing major hepatic resection.

Somatostatin inhibits bombesin-stimulated Gi-protein via its own receptor in rabbit colonic smooth muscle cells.

The effect of somatostatin on Bombesin-induced contraction of isolated rabbit colonic smooth muscle cells was examined. Preincubation of muscle cells with somatostatin 10(-6) M inhibited bombesin-induced contraction. To characterize somatostatin receptors, muscle cells (10(5) cells/tube) were incubated at 24 degrees C with 125I-Tyr0-SS-28. Binding reached a plateau at 60 sec and was reversible by addition of excess synthetic SS-28. Scatchard analysis revealed high and low affinity bindings sites (Ka = 0.48 +/- 0.01 and 40 +/- 13 (nM +/- S.E.), 1830 +/- 433 and 65820 +/- 13183 receptors/cell +/- S.E.). Inhibition of 125I-Tyr0-SS-28 binding was possible with biologically active analogs of somatostatin, indicating the specificity of the receptors to somatostatin. Binding of 125I-Tyr0-SS-28 was inhibited by GTP gamma s, a nonhydrolysable analog of guanosine 5'-triphosphate, whereas adenosine 5'-triphosphate at a high concentration (100 microM) slightly inhibited the binding. Further, pretreatment of muscle cells with pertussis toxin at 37 degrees C abolished binding of 125I-Tyr0-SS-28, although pretreatment of cells with cholera toxin had no effect. Inasmuch as Gi protein is postulated as a signal protein, muscle cells were labeled with 3H-methionine, before stimulation with Bombesin (10(-6) M), in the presence and absence of somatostatin (10(-6) M). The cells were then lysed and Gi was precipitated by a Gi specific antibody. Gi synthesis was stimulated by bombesin at 60 sec and somatostatin inhibited it (6114 +/- 986 vs. 2998 +/- 841 cpm +/- S.E., P < .05). These data suggest that colonic smooth muscle cells contain specific receptor for somatostatin-28 and that somatostatin reverses bombesin-induced contraction regulated by Gi-type G protein.

Preferential suppression of insulin-stimulated proliferation of cultured hepatocytes by somatostatin: evidence for receptor-mediated growth regulation.

The role of somatostatin (SS-14) in the regulation of rat liver regeneration was examined by using thymidine incorporation into hepatocyte DNA labeled with tritiated thymidine, a nuclear-labeling index, and the binding of 125I-tyr11-SS-14 to hepatocytes isolated at various times after partial hepatectomy. The data demonstrated no suppressive effect of SS-14 on insulin and glucagon-stimulated thymidine incorporation into hepatocyte DNA as early as 2 h after partial hepatectomy. These data were substantiated by a nuclear labeling index studies. At 2 h, 125I-tyr11-SS-14 binding to its specific sites on isolated hepatocytes was undetectable. There was a time-dependent increase in binding of 125I-tyr11-SS-14 to hepatocytes obtained at various times after partial hepatectomy. There was a significant decrease in the number of binding sites after partial hepatectomy as determined by Scatchard analysis. The data were supported by autoradiography analysis of affinity labeled 125I-tyr11-SS-14-binding protein complex followed by SDS-PAGE. SS-14 also inhibited intracellular cAMP in hepatocytes obtained at 18 h after hepatectomy. The data are consistent with the hypothesis that SS-14 participates via its own receptor in the regulation of the liver regeneration.

Impaired phorbol ester-induced hepatocyte proliferation in cirrhosis.

Cirrhotic livers are considered to regenerate less actively than normal livers after hepatic resection. Little is known about the mechanisms responsible for impaired capacity of regeneration in cirrhotic liver. In the present study, we investigated the effect of phorbol ester on hepatocyte proliferation in healthy and cirrhotic hepatocytes, using one of the phorbol esters, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which has a direct effect on activation of protein kinase C (PKC). Cirrhosis was established by the administration of carbon tetrachloride and phenobarbital to rats. Healthy and cirrhotic hepatocytes were isolated from Wistar male rats by a two-step collagenase perfusion technique. DNA synthesis was estimated by [3H]thymidine incorporation into DNA and by autoradiographic nuclear labeling index. [3H]Thymidine incorporation was measured 24 hr after hepatocytes were stimulated by appropriate reagents. TPA (50 nM) stimulated [3H]thymidine incorporation in healthy hepatocytes (control vs TPA, 991 +/- 247 vs 2569 +/- 766 mean +/- SEM cpm/microgram DNA; P < 0.05), whereas TPA (50 nM) failed to stimulate in cirrhotic hepatocytes (control vs TPA, 1144 +/- 184 vs 1304 +/- 187 cpm/microgram DNA; NS). Staurosporine, a specific PKC inhibitor, suppressed [3H]thymidine incorporation in TPA-stimulated healthy hepatocytes (806 +/- 263 cpm/microgram DNA; P < 0.05); however, it had no effect on cirrhotic hepatocytes (1295 +/- 180 cpm/microgram DNA; NS). An autoradiographic nuclear labeling index exhibited the same results with [3H]thymidine incorporation. We conclude that TPA stimulates hepatocyte proliferation in healthy rat hepatocytes but has no effect on cirrhotic hepatocytes.

Vasopressin stimulates DNA synthesis in cultured rat hepatocytes.

Liver regeneration following partial hepatectomy is significantly impaired in rats with hereditary vasopressin (AVP) deficiency. This suggested that AVP might have a direct effect on cultured rat hepatocytes. Hepatocytes from male Sprague-Dawley rats were isolated using a two-step collagenase perfusion technique and plated at a density of 10(5)/16-mm Primaria plate. After a suitable attachment period, hepatocytes were incubated with minimal essential media, AVP, AVP plus a specific AVP antagonist, or oxytocin. Hepatocyte proliferation was measured by [3H]thymidine incorporation ([3H]Thy) into hepatocyte DNA. AVP (10 nM) increased [3H]Thy significantly (and this effect was blocked by an AVP-specific antagonist (50 nM). Oxytocin had no effect on hepatocyte DNA synthesis. To further investigate the influence of AVP on hepatocyte proliferation, the effect of AVP on transforming growth factor-alpha (TGF-alpha)-stimulated hepatocyte proliferation was also studied. This combination was chosen based on the ability of AVP to inhibit the biologic effects of EGF (a TGF-alpha analog). There was significant attenuation of TGF-alpha (50 nM)-stimulated [3H]Thy in the presence of AVP (10 nM). In summary: (1) AVP stimulates proliferation of cultured rat hepatocytes. (2) The effect of AVP can be significantly abolished by a specific AVP antagonist. (3) The proliferative response of AVP is specific. (4) AVP significantly attenuates TGF-alpha-stimulated hepatocyte hepatic DNA synthesis. Further studies should elucidate the mechanisms for the effects of AVP on hepatic proliferation alone or in combination with other factors.

Molecular and functional studies of inhibitory G protein in RINm5F cells.

Inhibitory G proteins (Gi) play an important role in cell proliferation. In order to characterize Gi proteins in RINm5F (RIN) cells, we first established RIN cells in cell culture. Immunoblot analysis was performed on extracted G proteins using Western blot techniques and a Gi-specific antibody. We identified three prominent bands consistent with three distinct inhibitory alpha subunits of membrane-bound G protein (Gi) in RIN cells. In contrast, we identified only one prominent distinct inhibitory alpha subunit of G protein in an equal quantity of membrane-protein in our control (normal rat pancreas). In several cell types, Gi is known to mediate the inhibitory action of somatostatin on intracellular cyclic AMP (cAMP) accumulation. Therefore, we studied the action of the long-acting analogue of somatostatin, octreotide (SMS), on basal and 3-isobutyl-1-methylxanthine-stimulated cAMP accumulation in RIN cells. SMS did not inhibit cAMP accumulation or tritiated thymidine incorporation into DNA (TTID) in RIN cells. However, when treatment with SMS is supplemented with the nonhydrolyzable analogue of guanine nucleotide, Gpp(NH)p (Gpp), which is known to dissociate G proteins into its constitutive subunits, then SMS+Gpp induced an inhibitory action and significantly reduced cAMP accumulation and TTID. These data are consistent with the concept of qualitatively and functionally altered inhibitory G protein expression in the insulin-producing, islet cell (RINm5F) rat insulinoma tumor cell line. Further study of human tumors will lead to new insights into the clinical implications of G protein-mediated signal transduction in insulinoma.

The effects of transforming growth factor alpha and somatostatin on regenerating hepatocytes in the rat.

Transforming growth factor alpha (TGF alpha) stimulates DNA synthesis in adult rat hepatocytes, and plays a physiological role after partial hepatectomy by an autocrine mechanism. Somatostatin (SS-14) is a potent inhibitor of gastrointestinal function and inhibits proliferation in various cell types. We examined the proliferative effect of TGF alpha and the inhibitory effect of SS-14 on hepatocytes isolated at various times after partial hepatectomy. To study the mechanism of SS-14 further, we treated rats with the long acting SS-14 analog, octreotide, before or after 70% hepatectomy to determine whether or not a differential effect could be seen. We confirmed the proliferative effects of TGF alpha, and the inhibitory action of SS-14 in the early phase of liver regeneration in vitro. Regenerating hepatocytes isolated from hepatectomized livers respond to TGF alpha only at early time points (2 h) but do not respond to SS-14. In addition, the long acting SS-14 analog, octreotide, inhibited hepatic regeneration only when administered prior to hepatectomy. We conclude that exogenous peptide stimulation is effective only in the early phase of the hepatic proliferative response. After the initial changes brought about by hepatectomy, subsequent steps of the regenerative process appear refractory to external stimuli.

Hepatectomy impairs hepatic processing of somatostatin-14.

Somatostatin-14 (SS-14) inhibits the hepatotrophic effect of a variety of growth factors in cultured hepatocytes. We hypothesized that hepatic somatostatin processing might be altered during regeneration. Male Sprague-Dawley rats underwent the intraportal injection of radiolabeled SS-14 after sham or 70% hepatectomy. To study the mechanisms of hepatic SS-14 transport in the rat, the lysosomal enzyme inhibitors, chloroquine and leupeptin, and the microtubule inhibitors, vinblastine and colchicine, were administered 1 to 2 hours prior to the intraportal injection of SS-14. Bile was collected, organs were weighed, and radioactivity was quantitated. The analysis of serial timed collections of bile revealed that, for saline, chloroquine, and leupeptin, peak biliary radioactivity appeared at 20 minutes. Pretreatment with vinblastine and colchicine abolished the 20-minute peak of radioactivity. The appearance of biliary and hepatic iodine 125-SS-14 (125I-[tyr11]-SS-14) at various times after 70% hepatectomy showed a significant decrease starting at 2 hours, which persisted for up to 24 hours. In regenerating liver, both vinblastine and chloroquine decreased 125I-[tyr11]-SS-14 in bile and the liver. In summary, after sham or 70% hepatectomy, vinblastine and colchicine inhibit biliary and increase hepatic 125I-[tyr11]-SS-14 accumulation. After 70% hepatectomy was performed, chloroquine also inhibited 125I-[tyr11]-SS-14 accumulation. We concluded that an important mechanism for hepatic regeneration is decreased responsiveness to SS-14, by decreased SS-14 uptake and increased SS-14 degradation.

Identification and partial characterization of a somatostatin-14 binding protein on rat liver plasma membranes.

Binding of somatostatin-14 to rat liver plasma membranes was characterized with 125-labeled[tyr11] somatostatin-14. Binding at 24 degrees C reached a plateau at 50 min and was reversible by synthetic somatostatin-14. Scatchard analysis revealed a single class of binding sites (affinity constant = 2.4 +/- 0.2 nmol/L, binding capacity = 148 +/- 0.02 fmol/mg protein). Specificity for somatostatin-14 was demonstrated by the inhibition of 125I-[tyr11]somatostatin-14 binding by biologically active somatostatin analogs but not by a biologically inactive somatostatin analog or unrelated peptides. The radioiodinated binding site complex could be cross-linked with disuccinimidyl suberate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel autoradiography revealed a 70,000-Da band. Dithiothreitol, a reducing reagent, did not alter the mobility of the band, and the band could be abolished in the presence of 10 mumol/L synthetic somatostatin-14. Covalently cross-linked, iodinated binding protein complexes could be solubilized by the nonreducing detergents Zwittergent 3-12 and 3-([3-cholamidopropyl] diethylammonio)-1-propanesulfonic acid (CHAPS). Solubilized complex bound to wheat-germ agglutinin-agarose columns and was eluted by N,N',N"-triacetylchitotriose. Binding to wheat-germ agglutinin agarose columns was lost after pretreatment with endo-beta-N-acetylglucosaminidase F. Binding studies with liver plasma membranes, 125I-labeled[tyrosine11]somatostatin-14 and guanine nucleotides showed inhibition of binding in the presence of guanine nucleotides. These results indicate that the purified rat liver plasma membranes contain a specific binding protein for somatostatin-14, the binding protein appears to be glycosylated and somatostatin-14 binding to rat liver plasma membranes may be regulated by G proteins.

Transforming growth factor-alpha (TGF-alpha) improves hepatic DNA synthesis after hepatectomy in cirrhotic rats.

Impaired liver regeneration in cirrhosis complicates the surgical treatment of liver tumors which arise in this setting. We developed a rat model to investigate the regenerative response of cirrhotic liver after hepatectomy and studied the effect of exogenous transforming growth factor-alpha (TGF-alpha), a potent liver mitogen. Micronodular cirrhosis was established by the simultaneous administration of CCl4 and phenobarbital. Hepatic DNA synthesis ([3H]thymidine incorporation into DNA) 24 hr after partial hepatectomy in cirrhotic rats was 15.6 +/- 3.4 cpm/micrograms DNA (means +/- SEM), which was significantly lower than in normal rats (37.3 +/- 3.4 cpm/micrograms DNA, P less than 0.05). Exogenous TGF-alpha (30 nmol/kg, sc every 12 hr) significantly improved [3H]thymidine incorporation (35.6 +/- 8.2 cpm/micrograms DNA, P less than 0.05). An autoradiographic nuclear labeling index also confirmed increased DNA synthesis (6.7% vs 13.4%). TGF-alpha had no effect on normal regenerating liver (42.5 +/- 8.8 cpm/micrograms DNA, NS). Although the significance of TGF-alpha-enhanced liver regeneration in cirrhosis has yet to be assessed, this model may be useful for the study of mechanisms which control hepatic proliferation.

Inhibition of DNA synthesis by somatostatin in rat hepatocytes stimulated by hepatocyte growth factor or epidermal growth factor.

The antiproliferative effects of somatostatin on hepatocytes stimulated by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) were investigated using primary cultures of adult rat hepatocytes. Somatostatin inhibits HGF-induced (at a dose of 10 ng/mL) or EGF-induced (at a dose of 100 ng/mL) 3H-thymidine incorporation into hepatocytes in a dose-dependent manner (10(-10) to 10(-8) M). This inhibition was confirmed by autoradiography. The effect of somatostatin was nontoxic as judged by preserved albumin synthesis, a marker for differentiated hepatocyte function. In the presence or absence of somatostatin, neither HGF nor EGF significantly altered intracellular cyclic adenosine monophosphate (cAMP). We conclude that somatostatin is a potent inhibitor of HGF- or EGF-induced deoxyribonucleic acid synthesis in adult rat hepatocytes. The mechanism of this inhibition appears to be independent of cAMP. The significance of somatostatin in liver regeneration has yet to be assessed.

Inhibitory effects of somatostatin on rat hepatocyte proliferation are mediated by cyclic AMP.

Somatostatin (SS-14) is known as an antigrowth factor for a variety of cell types, including gastrointestinal mucosa, exocrine pancreas, lymphocytes, and some tumors. We have recently identified and biochemically characterized SS-14-binding protein on rat liver plasma membranes (S. E. Raper, P. C. Kothary, and J. DelValle, Gastroenterology 96: A408, 1989; P. C. Kothary et al., Digestion 46 (Suppl 1): 58, 1990). We hypothesized that SS-14 may affect liver growth as well and investigated cellular mechanisms of this phenomenon focusing on the second messenger cAMP. Freshly isolated rat hepatocytes were plated on tissue culture dishes coated with Matrigel (laminin, heparan sulfate, and type IV collagen). The medium was not supplemented with serum or hormones. Either dibutyryl-cAMP (1 mM) or isobutylmethylxanthine (IBMX, 0.1 mM) was added in the presence or absence of SS-14 (10 nM). DNA synthesis was estimated by the rate of [3H]thymidine incorporation into DNA and by the labeling index (an autoradiographic measurement of the number of labeled nuclei). SS-14 significantly inhibited both [3H]thymidine incorporation and labeling index of rat hepatocytes stimulated by dibutyryl-cAMP or IBMX. SS-14 also inhibited intracellular cAMP accumulation stimulated by IBMX. We conclude that SS-14 exerts at least part of its antiproliferative effects via the adenylate cyclase system. Further study using other signal transduction systems may yield more information about mechanisms of hepatocyte growth.

Somatostatin-14 blocks the hepatotrophic effects of insulin in the rat.

UNLABELLED: We hypothesized that somatostatin-14 (SS-14) might inhibit insulin-stimulated hepatic growth. Rat hepatocytes were isolated by a two-step collagenase perfusion technique and cultured on Matrigel. Differentiated hepatocyte function was documented by albumin synthesis. Hepatocytes were incubated with insulin in the presence or absence of SS-14. Hepatocyte proliferation was assessed by tritiated thymidine ([3H]thy) incorporation into DNA. [3H]thy incorporation was increased by 230% in the presence of insulin and was essentially abolished by the addition of SS-14. Insulin-stimulated cyclic-AMP accumulation was also decreased from 190 to 108% of control levels (P less than 0.05) by the addition of SS-14. Pretreatment with pertussis toxin, which inactivates the inhibitory G-protein, Gi, blocked the effect of SS-14. CONCLUSIONS: (i) In the rat, SS-14 effectively blocks insulin-stimulated [3H]thy incorporation into DNA, possibly by blocking intracellular cAMP accumulation. (ii) Pertussis toxin blocks the growth inhibitory effects of SS-14, suggesting that inhibitory G proteins are involved in the mechanism of SS-14 action. Somatostatin may be useful in studying the role of second messengers in cell growth.

The liver plays an important role in the regulation of somatostatin-14 in the rat.

Since little is known about the in vivo disposition of circulating somatostatin-14 (SRIF-14), we examined hepatic processing of SRIF-14 in the rat. Three minutes after the intraportal injection of iodine 125 (125I)-labeled SRIF-14, 16.0 +/- 2.0% of the injected dose is localized to the liver. In the presence of unlabeled SRIF-14, hepatic uptake can be decreased by 68%. Five minutes after the intraportal injection of 125I-SRIF-14, 9.5 +/- 1.4% of the tracer is localized to the liver, more than any other organ tested. Serial collections of bile reveal peak radioactivity at between 10 and 20 minutes. Simultaneous administration of unlabeled SRIF-14 decreases biliary radioactivity by 40%. HPLC analysis of radioactive bile reveals a chromatographic profile similar to that of intact SRIF and is 73% immunoprecipitable by an anti-SRIF antibody. Pretreatment with chloroquine, a lysosomal enzyme inhibitor, does not significantly decrease biliary radioactivity. We conclude that the data are consistent with saturable hepatic uptake and predominantly nonlysosomal transcellular transport.

NH2-terminal of gastrin-17 in duodenal ulcer disease: identification of progastrin-17.

Serum gastrin concentrations were measured using antisera with specificity for the carboxyl and amino terminus of gastrin-17 in 50 healthy subjects and 18 patients with active duodenal ulcer disease (DU). The amino terminal of gastrin-17 immunoreactivity was significantly higher in DU patients than in healthy subjects. NH2-terminus of gastrin-17 immunopurified material from serum of DU patients was subjected to Sephadex G50 column chromatography and eluates were monitored by an additional antiserum EG10 that recognizes COOH-terminally extended gastrin. Besides the NH2 terminal tridecapeptide of gastrin-17, COOH-terminally extended progastrin was found. This may reflect abnormal processing of gastrin in patients with active duodenal ulcer disease.

Identification of gastrin molecular variants in gastrinoma syndrome.

The molecular species of gastrin in the circulation and in tumor extracts were studied in two groups of patients: (1) with benign gastrinoma and (2) with gastrinoma with liver metastases. Radioimmunoassays (RIAs) and immunoaffinity chromatography for the amino (NH2)- and amidated COOH-terminus of gastrin-17 (antiserum G17) and the NH2-terminus of gastrin-34 (antiserum G34) were employed. In both benign and metastatic tumors the molecular forms of gastrin in boiling water extracts measured by the gastrin-17 NH2- and COOH-terminal assays were similar. In addition to a molecular component resembling the amidated gastrin-17, there were also significant amounts of larger molecular weight (mol. wt.) forms. The larger mol. wt. forms absorbed by the NH2-terminus of G17 antiserum corresponded to the COOH-terminus-extended forms of gastrin-17. Furthermore, larger mol. wt. gastrins immunopurified by antiserum to the NH2-terminus of gastrin-34 corresponded to gastrin-34 extended molecules. Sera of patients with liver metastases had higher concentrations of the NH2-terminal of gastrin-17 whereas sera of patients with benign gastrinoma contained predominantly gastrins detected by the COOH-terminal assay. These results suggest that: (a) there are differences in the molecular pattern of gastrin in the circulation of patients with benign and metastatic gastrinomas; (b) gastrins which are fully processed with carboxy-terminal amidation predominate in the circulation of patients with benign gastrinoma; and (c) gastrins containing the gastrin-17 and COOH-terminally extended gastrin-17 and gastrin-34 precursor molecules occur in high concentration in the circulation of gastrinoma patients with metastases to the liver.

Evaluation of NH2-terminus gastrins in gastrinoma syndrome.

Forty-six patients with the gastrinoma syndrome were divided into 2 categories: 1) benign sporadic gastrinoma (n = 30), and 2) gastrinoma with metastases to liver (n = 16). Thirteen of the 46 patients had multiple endocrine neoplasia type I syndrome. Serum gastrin levels in patients fasted overnight were determined by RIA using antisera directed toward the NH2- and COOH-terminals of heptadecapeptide gastrin (G17) and the NH2-terminus of the triacontatetrapeptide (G34). These results were compared with findings in 50 normal subjects. In the normal subjects, the mean COOH-terminal gastrin-17 level was higher [65 +/- 8 (+/- SEM) pg/ml] than the NH2-terminal gastrin-17 level (11 +/- 0.2 pg/ml) and lower than the NH2-terminal gastrin-34 level (134 +/- 20 pg/ml). The levels of NH2-terminal gastrin-17 were higher in patients with metastatic disease than in those with benign gastrinoma, whereas the COOH-terminal gastrin-17 and the NH2-terminal gastrin-34 levels were similarly high in both groups. The mean ratio of NH2-terminal gastrin-17 to COOH-terminal gastrin-17 was less than 1 in normal subjects (0.22 +/- 0.02) and benign gastrinoma patients (0.2 +/- 0.04), and it was 2.2 +/- 0.41 in the patients with metastatic gastrinoma. An NH2 to COOH gastrin-17 ratio greater than 1 was found in 13 of 16 patients with metastatic gastrinoma, but in none of the patients with benign gastrinoma or normal subjects. Similar results were found in multiple endocrine neoplasia type I patients with benign and metastatic disease. A high NH2 to COOH gastrin-17 ratio is suggestive of metastatic gastrinoma. In 4 patients with metastatic gastrinoma, the NH2 to COOH gastrin-17 ratio fell in parallel with the response to chemotherapy.